Simultaneous Targeting of RGD-Integrins and Dual Murine Double

Article Cite This: J. Med. Chem. 2018, 61, 4791−4809

pubs.acs.org/jmc

Simultaneous Targeting of RGD-Integrins and Dual Murine Double Minute Proteins in Glioblastoma Multiforme Francesco Merlino,†,∇ Simona Daniele,‡,∇ Valeria La Pietra,†,∇ Salvatore Di Maro,*,§ Francesco Saverio Di Leva,† Diego Brancaccio,† Stefano Tomassi,§ Stefano Giuntini,∥,⊥ Linda Cerofolini,∥,⊥ Marco Fragai,∥,⊥ Claudio Luchinat,∥,⊥ Florian Reichart,# Chiara Cavallini,‡ Barbara Costa,‡ Rebecca Piccarducci,‡ Sabrina Taliani,‡ Federico Da Settimo,‡ Claudia Martini,*,‡ Horst Kessler,# Ettore Novellino,† and Luciana Marinelli*,† Downloaded via UNIV OF LIVERPOOL on June 16, 2018 at 17:18:53 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Dipartimento di Farmacia, Università degli Studi di Napoli “Federico II”, via D. Montesano 49, 80131 Napoli, Italy Dipartimento di Farmacia, Università di Pisa, via Bonanno 6, 56126 Pisa, Italy § DiSTABiF, Università degli Studi della Campania “Luigi Vanvitelli”, via Vivaldi 43, 81100 Caserta, Italy ∥ Magnetic Resonance Center (CERM) University of Florence, via L. Sacconi 6, 50019 Sesto Fiorentino (FI), Italy ⊥ Department of Chemistry “Ugo Schiff”, University of Florence, via della Lastruccia 3-13, 50019 Sesto Fiorentino (FI), Italy # Institute for Advanced Study and Center for Integrated Protein Science, Department of Chemistry, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany ‡

S Supporting Information *

ABSTRACT: In the fight against Glioblastoma Multiforme, recent literature data have highlighted that integrin α5β1 and p53 are part of convergent pathways in the control of glioma apoptosis. This observation prompted us to seek a molecule able to simultaneously modulate both target families. Analyzing the results of a previous virtual screening against murine double minute 2 protein (MDM2), we envisaged that Arg-Gly-Asp (RGD)-mimetic molecules could be inhibitors of MDM2/4. Herein, we present the discovery of compound 7, which inhibits both MDM2/4 and α5β1/αvβ3 integrins. A lead optimization campaign was carried out on 7 with the aim to preserve the activities on integrins while improving those on MDM proteins. Compound 9 turned out to be a potent MDM2/4 and α5β1/αvβ3 blocker. In p53-wild type glioma cells, 9 arrested cell cycle and proliferation and strongly reduced cell invasiveness, emerging as the first molecule of a novel class of integrin/MDM inhibitors, which might be especially useful in subpopulations of patients with glioblastoma expressing a functional p53 concomitantly with a high level of α5β1 integrin.

■

INTRODUCTION

resection together with radiation and chemotherapy. The alkylating agent Temozolomide (TMZ) is the first line option treatment, especially in methylated MGMT-carrying patients. More recently, new hopes arise from cytomegalovirus cytotoxic T lymphocytes, dendritic cell vaccine, and immune checkpoint inhibitors that have entered diverse phases of clinical trials for patients with recurrent GBM.3 Due to its malignancy, chemotherapy resistance, and scarcity of therapeutic options, GBM has been the first cancer type

Glioblastoma Multiforme (GBM) is a malignant, fast-growing brain tumor and is the most common primary brain cancer in adults.1 It accounts for about 15% of all brain tumors and occurs in adults generally between the ages of 45 to 70 years. Patients with GBM have a poor prognosis and usually survive less than 12−15 months after diagnosis.2 Very few treated patients survive longer than 5 years, and molecular factors associated with long-term survival, beside the methylation of MGMT (O6-methylguanine DNA methyltransferase) gene, are still largely unknown. Currently, there are no effective long-term treatments for this disease, while the standard of care includes surgical © 2018 American Chemical Society

Received: January 2, 2018 Published: May 18, 2018 4791

DOI: 10.1021/acs.jmedchem.8b00004 J. Med. Chem. 2018, 61, 4791−4809

Journal of Medicinal Chemistry

Article

Chart 1. Chemical Structures of 1−7

target the Arg-Gly-Asp (RGD)-recognizing integrins α5β1 and αvβ3, although it turned out not to bind these receptors (unpublished data). These findings opened up the possibility that a unique compound could target simultaneously MDM proteins and RGD-integrins. This would be particularly important for GBM, as the expression profiling of high-grade glioma revealed that genes encoding for extracellular matrix components (e.g., fibronectin) and their regulators (e.g., integrins) are often affected in the gliomas patients.24,25 In fact, it is wellknown that fibronectin belongs to the cluster of genes associated with a more malignant glioblastoma phenotype. Main receptors for fibronectin are α5β1, αvβ6, αvβ8, and αvβ3 integrins that recognize the RGD motif in their ligands. Until recently, efforts of medicinal chemists were mainly directed toward αvβ3 and culminated in the discovery, by one of us, of Cilengitide (5),26,27 a superpotent, preferential αvβ3 ligand that reached phase III clinical trial for GBM. Recently, in an interesting work, Dontenwill et al. showed that adhesion to fibronectin by U87MG glioma cells primarily depends on the α5β1 integrin and that the simultaneous inhibition of α5β1 and αvβ3 integrins is a more successful approach with respect to the sole inhibition of αvβ3.28 Although few studies address the role and the importance of α5β1 integrin in glioma,29−32 its expression was clinically associated with a more aggressive phenotype in brain tumors and a decreased survival of patients.33 In line with this observation, perturbing the α5β1 function through a selective antagonist of this integrin receptor provokes cell cycle arrest, decreases cell aggressiveness,30 and sensitizes p53 wild-type (p53-wt) glioma cells to chemotherapeutic drugs.34 These results provide a robust rationale for a simultaneous targeting of RGD integrins and MDM proteins to increase the efficacy of anti-GBM therapeutic regimens. In this context, we sought to combine our expertise and available resources in the MDM19−23 and integrins field35−40 to develop multitarget MDM2/4-integrins ligands as potential tools for GBM treatment. In fact, a single molecule capable to hit multiple targets simultaneously might

investigated by The Cancer Genome Atlas (TCGA) research network, in 2008. Among the results, it was found that a reorganization of a region on chromosome 12 that contains the oncogenes cyclin-dependent kinase 4 (CDK4) and the murine double minute 2 protein (MDM2) has been frequently found in patients with GBM. Taking into account the role of MDM2 as primary cellular negative regulator of p53 protein,4 whose importance as genome guardian is well-known,5−7 and starting from the TCGA observation, in the past decade, the MDM2 has been extensively investigated for GBM as well as for other tumors. Accordingly, MDM2 gene amplification has been found in at least 19 tumor types as soft tissue tumors (20%), osteosarcomas (16%), esophageal carcinomas (13%), and neuroblastomas, where overexpression of MDM2 correlates with poor clinical prognosis and poor treatment response to current cancer therapies.8 At present, eight low-molecular weight MDM2 inhibitors are undergoing clinical-stage studies for the treatment of human cancers.9 However, the recent finding that MDM2-selective inhibitors may induce high levels of its homologue MDM4, thus impairing the final clinical response,10−13 is now prompting medicinal chemists to develop potent dual MDM2/4 inhibitors, which are supposed to give rise to more durable effects with respect to either MDM2- (e.g., Nutlin-3, 1 in Chart 1) or MDM4-selective inhibitors (e.g., SJ1722550, 2 in Chart 1). In pioneering studies, Walensky et al. developed a 14-mer α-helix stapled peptide, SAH-p53−8 with a 25-fold higher affinity for MDM4 over MDM2 (MDM2 Kd = 55 nM; MDM4 Kd = 2.3 nM);14 while some other dual inhibitors have been later reported with IC50 falling in micromolar range.15−18 As part of our ongoing efforts in identifying novel modulators of the p53 pathway, useful against GBM,19−22 we have recently reported the identification, through a virtual screening (VS) campaign against our in-house database (DB), of a small molecule (3, Chart 1) as dual MDM2/MDM4 binder (MDM2 IC50 = 93.7 nM; MDM4 IC50 = 4.6 nM) and its potent derivative 4 (MDM2 IC50 = 7.8 nM; MDM4 IC50 = 6.1 nM).23 Interestingly, the lead compound 3 was originally designed to 4792



Article

Scheme 1. Synthetic Strategy for Compounds 6−19a

Reagents and conditions: (a) DIEA, DMF, overnight; (b) 20:80 piperidine/DMF, 1 × 5 min, 1 × 25 min; (c) Fmoc-Gly, HBTU, HOBt, DIEA, 1:1 DCM/DMF, 2 h; (d) 3-(Fmoc-amino)benzoic acid, HBTU, HOBt, DIEA, 1:1 DCM/DMF, 2 h; (e) BocNC(SCH3)NHBoc, HgCl2, DIEA, DMF, 16 h; (f) Pd(PPh3)4, morpholine, 3:2 DCM/DMF, 2 h (×2); (g) R-NH2, PyAOP, HOAt, DIEA, 1:1 DCM/DMF, 16 h; (h) 95:5 TFA/H2O, 2 h. a

probably because of the higher flexibility of the two-methylene linker that allows the proper adaptation of the pendant aromatic moiety in the target protein. This would indicate that the size and nature of this moiety might have an impact on the binding of our compounds to MDM proteins. However, in RGD-mimetic integrin ligands the aromatic substituent, next to the carboxylic acid, can be variably substituted, with only minor changes in the receptor binding affinity as long as a certain flexibility remains.38,40 Thus, to enhance the potency toward the MDM proteins, while preserving the low nanomolar affinity observed toward α5β1 and αvβ3 integrins, we decided to explore the aromatic portion of 7. Hence, a library of compound 7 derivatives was designed and synthesized by replacing the phenylethyl group in the lead compound with chains of varying lengths combined with diverse aromatic substituents. Chemistry. From a structural point of view, compounds 6−19 are peptidomimetics of a general sequence X-Gly-Asp-Y, where X is a 3-guanidino benzoic acid (GBA) residue and Y is different aromatic moieties linked to the α-carboxy group of the aspartic acid. All the compounds (6−19) were fully synthesized on solid support integrating the conventional Fmoc/tBu solidphase peptide approach,41,42 required to build the tripeptidomimetic sequences, with the guanylation reaction and the aromatic linkage, which were carried out directly on the solid support (Scheme 1). Such pursued strategy limited the number of solution reaction steps to the sole allyl ester functionalization of the α-carboxyl group of aspartic acid (20), which was selected as a well-documented Fmoc/tBu orthogonal protecting group for the subsequent functionalization.43,44 As the first residue, 20 was attached to the 2-chlorotrityl chloride (2-CTC) resin in the presence of the diisopropylethylamine (DIEA) as base, yielding 21. Then, the sequences were elongated by sequentially introducing Fmoc-Gly-OH and Fmoc-3-amino benzoic acid, thus providing 22. Upon Fmoc removal of 22, the resulting resinbound aromatic primary amine was reacted with 1,3-bis(tertbutoxycarbonyl)-2-methyl-2-thiopseudourea, in the presence of mercury(II) chloride (HgCl2) and DIEA, to furnish the bis-Boc protected guanidine group in the N-terminus (23).

present several advantages compared to the coadministration of different drugs (e.g., lower risk of pharmacokinetic drug−drug interactions or reduced susceptibility to adaptive resistance). As briefly mentioned above, in a recent VS campaign, we found that a RGD-mimetic molecule can act as dual MDM2/4 inhibitors. This prompted us to test two further RGD mimetic compounds (6−7, Chart 1) from our in-house library as MDM2/4 binders. The molecules were already recognized as ligands of α5β1 and αvβ3 integrins (data not published). While 6 was totally inactive against both the MDM proteins, 7 turned out as a high nanomolar inhibitor for both the MDM proteins (MDM2 IC50 = 437 nM; MDM4 IC50 = 219 nM). Starting from these results, we have here developed a library of derivatives of 7 with the aim to preserve the activity on integrins while improving the affinity toward the MDM proteins. This lead optimization cycle resulted in the identification of compound 9 as a potent α5β1/αvβ3 integrin binder endowed with enhanced inhibitory activity on MDM2/4 (MDM2 IC50 = 72.0 nM; MDM4 IC50 = 77.4 nM). Extensive NMR and modeling studies were performed to investigate the molecular basis of the interaction of this derivative to its targets. Also, the newly identified compound was demonstrated to reactivate the p53 pathway. As a result, 9 induced cell cycle block and reduced GBM cell invasiveness, finally blocking the proliferation of p53 wild-type GBM cells.

■

RESULTS AND DISCUSSION Design Strategy. We have elsewhere demonstrated that an RGD-mimetic compound (3), albeit inactive on integrins, could inhibit both MDM proteins.23 This provided the rationale to test two molecules (6−7) that were previously developed as integrin ligands (α5β1, 6 IC50 = 13 nM; 7 IC50 = 28 nM; αvβ3, 6 IC50 = 0.21 nM; 7 IC50 = 2.8 nM), on MDM2/4. When tested on the MDM proteins, 6 was found inactive on both MDM2 and 4, while 7 displayed a high nanomolar affinity for both targets (MDM2 IC50 = 437 nM; MDM4 IC50 = 219 nM). Interestingly, as shown in Chart 1, the two compounds only differ in the length of the chain to the aromatic ring at the C-terminus. Compound 7 gains activity on MDM proteins 4793



Article

Table 1. Inhibition of Integrin Binding Fibronectin (α5β1) and Vitronectin (αvβ3)

a IC50 values were derived from a competitive ELISA using immobilized ECM protein and soluble integrin. bCompound 5, c(-RGDfMeV-), was used in ELISA as an internal reference compound for αvβ3 and α5β1 assays.

The conversion of the amino- into guanidine group was quantitatively monitored by analytical HPLC. At this stage, the allyl ester 23 was removed by treatments with catalytic amounts of Pd0 and morpholine as scavenger and the released acid 24 was coupled to a set of amines by using (7-azabenzotriazol-1yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) and 1-hydroxyazabenzotriazole (HOAt), which are known effective coupling reagents for the amide bond formation of hindered derivatives.45 Final products 6−19 were released from solid support by treatment with 5% water in TFA and purified using preparative HPLC. Finally, 6−19 were characterized by analytical HPLC in two different solvent systems (Figures S1−14, see Supporting Information), high-resolution mass spectrometry (HRMS), and NMR spectroscopy (1H and 13C). Biological Evaluation. Integrin Binding Assays. The affinity of compounds 6−19 toward the αvβ3 and α5β1 receptors was determined in a competitive solid-phase binding assay using coated extracellular matrix proteins and soluble integrins. Binding to integrin receptors was detected by specific antibodies in an enzyme-linked immune sorbent assay (ELISA). This assay was based on a previously reported method implemented with some modifications already described by us.46,47

As shown in Table 1, similar to the lead 7, all the newly synthesized compounds potently bind both αvβ3 and α5β1. Notably, all the tested ligands are more active against the former integrin subtype, displaying binding affinities in the low/ subnanomolar range. It is interesting to note that, in the case of the phenyl-substituted compounds, the elongation of the linker between the aspartic and aromatic moieties is detrimental for the αvβ3 potency (compare 6 vs 7 vs 8). An opposite effect is instead achieved by increasing the linker length in benzhydrylsubstituted derivatives (compare 9 vs 17). However, keeping constant the length of the linker, either the introduction of p-substituents on the phenyl ring (compounds 15 and 16) or the replacement of the latter with larger aromatic rings (compounds 10−12, 18, 19), with the lone exception of the benzhydryl moiety (17), does not significantly influence the ligand αvβ3 affinity. Similar effects are observed with the introduction of a methyl group on the linker (compounds 13 and 14). Contrary to αvβ3, chemical modifications on the lead 7 generally cause minor changes in the α5β1 binding affinities. Dissociation Studies of Native MDM2/p53 and MDM4/ p53 Complexes. Compounds 6−19 were then tested for their ability to dissociate p53 from MDM2 and MDM4. In parallel experiments, the standard αvβ3 ligand (5) was tested, too. 4794



Article

Table 2. Effect of New Compounds on the Dissociation of Human p53/MDM2 or p53/MDM4 Complex

a

Concentration (nM) leading to half-maximal inhibition of p53/MDM2 complex. bConcentration (nM) leading to half-maximal inhibition of p53/MDM4 complex. cData from ref 23. Data represent the mean values (±SEM) of three independent determinations. Inhibition percentages of p53/MDM2 and p53/MDM4 complex exerted by 5 (at 100 μM, corresponding to the maximum concentration tested) were 14.1 ± 0.3% and 2.6 ± 0.1%, respectively.

To this purpose, specific immune-enzymatic assays were employed, using cell lysates obtained from U87MG (for p53MDM2) or SHSY-5Y cells (for p53-MDM4).19,22 Compounds 6, 8, 17, and 18 did not bind with significant affinities either MDM2 or MDM4. In contrast, a moderate affinity for MDM2 was obtained for compounds 10−16 and 19 (Table 2). Moreover, although 10 did not show affinity toward MDM4, 11 and 12 showed a nanomolar binding to the latter protein, even higher than those elicited toward MDM2. Interestingly, 13 showed a nanomolar affinity toward MDM4 only, without any significant binding to the MDM2 subtype. As expected, the results obtained for 5 demonstrated its inability to dissociate p53/MDM2 and p53/MDM4 complex (Table 2). In Table 2, the previously published IC50 values of 1 and 2 (standard inhibitors of MDM2 and MDM4, respectively) are also reported. Among all, compound 9 seems to be the most potent in inducing the dissociation of p53 from both MDM2 and MDM4

(MDM2 IC50 = 72.0 nM; MDM4 IC50 = 77.4 nM, Figure 1 and Table 2). Considering its good affinity to both MDM2 and MDM4 and its ability to inhibit both the integrin receptors (α5β1 IC50 = 22 nM; αvβ3 IC50 = 1.4 nM), 9 was evaluated in further assays aimed at exploring the cross-talk between integrins and p53 pathway in GBM cells. Also, the standard MDM4 inhibitor 248,49 and the MDM2 inhibitor 150,51 or the integrin blocker 5, alone or in combination, were employed in these assays. All the compounds were tested at a concentration corresponding to their IC50 values toward the respective target. The human U87MG cells were chosen as representative GBM cell line because they express a wild-type p53, whose function is inhibited by high MDM2 levels,51 as well as the α5β1 and αvβ3 integrins.52 First, the reactivation of the p53 pathway was assessed by evaluating p53 protein accumulation and the transcription induction of p53 target genes after 8 and 24 h of incubation. As expected, the standard MDM2 inhibitor accumulated 4795



Article

Figure 1. Dissociation of human p53/MDM2 or p53/MDM4 complex. U87MG (for p53/MDM2) or SHSY-5Y (for p53/MDM4) cell lysates were incubated with 9 for 10 min, and the percentage of residual p53/MDM2 or p53/MDM4 complex was quantified as reported in the Experimental Section. The data are expressed as a percentage with respect to control cells (mean ± SEM, N = 3). Control represents cellular samples in the presence of the solvent DMSO used to solubilize the tested compounds.

significantly the p53 protein (Figure 2A,B). When cells were incubated simultaneously with the MDM2 and MDM4 blockers, a significant greater p53 accumulation was evidenced (Figure 2A,B), as reported previously.23 Similarly, compound 5 showed a significant increase in p53 levels (Figure 2A,B), demonstrating that an integrin block is enough to induce p53 accumulation. Such protein accumulation was not due to a direct inhibition of the two MDM isoforms, as 5 was not able to dissociate the p53/MDM2 and p53/MDM4 complex (Table 2). Other molecular mechanisms underlying the link between integrins and p53 activation have to be supposed. In fact, proteins involved in integrin signaling (i.e., FAK, the main kinase activated by integrins) have been shown to shuttle between plasma membrane and nucleus interfering with p53 activity.53 Combined cell treatment with 5 and the MDM2 standard inhibitor 1 was effective in further increasing p53 accumulation compared to treatment with 5 or 1 alone. The p53 accumulation did not further increase upon combined treatment with 5 and both inhibitors of the two MDM isoforms (Figure 2A,B). Interestingly, 9 induced a high statistically significant enhancement of p53 protein levels vs control, even greater with respect to that elicited by the cotreatment of the single-target agents (Figure 2A,B). Compound 9 has no greater effects than the 1 + 2 + 5 combination, suggesting that it specifically acts through the same action mechanisms activated by the combined treatment induced by each single-target compound. These data suggest that the multitarget compound can provide a higher efficacy with respect to formulations containing mixtures of singletarget agents. Then, the transcription activity of p53 was investigated. In particular, we evaluated the expression of the following genes: MDM2, physiological inhibitor of p53 and its main transcriptional target; PUMA, a gene product required for p53-mediated apoptosis; p21, a cell cycle inhibitor.21 Challenging U87MG cells with the standard MDM2 inhibitor 1 for 8 h caused a significant induction of MDM2 (Figure 3A), consistent with the data obtained in different tumor cell lines.21,51 At this time point, neither 2 nor 5 modulated the

Figure 2. Accumulation of p53 protein. U87MG cells were treated with 9 (100 nM), MDM2 inhibitor 1 (100 nM), MDM4 inhibitor 2 (100 nM), or 5 (100 nM), alone or in combination, for 24 h. Following treatment, p53 accumulation was evaluated by Western blot analysis, as reported in the Experimental Section. (A) Representative blot is shown. Control represents cell sample in the presence of DMSO (solvent used to solubilize the tested compounds). (B) Relative quantification of optical density (OD) was evaluated by densitometric analysis. The data are expressed as percentage versus the levels of the control (mean values ± SEM, N = 3). **P < 0.01, ***P < 0.001 vs control; #P < 0.05, ##P < 0.01 vs cells treated with 1; §§§P < 0.001 vs cells treated with 2; +++P < 0.01 vs cells treated with 5. The P value obtained by the comparisons between the combination 1 + 2 + 5 or 9 vs the combination 1 + 2, 1 + 5, or 2 + 5 are shown using the symbol Λ; ΛP < 0.05, ΛΛΛP < 0.001. The comparison between the cell treatment with 9 and the combination 1 + 2 + 5 did not give statistically significant differences.

mRNA levels of MDM2 (Figure 3A). Then, the standard compounds (1, 2, and 5) were tested using various combinations (1 + 2, 1 + 5, 2 + 5, or 1 + 2 + 5). A significant increase of MDM2 mRNA levels was evidenced only for the combinations 1 + 2 and 1 + 2 + 5, suggesting that MDM2 inhibition plays a pivotal role in regulating the transcriptional controls of p53. The new compound 9 exhibited a significant induction of MDM2 gene transcription (Figure 3A), which was even higher than that observed in cells simultaneously treated with the three single-target inhibitors. In contrast, MDM2 transcription failed to be modulated after 24 h of treatment (data not shown), consistent with a feedback regulation exerted by a reactivated p53. Concerning PUMA mRNA levels, no significant changes were observed, both after 8 and 24 h of incubation with 1 (data not shown and Figure 3B), consistent with the lack of apoptosis 4796



Article

Figure 3. Reactivation of p53 pathway. U87MG cells were treated with 9 (100 nM), MDM2 inhibitor 1 (100 nM), MDM4 inhibitor 2 (100 nM), or 5 (100 nM), alone or in combination, for 8 h (A) or 24 h (B,C). Following treatment, total RNA was extracted, and the relative mRNA quantification of p53 target genes MDM2 (A), PUMA (B), and p21 (C) was performed by real-time RT-PCR as reported in the Experimental Section. The data are expressed as the fold change versus the levels of the control (mean values ± SEM, N = 3). Control represents sample cells in the presence of DMSO (used to solubilize the tested compounds). *P < 0.05, **P < 0.01, ***P < 0.001 vs control; §P < 0.05 comparison between cells treated with 9 and 1 + 2 + 5.

induction by 1 at shorter times of incubation.21,50,51 In contrast, after 24 h of treatment, the MDM4 blocker 2 significantly increased PUMA gene transcription (Figure 3B), suggesting that the inhibition of MDM4 may play a major role in driving cells to apoptosis. As concerns 5, a greater effect than 2 was evidenced (Figure 3B). Consistently, α5β1 integrin antagonists have been demonstrated to modulate effectively p53 target gene expressions and to facilitate apoptosis in U87MG cells.34 Challenging cells simultaneously with 1 and 2 induced significant modulated PUMA transcription, with higher effects with respect to singly treated cells (1 + 2 vs 1, P < 0.01, Figure 3B). The combinations 1 + 5, 2 + 5, and 1 + 2 + 5 did not produce additive effects (Figure 3B). In contrast, a significant reduction of PUMA mRNA levels was observed in the same conditions (for example, reduced PUMA mRNA levels were evidenced in combination 1 + 2 + 5 vs compound 5, P < 0.01, and combination 1 + 2 + 5 vs combination 1 + 2, P < 0.01). Such a reduction suggests that the maximal effect on the induction of PUMA gene transcription could be reached in shorter time of treatment. Finally, the cell treatment with the compound 9 significantly increased PUMA transcription levels (Figure 3B). Concerning the p21 mRNA levels, the cell treatments determined similar results already observed for the PUMA transcript. Compound 1 did not affect p21 gene transcription in agreement with previously published data.51 A significant induction of p21 mRNA levels was noticed in the presence of 2 (Figure 3C). For compound 5, a higher effect than 2 was evidenced. According to literature data, the blockade of integrin activity has been shown to inhibit effectively cell cycle progression by stimulating expression of cell cycle key proteins.30,54 In general,

the various combinations did not further increase p21 mRNA levels with respect to the single-treated samples, probably because the maximal effect had been already achieved. Finally, 9 was able to enhance significantly p21 mRNA levels (Figure 3C). All together these results confirm the ability of the new compound to reactivate effectively p53 functionality. Then, the effects of MDM2, MDM4, and integrin blockers were tested on GBM cell proliferation. Following 72 h of cell incubation, each standard compound, tested individually, did not show a significant inhibition of GBM proliferation (Figure 4). The combined treatment of 1 and 5 showed synergic/additive antiproliferative effects with respect to single treatments; the antiproliferative activity was even higher in the concomitant presence of 2 (Figure 4). Of note, 9, tested at the same concentration (i.e., 100 nM), induced a significant arrest of U87MG cell proliferation (Figure 4), with a percentage of inhibition of cell proliferation similar to that obtained in the presence of 1 + 2 + 5. Moreover, 9 induced effects were concentration dependent (Figure 5A) and yielded to an IC50 value of 116 ± 10 nM. All together, these results suggest that the simultaneous inhibition of MDM2/4 and α5β1 integrin can represent an effective strategy to arrest GBM cell proliferation. Consistent with our findings, recent literature data have highlighted that integrin α5β1 and p53 are convergent pathways in the control of glioma apoptosis.32,33 To further explore the contribution of the p53 pathway in 9 elicited effects, the experiments were repeated in human T98G cells, exhibiting a mutated p53.55 As depicted in Figure 5B, 9 inhibited significantly cell proliferation at micromolar concentrations, while nanomolar concentrations of the compound did 4797



Article

Figure 4. Antiproliferative effects of MDM2/4 and integrin inhibitors on U87MG cells. U87MG cells were challenged for 72 h with the indicated concentrations of 9 (1, 10, 100 nM), 1 (100 nM), 2 (100 nM), or 5 (100 nM), alone or in combination. Following incubation, cell proliferation was determined by MTS assay as reported in the Experimental Section. The data are reported as percentage with respect to control, set to 100% (mean ± SEM, N = 3). Control represents sample cells in the presence of the solvent DMSO used to solubilize the tested compounds. *P < 0.05, ***P < 0.001 vs control; ### P < 0.001 vs cells treated with 1 alone; §§§P < 0.001 vs cells treated with 2 alone; +++P < 0.001 vs cells treated with 5 alone. &&&P < 0.001 vs combined treatment with 2 + 5 or 1 + 2.

Figure 6. Cell cycle analysis upon treatment with 9, MDM2/4, or integrin inhibitors or combined compounds. U87MG cells were treated for 72 h with the solvent DMSO used to solubilize the compounds (control), 9, 1, 2, and 5, alone or in combination, and the cell cycle was analyzed. The data were expressed as percentage of cell in the different phases (G0/G1, G2, or S) versus total cell number (mean ± SEM; N = 3). *P < 0.05 versus control.

produced similar results, suggesting that the MDM4 block is not sufficient to efficiently arrest cell cycle. However, the combination of 1 and 2 leads to a significant block of U87MG cells in G0/G1 phase (Figure 6), thus confirming the additive effects of MDM2/4 inhibition. Consistent with its great effect on p21 mRNA levels, challenging cells with 5 alone was sufficient to induce a G0/G1 cell cycle block (Figure 6). Compound 9 alone caused a cell cycle arrest in G0/G1 phase, with comparable percentage with respect to cells treated simultaneously with the three inhibitors (Figure 6). Finally, 9 was tested for its ability to block cell invasiveness of GBM cells using the Matrigel assay. Consistent with the role of integrins in the invasive potential of tumor cells,59,60 our results showed that 9 significantly inhibited the invasive potential of U87MG cells after 24 h of cell treatment (Figures 7A,B). In particular, the number of invaded cells decreased to 80% upon U87MG cell treatment with the new derivative, thus suggesting that 9 can be a useful tool not only to block proliferation but also to control migration/invasiveness of glioma cells.

not affect cellular viability. Our data suggest that 9 requires a wild-type p53 to exert its antiproliferative action on human GBM cells. Moreover, such findings are consistent with literature reporting that nonmutated p53 is needed for the pharmacological functionality of MDM2/4 inhibitors.56−58 In addition, due to the great interconnection between the two pathways, α5β1 ligands are not effective in tumor cells not expressing p53 or expressing a mutated form of the protein.32−34 Also, our results are consistent with literature data reporting that an effective p53 pathway is essential to control antiproliferative and apoptotic signaling in glioma, including those relating to integrin α5β1.32,33 Then, a cell cycle analysis was performed on U87MG cells, in order to assess if the antiproliferative effects of MDM2, MDM4, and integrins inhibitors could be associated with cell cycle disturbance. As depicted in Figure 6, 1 failed to induce cell cycle disturbance, consistent with the data obtained for p21 mRNA levels. Compound 2

Figure 5. Antiproliferative effects of 9 on different GBM cells. U87MG (A) or T98G (B) cells were challenged for 72 h with different concentrations of 9. Following incubation, cell proliferation was determined by MTS assay as reported in the Experimental Section. The data are reported as percentage with respect to control, set to 100% (mean ± SEM, N = 3). Control represents sample cells in the presence of the solvent DMSO used to solubilize the tested compounds. A sigmoid dose−response analysis (GraphPad Prism 5 software) was derived. **P < 0.01, ***P < 0.001 versus control. 4798



Article

with the new derivative 9, thus suggesting that the latter compound can be a useful tool not only to arrest proliferation but also to control migration/invasiveness of glioma cells. A similar high inhibition of cell invasiveness was observed upon cell incubation with the triple treatment (1 + 2 + 5, Figure 7A,B). However, it is important to highlight that the observed effects were obtained with a single dose of 9 (10 μM) but with three 10 μM doses in the triple treatment. Structural Studies. Binding of 9 to Integrin Receptors. To get insights into the binding mode of 9 to the αvβ3 and α5β1 integrins, standard docking calculations of this compound were performed in the crystal structures61,62 of the latter receptors. In the present case, the choice of the docking pose would be indeed straightforward since 9 features a typical RGDmimetic structure and the RGD binding pockets of both αvβ3 and α5β1 integrins have been well characterized.35,37 Docking results show that 9 can interact with both αvβ3 and α5β1 through the canonical RGD binding pattern (Figure 8).35−40 In both receptors, indeed, the ligand carboxylate group coordinates the metal ion at the MIDAS and forms multiple H-bonds with the backbone of (β3)-Y122, (β3)-S123, and (β3)-N215 in αvβ3 or (β1)-Y133 (β1)-S134, and (β1)-N224 in α5β1. However, the guanidinium group of 9 establishes tight polar contacts with the side chains of (αv)-D150 and (αv)D218 in αvβ3 and (α5)-Q221 and (α5)-D227 in α5β1, respectively. The same group is also engaged in a cation−π interaction with the aromatic ring of either (αv)-Y178 in αvβ3 or (α5)-F187 in α5β1. Finally, the ligand benzhydryl group, albeit differently oriented, can establish favorable interactions in the region defined by the specificity determining loop (SDL) of both receptors. In particular, it can form π-stacking interactions with the aromatic chains of (β3)-Y122 and (β3)-Y166 in αvβ3 and (α5)-W157 and (β1)-Y133 in α5β1, respectively. Nevertheless, in the former receptor, the benzhydryl group can establish additional cation−π interactions with the positively charged (β3)-R214 and (β3)-R216, which might be the reason for the higher affinity of 9, and more generally of our derivatives, toward αvβ3 compared to α5β1. However, it is likely that the strength of the latter interactions lowers as much as the distance between the arginine residues and the ligand aromatic ring increases, specifically when the linker between the ligand

Figure 7. U87MG cell invasiveness upon treatment with 9. U87MG cells were incubated with each standard compound alone (10 μM), or combined (each at 10 μM), or 9 (10 μM) for 24 h, and cell invasion was analyzed using the Matrigel basement membrane transwell system, as described in the Experimental Section. Control represents sample cells in the presence of the solvent DMSO used to solubilize the tested compounds. (A) Representative images are shown. (B) Cell migration was quantified by counting the number of cells that migrated into the lower surface of the transwell membrane. *P < 0.05, ***P < 0.001 versus control. ###P < 0.001 vs cells treated with 1; §§§P < 0.001 vs cells treated with 2; ++P < 0.01, +P > 0.05 vs cells treated with 5.

Finally, the standard compounds and 9 were tested for their ability to arrest cell invasiveness of GBM cells using the Matrigel assay. As expected, MDM2/4 inhibitors were slightly able to affect cell invasiveness. In particular, only the MDM4 inhibitor caused a significant reduction in cell invasiveness after 24 h of cell treatment. Consistent with the role of integrins in the invasive potential of tumor cells,59,60 our results showed that both 5 and 9 significantly inhibited the invasive potential of U87MG cells (Figure 7A,B). In particular, the number of invaded cells decreased to 80% upon U87MG cell treatment

Figure 8. Docking poses of 9 (magenta sticks) at the (A) αvβ3 (PDB code: 4MMX) and (B) α5β1 (PDB code: 4WK4) integrins. The αv and β3 subunits are depicted as wheat and violet surfaces, while the α5 and β1 subunits are depicted as yellow and light blue surfaces, respectively. Receptor amino acid side chains important for ligand binding are represented as sticks. The metal ion at the MIDAS is represented as a green sphere. 4799



Article

L33 or R97, L34 or K45, L35 or Y56, Q44 or N79, D46 or C77, L54 or Q72) (Figure 10).

carboxylate group and its aromatic moiety is elongated. This could explain why compounds endowed with shorter linkers but not much bulky aromatic moieties (compare 6 vs 7 and 8) generally display lower αvβ3 IC50s. Conversely, a slight elongation of the linker in compounds carrying a bulkier aromatic function can increase the binding potency by allowing the latter to better accommodate the sterically hindered SDL pocket of αvβ3 (compare 9 vs 17). For similar reasons, namely, the strict αvβ3 steric requirements, the benzyl derivative 8 is more potent than the benzhydryl analogue 9 toward this integrin subtype, although featuring a reduced stacking surface (Figure S15, see Supporting Information). These trends are not maintained in the case of α5β1, where no significant change is observed in the ligand binding affinity by varying the type of the aromatic moiety as well as the linker length. This can be ascribed to the presence of a wider and less hindered SDL region in α5β1 compared to αvβ3, which can thus more easily hosts lipophilic substituents of various size and nature. Binding of 9 to MDM2 Protein. Although it is broadly acknowledged that the inhibition of the MDM4 is important for a lasting and effective clinical response, the MDM2 is the main actor of the p53 control.7 For this reason, NMR and modeling studies were solely focused on MDM2. Considering the significant structural differences between compound 9 and known MDM2 inhibitors, we could not assume a priori that the p53 binding pocket was the preferred region of binding of this ligand. A blind docking comprising the entire protein (see details in Supporting Information) showed that three sites were mostly bound by 9: the canonical binding site; a site located on the back of N-terminus region; and a site located on the bottom of the protein (herein named the “bottom pocket”) (Figure S16, see Supporting Information). Thus, NMR studies were carried out to discriminate between the different sites. The amino acids forming the binding site for 9 on MDM2 have been identified by monitoring the chemical shift perturbation and intensity decreases in the 2D 1H−15N HSQC NMR spectrum of the 15N isotopically enriched protein upon the addition of increasing amounts of the ligand.63 Progressive shifts are characteristic of a ligand in fast-exchange regime on the NMR time scale, while a decrease in signal intensity is indicative of an intermediate or slow exchange phenomenon.64 The residues exhibiting the largest chemical shift perturbation in the presence of compound 9 at a concentration of 200 μM are highlighted in Figure 9 (A13, S22, R29, K51, F55, Y56, G58, Y60, M62, F91, S92, V93, K94, I103).

Figure 10. Intensity decreases of the signals of MDM2 (200 μM) in the presence of 9 (200 μM); the residues exhibiting the largest decreases are highlighted in red, and the overlapping signals have been marked with a star.

To better shed light on the binding region of 9 on the protein surface, a competitive binding experiment in the presence of 1 has been carried out. The high affinity binder of the canonical pocket, 1 (ligand in slow exchange), was added to the complex of MDM2 protein with 9. After the addition of a stoichiometric amount of 1, the spectrum of the protein is largely superimposable with that of the protein in the presence of 1 alone (Figure 11). Therefore, 1 largely prevents binding of 9,

Figure 11. Two-dimensional 1H−15N HSQC spectrum of MDM2 (200 μM) in the presence of 1 (200 μM) (red) and in the presence of 1 (200 μM) and 9 (400 μM) (blue).

showing that 9 mainly binds in the canonical pocket. However, some resonances experience small chemical shift variations. Unfortunately, the assignment of the protein in complex with 1 is not available, making it impossible to identify a possible second binding site different from the canonical pocket for 9. Also, mapping the residues exhibiting the largest chemical shift perturbation and those isolated signals that exhibit a decrease in intensity on the MDM2 structure, it is evident that the canonical site is the pocket mainly involved in the binding with 9. In fact, almost all these residues are located on the α2 helix, L2 and L5 loops, and β1 and β2 strands, which are all segments lining the p53-binding cavity (Figure 12A). However, considering the residues presenting overlapping signals, it emerges that in four cases (L34 or K45, L35 or Y56, Q44 or N79, D46 or C77), although an assignment could not be obtained, both residues of each couple are spotted in the region forming the bottom pocket (Figure 12B), thus a binding with this other site could be also possible. From these data, we can speculate that a “primary” and a possible “secondary” binding pocket may exist. This because, comparing the abovementioned data with those obtained with p53 helix65 or with unambiguous binders23 of p53 pocket, a novel set of amino acids, mostly located in the bottom part of the protein, displaying changes in chemical shifts or signal intensities appeared.

Figure 9. Chemical shift perturbation (CSP) between the uninhibited MDM2 (200 μM) and MDM2 in the presence of compound 9 (200 μM) evaluated according to the formula

Δδ = 1/2 Δδ H 2 + (Δδ N/5)2 ; the residues exhibiting the largest CSP are highlighted in blue.

The isolated signals of residues (G12, S22, G58, I61, K70, I74, S78, V110), instead, displayed a decrease in intensity. A change in intensity is observed also for some other signals corresponding to overlapping residues, but in this case, an unambiguous assignment could not be obtained (D11 or D117, 4800



Article

MDM2. Indeed, it is well-known that the full occupancy of the three subpockets, especially F19 and W23, is important to reach a high inhibitor potency toward this protein, and 9, which is the only derivative possessing two aromatic moieties at the C-terminus, is able to fulfill this requirement, showing the lowest IC50 among the series, while the others, evidently able to occupy just one of the two subpockets, are all less potent. Blind docking on 9 within MDM2 showed that more than 52% of the poses bind the canonical pocket, while ∼13% of the poses would bind the “bottom pocket”. Glide gave the best score to the pose having 9 in the canonical site (“p53 pocket”). However, from NMR study, it clearly emerges that the majority of amino acid experiencing the largest CSP or signal intensity decrease is mostly located in the “p53 pocket”. So, based on computational and NMR data, we envisage that “p53 pocket” is the main binding site for 9, although the existence of a “secondary ” binding site seems to be plausible and cannot be ruled out.

Figure 12. (A) Residues showing the largest CSP (blue sticks) and residues (isolated signals) exhibiting the largest decreases (red sticks), mapped onto the MDM2 structure (PDB: 3LBL), are located around the canonical binding site, which is represented in green dots (space possibly occupied by the ligand). (B) Four of the couples of residues, presenting overlapping signals, exhibiting the largest decreases (deep red sticks), mapped onto the MDM2 structure, are located below the canonical site, lining the “bottom pocket”, which is represented in yellow dots.

■

CONCLUSION Recent literature data have highlighted that integrin α5β1 and p53 are part of convergent pathways in the control of glioma apoptosis.28 In a recent VS campaign, we found that a RGD mimetic molecule can act as dual MDM2/4 inhibitor. This prompted us to test two further RGD mimetic compounds (6−7) active on α5β1 and αvβ3 integrins that where present in our in-house library. While 6 was totally inactive against both the MDM proteins, 7 turned out to be somehow active (MDM2 IC50 = 437 nM; MDM4 IC50 = 219 nM). Here, to enhance the potency toward the MDM proteins, while preserving the low nanomolar affinity toward integrins, a small library of derivatives of 7 was designed and synthesized by replacing its phenylethyl group with chains of varying lengths combined with diverse aromatic substituents. This optimization cycle resulted in the identification of 9 as a potent α5β1/αvβ3 integrin ligand endowed with enhanced inhibitory activity on MDM2/4 (MDM2 IC50 = 72.0 nM; MDM4 IC50 = 77.4 nM). In p53-wild type glioma cells, 9 arrested cell cycle and proliferation while strongly reducing cell invasiveness. Compound 9 represents the first molecule of a novel class of integrin/MDM inhibitors, which might be especially useful in the subpopulation of patients with glioblastoma expressing a functional p53

With these data in our hands, further docking runs focused on the canonical site were carried out (details of molecular docking on the bottom pocket can be found in Supporting Information). To better sample the possible poses of binding for 9, two docking softwares, Autodock 4.2 and Glide 5.5 program,66,67 were used (see Experimental Section for further details). Analyzing the results, the two softwares suggested essentially two solutions. In both cases, the biphenyl moiety of 9 is well anchored into the W23 and F19 pockets, while the rest of the molecule is oriented toward the L2 loop (binding mode A) or flip into the N-terminus region (binding mode B, see Supporting Information). Bearing in mind the residues exhibiting the largest decreases, with three of them located exactly on the L2 loop, it could be said that the binding mode A, depicted in Figure 13, is surely in better accordance with the NMR data. Particularly, in this pose, the carboxylic group is found into the L26 pocket, in close proximity with H96, with which, in a dynamical context, a fruitful interaction could be expected. The terminal phenyl-guanidine branch interacts with the H73 and E69 side chains of the L2 loop. It is worth noticing that these theoretical results are consistent with the IC50 found for the synthesized ligands toward

Figure 13. Binding mode of 9, as found by Autodock and Glide softwares, in the canonical MDM2 binding cleft. The ligand is shown as magenta sticks, and the protein as gray surface/ribbon. (A) Residues exhibiting the largest signal intensity decreases are depicted as red sticks; (B) ligand binding mode showing the interacting residues as gray sticks. 4801



Article

(0.1% TFA) in H2O (0.1% TFA). Compounds examined for biological activity were purified to >98% (Table S1, see Supporting Information). Solid-Phase Synthetic Strategy. Construction of Peptidomimetic Sequences on Solid Support. Compounds 6−19 were synthesized on a 2-CTC resin as solid support and by using the Fmoc/tBu orthogonal strategy. The resin (0.3 mmol) was placed into a plastic syringe tube equipped with Teflon filter, stopper, and stopcock, and preswollen in DCM on an automated shaker at rt for 30 min. The Fmoc-Asp-OAll amino acid 20 (118.6 mg, 0.3 mmol) along with DIEA (1 equiv) was dissolved in DMF (3 mL) and added to the resin and shaken at rt for 10 min, and then the mixture was treated with additional DIEA (1.5 equiv) and shaken for 16 h. The loaded resin was washed with DCM (2 mL × 3) and DMF (2 mL × 3). To end-cap any remaining reactive 2-CTC groups, a solution of 85:10:5 DCM/ MeOH/DIEA (v/v/v) was added to the resin and shaken at rt for 30 min. The Fmoc group removal was performed using 20% solution of piperidine in DMF (1 × 5 min, 1 × 25 min). Coupling of Fmoc-Gly (3 equiv) was then accomplished following general solid-phase peptide synthesis (SPPS) protocols,41,42,70 by using HBTU (3 equiv) and HOBt (3 equiv) as coupling reagents and DIEA (6 equiv) as base, in 1:1 DCM/DMF (v/v) at rt for 2 h. Accordingly, Fmoc-deprotection from glycine residue and peptide bond formation with 3-(Fmocamino)benzoic acid were both performed as described above. The resins were washed after each coupling and Fmoc group removal step with DMF (2 mL × 3) and DCM (2 mL × 3). The progression of coupling and Fmoc-deprotection reactions were monitored by the observation of colorimetric tests such as Kaiser and p-chloranil tests for aliphatic and aromatic primary amines, respectively.71 Guanylation Procedure. Upon removal of Fmoc group of 3-aminobenzoic residue 22, the guanylation of the resulting resinbound aromatic primary amine was carried out via treatment with 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (2 equiv), in the presence of HgCl2 (2 equiv), and DIEA (2 equiv). The resulting suspension in DMF was shaken at rt for 16 h. The bis-Boc protected resin-bound guanidine 23 was further washed with DMF (2 mL × 3), MeOH (2 mL × 3), and THF (2 mL × 3). The quantitative formation of the guanidine group was confirmed by LC−MS analysis of the residue after an aliquot of resin (5 mg) was treated with a solution of 95:5 TFA/H2O (v/v) at rt for 1 h, filtered, and evaporated. Allyl Ester Removal and Coupling to Amines. The resin-bound allyl ester compound 23 was first washed with DCM (2 mL × 3) and dried for 30 min, then treated with a solution of Pd(PPh3)4 (0.15 equiv) and morpholine (2 equiv) in 3:2 dry DCM/DMF (v/v) and shaken gently for 2 h under argon. The resin was filtered and washed with DMF (2 mL × 3) and DCM (2 mL × 3), and the allyl deprotection procedure was repeated with the same conditions. The resin was finally filtered and washed with DMF (2 mL × 3) and DCM (2 mL × 3). After complete removal of the allyl group was ascertained by LC−MS of the residue from cleavage of an aliquot of resin (5 mg), acid 24 was treated with the appropriate amine. Amines (2 equiv) were dissolved in 4 mL of a solution of 1:1 DCM/DMF (v/v), treated with PyAOP (2 equiv), HOAt (2 equiv), and DIEA (4 equiv), and the resulting mixture was transferred to the plastic syringe tube containing resin 24. The reaction was carried out at rt for 16 h. The resins 25−38 were filtered, washed with DMF (2 mL × 3), MeOH (2 mL × 3), and DCM (2 mL × 3), and dried under vacuum. At this stage, a 5 mg aliquot of each resin was removed and treated with 1 mL of 95:5 TFA/ H2O (v/v) and filtered to provide the filtrates, which were analyzed by LC−MS to confirm that couplings had quantitatively been achieved. Cleavage of Peptidomimetics from the Resins 25−38. To afford final compounds 6−19, the Boc protecting groups were removed, and the peptidomimetics were released from the solid support by treating the resin with a solution of 95:5 TFA/H2O (v/v) at rt for 2 h. The filtrates were precipitated in chilled diethyl ether and centrifuged to yield a pellet, which was decanted from the solution. The crude compounds were dissolved in the appropriate solvent and purified by preparative HPLC. Finally, pure compounds 6−19 were yielded as white powders (4−15% overall yields, Table S1, see Supporting Information) by freeze-drying appropriate fractions, prior to removal of MeCN or MeOH by rotary evaporation under reduced pressure.

(15 to 31% glioblastoma have a mutated p53) concomitantly with a high level of α5β1 integrin (detected in 70% of a preliminary cohort of glioblastoma biopsies).34 A unique molecule that hit multiple targets at once should present a number of advantages if compared to the coadministration of different drugs (e.g., lower risk of pharmacokinetic drug−drug interactions or reduced susceptibility to adaptive resistance). However, further biological studies are needed to deepen the anticancer properties of such novel class of compounds, which hopefully might be a useful support for the GBM first line therapy.

■

EXPERIMENTAL SECTION

Materials and General Procedures. Nα-Fmoc-Asp-OAll (20) was synthesized from its corresponding Fmoc-Asp(OtBu), according to reported procedures.68 Briefly, Fmoc-Asp(OtBu) was reacted with allyl bromide and DIEA as base, followed by a treatment with 1:1 TFA/DCM (v/v) solution. 3-(Fmoc-amino) benzoic acid was synthesized following a literature procedure as well.69 Allyl bromide, diisopropylethylamine (DIEA), morpholine, 1,3bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, mercury(II) chloride (HgCl2), all were purchased from Sigma-Aldrich and used as received. Amines used, such as aniline, benzylamine, 2-phenylethylamine, tryptamine, 1-naphthylamine, 2-(2-naphthyl)ethylamine hydrochloride, 4-chlorobenzylamine, 4-methoxybenzylamine, and benzhdrylamine, all were purchased by Sigma-Aldrich-Merck. The remaining amines, such as (S)-(−)-1-phenylethylamine, (R)-(+)-1-phenylethylamine, 2,2-diphenylethylamine, 4-phenylbenzylamine, and 3-phenylbenzylamine, were purchased by abcr GmbH (Karlsruhe, Germany). Piperidine and trifluoroacetic acid (TFA) were purchased from Iris Biotech GmbH. Tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3)4] was purchased from Carbosynth. Anhydrous solvents [N,N-dimethylformamide (DMF) and dichloromethane (DCM)] were obtained by commercial sources such as Sigma-Aldrich and VWR. The amino acid Fmoc-Gly and coupling reagents N,N,N′,N′tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), 1-hydroxybenzotriazole (HOBt), and 1-hydroxyazabenzotriazole (HOAt) were purchased from GL Biochem (Shangai, China) and used as received. 2-Chlorotrityl chloride (2-CTC) resin (1.60 mmol/g as loading) was purchased from Chem-Impex, and the manufacturer’s reported loading of the resin was used in the calculation of final product yields. LC−MS analyses were performed on a LC−MS instrument from Agilent technologies equipped with an analytical C18 column and 6110 Quadrupole, in positive electrospray ionization (ESI) mode, to confirm that reactions had achieved >80% conversion. High-resolution mass (HRMS) measurements were recorded on a LTQ Orbitrap mass spectrometer in ESI mode, and proton adducts, [M + H]+, were used for empirical formula confirmation. 1H NMR and 13C NMR spectra were recorded on a Varian INOVA 500 MHz spectrometer and measured in 600 μL of CD3OD (3.31/49.00 ppm) or DMSO (2.50/39.52 ppm). Multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet, and bs = broad signal. Coupling constant J values are measured in Hertz (Hz), and chemical shift values are in parts per million (ppm). Analytical HPLC analyses were performed on a Phenomenex Kinetex reverse-phase column (C18, 150 mm × 4.6 mm, 5 μm, 100 Å) with a flow rate of 1 mL/min using a gradient of MeOH (0.1% TFA) or MeCN (0.1% TFA) in water (0.1% TFA) and UV detection at 220 and 254 nm. Purification of compounds 6−19 was performed on a preparative column (Phenomenex Kinetex C18 column, 150 × 21.2 mm, 5 μm, 100 Å) using specified linear gradients of MeOH (0.1% TFA) in water (0.1% TFA) with a flow rate of 10 mL/min and UV detection at 220 nm. The purity of compounds 6−19 was ascertained using analytical HPLC analysis performed on a C18-bonded Kinetex column from Phenomenex (150 mm × 4.6 mm, 5 μm, 100 Å) with a flow rate of 1 mL/min using gradients of MeCN (0.1% TFA) or MeOH 4802



Article

(S)-4-((2-(1H-Indol-3-yl)ethyl)amino)-3-(2-(3guanidinobenzamido)acetamido)-4-oxobutanoic Acid (10). Purity > 98%, tR = 14.6 min [analytical HPLC/Gradient#1, 10−70% MeCN (0.1% TFA) in H2O (0.1% TFA) over 20 min, flow rate of 1.0 mL/ min], and tR = 16.0 min [analytical HPLC/Gradient#2, 10−90% MeOH (0.1% TFA) in H2O (0.1% TFA) over 15 min, flow rate of 1.0 mL/min] on a Phenomenex Kinetex analytical column (150 mm × 4.6 mm, 5 μm, 100 Å); 1H NMR (CD3OD, 500 MHz) δ 7.85 (1H, d, J = 7.8 Hz), 7.80 (1H, s), 7.59−7.56 (2H, m), 7.46 (1H, d, J = 7.8 Hz), 7.32 (1H, d, J = 8.1 Hz), 7.09−7.06 (2H, m), 7.00 (1H, t, J = 7.4 Hz), 4.75 (1H, t, J = 6.3 Hz), 4.08 (1H, d, J = 16.2 Hz), 4.00 (1H, d, J = 16.3 Hz), 3.50 (2H, t, J = 7.3 Hz), 2.97 (2H, t, J = 7.4 Hz), 2.84 (1H, dd, J = 5.4, 16.8 Hz), 2.74 (1H, dd, J = 7.4, 16.8 Hz); 13C NMR (CD3OD, 167 MHz) δ 174.1, 172.8, 171.7, 169.5, 158.0, 138.0, 136.8, 136.7, 135.1, 133.7, 131.3, 129.6, 129.1, 128.6, 128.4, 128.1, 127.3, 126.9, 126.4, 125.5, 51.4, 44.4, 42.2, 36.5, 23.5; HRMS (ESI) m/z calculated for molecular formula, C24H28N7O5+ [M + H]+ 494.2147, found 494.2141 (Δ = 1.21 ppm). (S)-3-(2-(3-Guanidinobenzamido)acetamido)-4-(naphthalen-1ylamino)-4-oxobutanoic Acid (11). Purity > 98%, tR = 14.6 min [analytical HPLC/Gradient#1, 10−70% MeCN (0.1% TFA) in H2O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and tR = 16.0 min [analytical HPLC/Gradient#2, 10−90% MeOH (0.1% TFA) in H2O (0.1% TFA) over 15 min, flow rate of 1.0 mL/min] on a Phenomenex Kinetex analytical column (150 mm × 4.6 mm, 5 μm, 100 Å); 1 H NMR (CD3OD, 500 MHz) δ 8.03 (1H, d, J = 8.2 Hz), 7.89 (1H, d, J = 7.8 Hz), 7.81−7.79 (2H, m), 7.65 (1H, s), 7.58−7.53 (2H, m), 7.51−7.43 (4H, m), 5.07 (1H, t, J = 6.1 Hz), 4.19 (1H, d, J = 16.2 Hz), 4.10 (1H, d, J = 16.3 Hz), 3.05 (1H, dd, J = 5.9, 16.9 Hz), 2.97 (1H, dd, J = 6.6, 16.9 Hz); 13C NMR (CD3OD, 167 MHz) δ 174.2, 172.5, 172.2, 169.6, 158.0, 136.7, 136.6, 135.7, 134.0, 131.3, 130.4, 129.7, 129.2, 128.0, 127.4, 127.3, 127.2, 126.4, 125.7, 124.4, 123.9, 52.0, 44.5, 36.6; HRMS (ESI) m/z calculated for molecular formula, C24H25N6O5+ [M + H]+ 477.1881, found 477.1746 (Δ = 2.83 ppm). (S)-3-(2-(3-Guanidinobenzamido)acetamido)-4-((2-(naphthalen2-yl)ethyl)amino)-4-oxobutanoic Acid (12). Purity > 98%, tR = 16.8 min [analytical HPLC/Gradient#1, 10−70% MeCN (0.1% TFA) in H2O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and tR = 18.4 min [analytical HPLC/Gradient#2, 10−90% MeOH (0.1% TFA) in H2O (0.1% TFA) over 15 min, flow rate of 1.0 mL/min] on a Phenomenex Kinetex analytical column (150 mm × 4.6 mm, 5 μm, 100 Å); 1H NMR (CD3OD, 500 MHz) δ 7.84−7.82 (1H, m), 7.80− 7.76 (4H, m), 7.68 (1H, s), 7.58−7.54 (1H, m), 7.49−7.36 (4H, m), 4.74 (1H, t, J = 6.3 Hz), 4.08 (1H, d, J = 16.3 Hz), 4.00 (1H, d, J = 16.3 Hz), 3.57−3.53 (1H, m), 3.51−3.47 (1H, m), 2.98 (2H, t, J = 7.3 Hz), 2.82 (1H, dd, J = 5.4, 17.0 Hz), 2.73 (1H, dd, J = 7.3, 16.9 Hz); 13C NMR (CD3OD, 167 MHz) δ 174.1, 172.8, 171.7, 169.5, 158.0, 138.0, 136.7, 135.1, 133.7, 131.4, 131.3, 129.6, 129.1, 128.5, 128.4, 128.2, 128.1, 127.3, 126.9, 126.3, 125.5, 51.4, 44.4, 42.2, 36.6, 36.5; HRMS (ESI) m/z calculated for molecular formula C26H29N6O5+ [M + H]+ 505.2194, found 505.2182 (Δ = 2.38 ppm). (S)-3-(2-(3-Guanidinobenzamido)acetamido)-4-(((R)-1phenylethyl)amino)-4-oxobutanoic Acid (13). Purity > 98%, tR = 13.9 min [analytical HPLC/Gradient#1, 10−70% MeCN (0.1% TFA) in H2O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and tR = 15.7 min [analytical HPLC/Gradient#2, 10−90% MeOH (0.1% TFA) in H2O (0.1% TFA) over 15 min, flow rate of 1.0 mL/min] on a Phenomenex Kinetex analytical column (150 mm × 4.6 mm, 5 μm, 100 Å); 1H NMR (CD3OD, 500 MHz) δ 7.83 (1H, d, J = 7.8 Hz), 7.74 (1H, s), 7.57 (1H, t, J = 7.9 Hz), 7.46 (1H, d, J = 8.0 Hz), 7.36− 7.31 (2H, m), 7.28−7.24 (2H, m), 7.21−7.16 (1H, m), 5.01 (1H, q, J = 7.0 Hz), 4.78 (1H, t, J = 6.3 Hz), 4.10 (1H, d, J = 16.0 Hz), 4.00 (1H, d, J = 16.0 Hz), 2.86 (1H, dd, J = 5.3, 16.8 Hz), 2.78 (1H, dd, J = 7.4, 16.8 Hz), 1.52 (3H, d, J = 7.0 Hz); 13C NMR (CD3OD, 167 MHz) δ 174.0, 171.9, 171.8, 169.4, 158.0, 145.0, 136.7, 136.6, 131.3, 129.7, 129.4, 127.9, 127.4, 127.1, 125.7, 51.4, 50.5, 44.7, 36.6, 22.6; HRMS (ESI) m/z calculated for molecular formula C22H27N6O5+ [M + H]+ 455.2043, found 455.2034 (Δ = 1.98 ppm). (S)-3-(2-(3-Guanidinobenzamido)acetamido)-4-(((S)-1phenylethyl)amino)-4-oxobutanoic Acid (14). Purity > 98%,

(S)-3-(2-(3-Guanidinobenzamido)acetamido)-4-oxo-4(phenylamino)butanoic Acid (6). Purity > 98%, tR = 12.8 min [analytical HPLC/Gradient#1, 10−70% MeCN (0.1% TFA) in H2O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and tR = 14.7 min [analytical HPLC/Gradient#2, 10−90% MeOH (0.1% TFA) in H2O (0.1% TFA) over 15 min, flow rate of 1.0 mL/min] on a Phenomenex Kinetex analytical column (150 mm × 4.6 mm, 5 μm, 100 Å); 1 H NMR (CD3OD, 500 MHz) 7.85 (1H, d, J = 7.8 Hz), 7.79 (1H, s), 7.62 (2H, d, J = 7.9 Hz), 7.57 (1H, t, J = 8.0 Hz), 7.46 (1H, d, J = 8.0 Hz), 7.30 (2H, t, J = 7.9 Hz), 7.11 (1H, t, J = 7.4 Hz), 4.93−4.90 (1H, m), 4.13 (1H, d, J = 16.2 Hz), 4.06 (1H, d, J = 16.2 Hz), 2.96 (1H, dd, J = 5.8, 16.8 Hz), 2.85 (1H, dd, J = 7.2, 16.8 Hz); 13C NMR (CD3OD, 167 MHz) δ 174.0, 171.9, 171.1, 169.6, 158.0, 139.4, 136.7, 131.3, 129.8, 129.6, 127.3, 125.6, 125.5, 121.8, 121.7, 52.1, 44.5, 36.7; HRMS (ESI) m/z calculated for molecular formula, C20H23N6O5+ [M + H]+ 427.1725, found 427.1720 (Δ = 1.17 ppm). (S)-3-(2-(3-Guanidinobenzamido)acetamido)-4-oxo-4(phenethylamino)butanoic Acid (7). Purity > 98%, tR = 13.0 min [analytical HPLC/Gradient#1, 10−70% MeCN (0.1% TFA) in H2O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and tR = 15.8 min [analytical HPLC/Gradient#2, 10−90% MeOH (0.1% TFA) in H2O (0.1% TFA) over 15 min, flow rate of 1.0 mL/min] on a Phenomenex Kinetex analytical column (150 mm × 4.6 mm, 5 μm, 100 Å); 1 H NMR (DMSO, 500 MHz) δ 9.92 (1H, s), 8.88 (1H, t, J = 5.6 Hz), 8.35 (1H, d, J = 8.2 Hz), 8.29 (1H, t, J = 6.0 Hz), 7.74 (1H, d, J = 7.8 Hz), 7.71 (1H, s), 7.55 (5H, s), 7.52 (1H, d, J = 7.8 Hz), 7.39 (1H, d, J = 7.6 Hz), 7.17 (2H, d, J = 8.5 Hz), 6.84 (2H, d, J = 8.6 Hz), 4.62 (1H, q, J = 7.6 Hz), 4.21 (2H, d, J = 5.8 Hz), 3.96 (1H, dd, J = 5.6, 16.5 Hz), 3.87 (1H, dd, J = 5.7, 16.5 Hz), 3.71 (2H, s), 2.72 (1H, dd, J = 5.5, 16.5 Hz), 2.55 (1H, dd, J = 7.7, 16.0 Hz); 13C NMR (DMSO, 167 MHz) δ 171.9, 170.4, 169.0, 166.1, 158.2, 155.9, 135.6, 135.3, 131.2, 129.8, 128.4, 127.5, 125.3, 123.6, 113.7, 55.1, 49.6, 42.9, 41.7, 36.3; HRMS (ESI) m/z calculated for molecular formula, C22H27N6O5+ [M + H]+ 455.2038, found 455.2031 (Δ = 1.54 ppm). (S)-4-(Benzylamino)-3-(2-(3-guanidinobenzamido)acetamido)-4oxobutanoic Acid (8). Purity > 98%, tR = 12.5 min [analytical HPLC/ Gradient#1, 10−70% MeCN (0.1% TFA) in H2O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and tR = 14.7 min [analytical HPLC/Gradient#2, 10−90% MeOH (0.1% TFA) in H2O (0.1% TFA) over 15 min, flow rate of 1.0 mL/min] on a Phenomenex Kinetex analytical column (150 mm × 4.6 mm, 5 μm, 100 Å); 1H NMR (CD3OD, 500 MHz) δ 7.78 (1H, d, J = 7.6 Hz), 7.69 (1H, s), 7.55 (1H, t, J = 7.8 Hz), 7.45 (1H, d, J = 7.8 Hz), 7.28−7.20 (5H, m), 4.82 (1H, t, J = 6.1 Hz), 4.42 (2H, s), 4.10 (1H, d, J = 16.2 Hz), 4.01 (1H, d, J = 16.2 Hz), 2.90 (1H, dd, J = 5.2, 17.0 Hz), 2.82 (1H, dd, J = 7.1, 16.8 Hz); 13C NMR (CD3OD, 167 MHz) δ 174.1, 172.8, 171.8, 169.5, 158.0, 139.7, 136.6, 136.6, 131.3, 129.7, 129.4, 128.3, 128.1, 127.3, 125.6, 51.4, 44.5, 44.2, 36.6; HRMS (ESI) m/z calculated for molecular formula, C21H25N6O5+ [M + H]+ 441.1881, found 441.1876 (Δ = 1.13 ppm). (S)-4-(Benzhydrylamino)-3-(2-(3-guanidinobenzamido)acetamido)-4-oxobutanoic Acid (9). Purity > 98%, tR = 16.8 min [analytical HPLC/Gradient#1, 10−70% MeCN (0.1% TFA) in H2O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and tR = 18.2 min [analytical HPLC/Gradient#2, 10−90% MeOH (0.1% TFA) in H2O (0.1% TFA) over 15 min, flow rate of 1.0 mL/min] on a Phenomenex Kinetex analytical column (150 mm × 4.6 mm, 5 μm, 100 Å); 1 H NMR (DMSO, 500 MHz) δ 12.34 (1H, s), 9.82 (1H, bs), 8.92 (1H, t, J = 5.5 Hz), 8.66 (1H, d, J = 8.7 Hz), 8.39 (1H, d, J = 8.0 Hz), 7.73 (1H, d, J = 7.8 Hz), 7.67 (1H, s), 7.52 (1H, t, J = 7.9 Hz), 7.49 (4H, bs), 7.39 (1H, d, J = 7.8 Hz), 7.34−7.30 (4H, m), 7.29−7.27 (4H, m), 7.25−7.20 (2H, m), 6.10 (1H, d, J = 8.6 Hz), 4.75−4.71 (1H, m), 3.93 (1H, dd, J = 5.6, 16.2 Hz), 3.87 (1H, dd, J = 5.8, 16.2 Hz), 2.71 (1H, dd, J = 5.4, 16.6 Hz), 2.56 (1H, dd, J = 8.2, 16.3 Hz); 13 C NMR (CD3OD, 167 MHz) δ 174.0, 172.0, 172.0, 169.3, 158.0, 142.9, 142.8, 136.6, 136.5, 131.3, 129.8, 129.5, 129.5, 129.0, 128.7, 128.3, 128.3, 127.4, 125.9, 58.4, 51.5, 44.6, 36.6; HRMS (ESI) m/z calculated for molecular formula, C27H29N6O5+ [M + H]+ 517.2194, found 517.2188 (Δ = 1.16 ppm). 4803



Article

1.0 mL/min] on a Phenomenex Kinetex analytical column (150 mm × 4.6 mm, 5 μm, 100 Å); 1H NMR (CD3OD, 500 MHz) δ 7.76 (1H, d, J = 7.8 Hz), 7.69 (1H, s), 7.63−7.61 (1H, m), 7.60 (1H, s), 7.57−7.56 (1H, m), 7.53−7.47 (2H, m), 7.44−7.35 (4H, m), 7.31−7.28 (2H, m), 4.83 (1H, bs), 4.50 (2H, s), 4.10 (1H, d, J = 16.2 Hz), 4.00 (1H, d, J = 16.2 Hz), 2.92 (1H, dd, J = 5.4, 16.8 Hz), 2.83 (1H, dd, J = 7.2, 17.3 Hz); 13C NMR (CD3OD, 167 MHz) δ 174.0, 172.9, 171.8, 169.5, 158.0, 142.7, 142.2, 140.4, 136.7, 136.6, 131.3, 130.0, 129.8, 129.6, 128.3, 128.0, 127.5, 127.4, 127.0, 126.8, 125.5, 51.5, 44.5, 44.2, 36.6; HRMS (ESI) m/z calculated for molecular formula, C27H29N6O5+ [M + H]+ 517.2199, found 517.2197 (Δ = 0.39 ppm). (S)-4-(([1,1′-Biphenyl]-4-ylmethyl)amino)-3-(2-(3guanidinobenzamido)acetamido)-4-oxobutanoic Acid (19). Purity > 98%, tR = 17.5 min [analytical HPLC/Gradient#1, 10−70% MeCN (0.1% TFA) in H2O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and tR = 18.7 min [analytical HPLC/Gradient#2, 10−90% MeOH (0.1% TFA) in H2O (0.1% TFA) over 15 min, flow rate of 1.0 mL/min] on a Phenomenex Kinetex analytical column (150 mm × 4.6 mm, 5 μm, 100 Å); 1H NMR (CD3OD, 500 MHz) δ 7.79 (1H, d, J = 7.8 Hz), 7.75 (1H, s), 7.57−7.52 (5H, m), 7.44−7.40 (3H, m), 7.38−7.36 (2H, m), 7.32 (1H, t, J = 7.4 Hz), 4.84−4.82 (1H, m), 4.47 (2H, bs), 4.12 (1H, d, J = 16.2 Hz), 4.02 (1H, d, J = 16.2 Hz), 2.91 (1H, dd, J = 5.4, 16.8 Hz), 2.83 (1H, dd, J = 7.2, 17.1 Hz); 13C NMR (CD3OD, 167 MHz) δ 174.1, 172.9, 171.8, 169.5, 158.0, 142.1, 141.3, 138.9, 136.7, 136.6, 131.3, 129.9, 129.8, 129.5, 129.0, 128.8, 128.4, 128.1, 128.0, 127.8, 127.4, 125.5, 125.5, 51.6, 44.4, 43.9, 36.6; HRMS (ESI) m/z calculated for molecular formula, C27H29N6O5+ [M + H]+ 517.2199, found 517.2197 (Δ = 0.39 ppm). Biology. Competitive Integrin Binding Assay. The affinity and selectivity of the integrin inhibitors were determined in a competitive solid-phase binding assay using coated extracellular matrix proteins and soluble integrins. Binding of the integrins was detected by specific antibodies in an enzyme linked immune sorbent assay (ELISA). The in vitro assay was based on a previously reported method with some modifications.46,47 Vitronectin-αvβ3 Assay. Flat-bottom 96-well ELISA plates (BRAND, Wertheim, Germany) were coated overnight at 4 °C with 100 μL/well of 1.0 μg/mL vitronectin (Merck Millipore, Schwalbach/Ts., Germany) in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). Each well was then washed with PBST buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.01% Tween 20, pH 7.4, 3 × 200 μL) and blocked for 1 h at room temperature with 150 μL/well of TSB-buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, pH 7.5, 1% BSA). After being washed three times with PBST, equal volumes of internal standard (5) or test compounds were mixed with 2.0 μg/mL human integrin αvβ3 (R&D Systems, Wiesbaden, Germany) giving a final dilution in TSB buffer of 0.128−400 nM for the inhibitors and 1.0 μg/mL for integrin αvβ3. These solutions (100 μL/well) were incubated for 1 h at room temperature. The plate was washed three times with PBST buffer and 100 μL/well of 2.0 μg/mL primary antibody (mouse antihuman CD51/61, BD Biosciences, Heidelberg, Germany) was added to the plate. After incubation for 1 h at room temperature, the plate was washed three times with PBST, and 100 μL/well of 2.0 μg/mL of secondary peroxidase-labeled antibody (antimouse IgG-POD, SigmaAldrich, Taufkirchen, Germany) was added to the plate and incubated for 1 h at room temperature. After the plate was washed three times with PBST, it got developed by adding 50 μL/well of SeramunBlau fast (Seramun Diagnostic GmbH, Heidesee, Germany) and incubated for 1 min at room temperature. The reaction was stopped with 50 μL/ well of 3 M H2SO4, and the absorbance was measured at 405 nm with a plate reader (Tecan SpectraFLUOR plus, Männedorf, Switzerland). Each compound concentration was tested in duplicate, and the resulting inhibition curves were analyzed using OriginPro 7.5G software; the inflection point describes the IC50 value. Each plate contained 5 as an internal standard. Fibronectin-α5β1 Assay. Experimental procedure was as described for the αvβ3 assay, except for the following modifications. The plates were coated with 100 μL/well of 0.5 μg/mL fibronectin (SigmaAldrich, Taufkirchen, Germany) in carbonate buffer, washed, and

tR = 13.7 min [analytical HPLC/Gradient#1, 10−70% MeCN (0.1% TFA) in H2O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and tR = 15.6 min [analytical HPLC/Gradient#2, 10−90% MeOH (0.1% TFA) in H2O (0.1% TFA) over 15 min, flow rate of 1.0 mL/min] on a Phenomenex Kinetex analytical column (150 mm × 4.6 mm, 5 μm, 100 Å); 1H NMR (CD3OD, 500 MHz) δ 7.84 (1H, d, J = 8.0 Hz), 7.78−7.74 (1H, m), 7.58 (1H, t, J = 7.9 Hz), 7.48−7.45 (1H, m), 7.36−7.34 (2H, m), 7.32−7.24 (2H, m), 7.21−7.18 (1H, m), 5.05− 4.99 (1H, m), 4.80 (1H, t, J = 5.7 Hz), 4.06 (1H, d, J = 16.4 Hz), 4.00 (1H, d, J = 16.4 Hz), 2.86 (1H, dd, J = 5.6, 16.9 Hz), 2.76 (1H, dd, J = 5.6, 16.8 Hz), 1.48 (3H, d, J = 7.0 Hz); 13C NMR (CD3OD, 167 MHz) δ 174.0, 171.8, 171.7, 169.4, 158.0, 144.9, 136.7, 136.7, 131.4, 129.7, 129.4, 128.0, 127.4, 127.2, 125.7, 51.4, 50.4, 44.5, 36.6, 22.5; HRMS (ESI) m/z calculated for molecular formula, C22H27N6O5+ [M + H]+ 455.2043, found 455.2034 (Δ = 1.98 ppm). (S)-4-((4-Chlorobenzyl)amino)-3-(2-(3-guanidinobenzamido)acetamido)-4-oxobutanoic Acid (15). Purity > 98%, tR = 14.7 min [analytical HPLC/Gradient#1, 10−70% MeCN (0.1% TFA) in H2O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and tR = 16.5 min [analytical HPLC/Gradient#2, 10−90% MeOH (0.1% TFA) in H2O (0.1% TFA) over 15 min, flow rate of 1.0 mL/min] on a Phenomenex Kinetex analytical column (150 mm × 4.6 mm, 5 μm, 100 Å); 1 H NMR (CD3OD, 500 MHz) δ 7.76 (1H, d, J = 7.8 Hz), 7.71 (1H, s), 7.55 (1H, t, J = 7.9 Hz), 7.45 (1H, d, J = 7.9 Hz), 7.28−7.24 (4H, m), 4.80 (1H, t, J = 6.2 Hz), 4.40 (2H, s), 4.10 (1H, d, J = 16.2 Hz), 4.01 (1H, d, J = 16.2 Hz), 2.89 (1H, dd, J = 5.5, 17.2 Hz), 2.83 (1H, dd, J = 7.0, 16.9 Hz); 13C NMR (CD3OD, 167 MHz) δ 174.1, 172.9, 171.9, 169.5, 158.0, 138.6, 136.6, 136.6, 133.8, 131.3, 129.9, 129.6, 129.5, 127.3, 125.4, 51.5, 44.5, 43.4, 36.5; HRMS (ESI) m/z calculated for molecular formula, C21H24ClN6O5+ [M + H]+ 475.1497, found 475.1489 (Δ = 1.68 ppm). (S)-3-(2-(3-Guanidinobenzamido)acetamido)-4-((4methoxybenzyl)amino)-4-oxobutanoic Acid (16). Purity > 98%, tR = 13.1 min [analytical HPLC/Gradient#1, 10−70% MeCN (0.1% TFA) in H2O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and tR = 15.0 min [analytical HPLC/Gradient#2, 10−90% MeOH (0.1% TFA) in H2O (0.1% TFA) over 15 min, flow rate of 1.0 mL/min] on a Phenomenex Kinetex analytical column (150 mm × 4.6 mm, 5 μm, 100 Å); 1H NMR (CD3OD, 500 MHz) δ 7.86 (1H, d, J = 7.8 Hz), 7.80 (1H, s), 7.59 (1H, t, J = 7.9 Hz), 7.47 (1H, d, J = 7.9 Hz), 7.28− 7.16 (4H, m), 4.72 (1H, t, J = 7.8 Hz), 4.09 (1H, d, J = 16.3 Hz), 4.01 (1H, d, J = 16.3 Hz), 3.47−3.37 (2H, m), 2.86−2.80 (4H, m), 2.73 (1H, dd, J = 7.4, 17.0 Hz); 13C NMR (CD3OD, 167 MHz) δ 174.0, 172.7, 171.6, 169.4, 158.0, 140.4, 136.8, 136.7, 131.4, 129.8, 129.7, 129.6, 129.5, 127.3, 125.6, 51.4, 44.4, 42.4, 36.6, 36.5; HRMS (ESI) m/z calculated for molecular formula, C22H27N6O6+ [M + H]+ 471.1992, found 471.1983 (Δ = 1.91 ppm). (S)-4-((2,2-Diphenylethyl)amino)-3-(2-(3-guanidinobenzamido)acetamido)-4-oxobutanoic Acid (17). Purity > 98%, tR = 17.4 min [analytical HPLC/Gradient#1, 10−70% MeCN (0.1% TFA) in H2O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and tR = 18.7 min [analytical HPLC/Gradient#2, 10−90% MeOH (0.1% TFA) in H2O (0.1% TFA) over 15 min, flow rate of 1.0 mL/min] on a Phenomenex Kinetex analytical column (150 mm × 4.6 mm, 5 μm, 100 Å); 1H NMR (CD3OD, 500 MHz) δ 7.86 (1H, d, J = 7.8 Hz), 7.81 (1H, s), 7.60 (1H, t, J = 7.9 Hz), 7.48 (1H, d, J = 7.9 Hz), 7.29−7.26 (8H, m), 7.19−7.16 (2H, m), 4.64−4.61 (1H, m), 4.31 (1H, t, J = 8.0 Hz), 4.01 (1H, d, J = 16.2 Hz), 3.95 (1H, d, J = 16.3 Hz), 3.88−3.79 (2H, m), 2.66 (1H, dd, J = 5.2, 16.9 Hz), 2.55 (1H, dd, J = 7.8, 16.9 Hz); 13 C NMR (CD3OD, 167 MHz) δ 173.9, 172.7, 171.5, 169.3, 158.0, 143.7, 136.8, 136.7, 131.4, 131.3, 129.6, 129.6, 129.5, 129.3, 129.1, 127.8, 127.6, 127.4, 125.6, 51.6, 51.3, 45.2, 44.2, 36.67; HRMS (ESI) m/z calculated for molecular formula, C28H31N6O5+ [M + H]+ 531.2356, found 531.2350 (Δ = 1.13 ppm). (S)-4-(([1,1′-Biphenyl]-3-ylmethyl)amino)-3-(2-(3guanidinobenzamido)acetamido)-4-oxobutanoic Acid (18). Purity > 98%, tR = 17.3 min [analytical HPLC/Gradient#1, 10−70% MeCN (0.1% TFA) in H2O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t R = 18.6 min [analytical HPLC/Gradient#2, 10−90% MeOH (0.1% TFA) in H2O (0.1% TFA) over 15 min, flow rate of 4804



Article

blocked as described above. Soluble integrin α5β1 (R&D Systems, Wiesbaden, Germany) was mixed with an equal volume of serial diluted inhibitors resulting in a final integrin concentration of 1.0 μg/mL, 0.00064−2 μM for 5, and 0.0032−10 μM for the inhibitors. As a primary antibody, 100 μL/well of 1.0 μg/mL primary antibody (mouse antihuman CD49e, BD Biosciences, Heidelberg, Germany) was used. Secondary antibody is the same as in integrin αvβ3 assay. Visualization and analysis was performed as described above. Each plate contained 5 as internal standard. Dissociation Studies of Native MDM2/p53 or MDM4/p53 Complexes. The ability of the new derivatives to dissociate the native MDM2/p53 or MDM4/p53 complex, quantitative sandwich immuneenzymatic assays19,20,22 were performed on cell lysates obtained from U87MG cells (for p53/MDM2) or from SHSY-5Y (for p53/MDM4). Wells were precoated with full-length anti-MDM2 (sc-965, Santa Cruz Biotechnology, in 0.05% poly-L-ornithine) or anti-MDM4 (sc-74468 Santa Cruz Biotechnology, in 0.05% poly-L-ornithine) antibody overnight at room temperature. Compound 9 was incubated, at different concentrations, with cell lysates (10 min at room temperature) and then transferred to the precoated wells for 90 min. After extensive washes, nonspecific sites were blocked with 1% BSA, and the wells were incubated for 90 min with an anti-p53 antibody (sc-6243, Santa Cruz Biotechnology, 1:250). Afterward, samples were washed and incubated for 60 min with a specific HRP-conjugate antibody. The colorimetric quantification (450 nm) of the p53/MDM2 or p53/MDM4 complexes was reached by the addition of a TMB substrate kit (Thermo Fisher Scientific). Blanks were in the absence of the primary anti-p53 antibody. Dose−response curves were derived with Graph Pad Prism 4 software, from which IC50 values were obtained.19,20,22 Human Cell Lines. Human glioblastoma (U87MG and T98G) and human neuroblastoma (SHSY-5Y) cells were obtained from the National Institute for Cancer Research of Genova (Italy). Each cell line was monitored for DNA profiling and cultured as described.51,72 For cell treatments with the synthetic compounds, the human cell lines were seeded at approximately 5000 cells/cm2. After 24 h, the culture medium was replaced with fresh medium containing compound solubilized in DMSO for the indicated incubation times. DMSO was added to control cells (1% v/v). Cell Proliferation Assays. U87MG cells were incubated with 9 (ranging from 1 to 100 nM), MDM2 inhibitor 1 (100 nM), MDM4 inhibitor 2 (100 nM), or 5 (100 nM), alone or in combination, for 72 h. After treatment period, cell proliferation was determined using the MTS assay, as described.73 The same experiment was performed on U87MG and T98G cells with 9 alone at different concentrations for 72 h. After the incubation time, cell proliferation was evaluated with the MTS assays, as described. RNA Extraction and Real Time PCR Analysis in U87MG Cells. U87MG cells were incubated with compound 9, or compounds 1, 2 or 5, for 8 or 24 h at the concentration of 100 nM, alone or in combination. Following treatments, cells were collected, and total RNA was extracted using Rneasy Mini Kit (Qiagen, Hilden, Germany). cDNA synthesis was performed with 500 ng of RNA (BioRad, Hercules, USA). The mRNA levels of p53 targets (MDM2, PUMA, and p21) were evaluated by quantitative real-time RT-PCR using Fluocycle II SYBR (Euroclone, Milan, Italy). The nucleotide sequences, annealing temperature, and product size of the primers have been previously reported.51,74,75 p53 Protein Accumulation. p53 protein levels were analyzed by Western blotting. In brief, U87MG cells were treated with compound 9, or compounds 1, 2 or 5, for 24 h. Then, cells were lysed, and an equal amount of the cell extracts (40 μg of proteins) were diluted in Laemmli solution, resolved by SDS-PAGE21,22 and blotted using a specific antibody for p53. GAPDH was used as the loading control. The densitometric analysis of immunoreactive bands was performed using ImageJ software (3.0 version). Cell Cycle Analysis in U87MG Cells. U87MG were treated with DMSO, 9 (100 nM), 1 (100 nM), 2 (100 nM), and 5 (100 nM), alone or in combination, for 24 h. The measurement of the percentage of cells in the different cell phases was performed using the Muse Cell Analyzer (Merck KGaA, Darmstadt, Germany) as described previously.22,75

Invasion Assay of U87MG Cells. The effect of 9 on cell invasion through Matrigel was evaluated using human GBM cells as follows: the surface of the transwell was coated with Matrigel basement membrane matrix (BD Biosciences) (0.32 mg/mL) at room temperature, as reported previously.76 The U87MG cells were suspended in serum free medium (60,000/400 μL), treated with 10 μM 9 and added to the upper compartment of the transwell, while 200 μL of RPMI containing 10% fetal bovine serum was added to the lower compartment. The cells were allowed to invade through the matrixes at 37 °C for 24 h.76,77 The number of invading cells was quantified by counting the cells on the lower surface of the transwell membrane after fixing with p-formaldehyde and staining with crystal violet. Nonmigrating cells on the upper surface were removed with a cotton bud. Pictures of randomly picked light microscope fields were taken (five fields for each filter), and cells were counted using ImageJ Software.76,77 Statistical Analysis. Graph-Pad Prism (GraphPad Software Inc., San Diego, CA) was used for data analysis and graphic presentations. All data are presented as the mean ± SEM. One-way analysis of variance (ANOVA) with Bonferroni’s corrected t test for posthoc pairwise comparisons was used to perform statistical analysis.74,75 Molecular Modeling. For our study, 8 and 9 were prepared using LigPrep (LigPrep, version 2.5, Schrödinger, LLC, New York, NY, 2011), which suggested to consider their zwitterion protonation state for docking calculations. As per the MDM2 X-ray selection, multiple 3D structures of MDM2 can be found in the Protein Data Bank (PDB). Among them, we took into consideration just the ones containing the N-terminus residues 16−24 (e.g., 3LBL, 4HBM, 4DIJ, 4JVR, 4JVE, 1T4E, 4ERF, etc.), which folds into an ordered helix that changes shape and size of the catalytic pocket and provides additional interaction points. So, among the nontruncated X-ray structures, only those cocrystallized with an organic compound were considered, and the one with the highest resolution (1.60 Å) (PDB: 3LBL) containing a spirooxindole derivative was chosen for our docking experiments. As for the integrin receptors, the crystal structures of αvβ3 in complex with fibronectin (Fn) (PDB: 4MMX)61 and of α5β1 in complex with a cyclic RGD peptide (PDB: 4WK4)62 were selected for docking studies. The three proteins were prepared using the Protein Preparation Wizard implemented in Maestro Suite 2011 (Maestro, version 9.0.211; Schrodinger, LLC, New York, 2009). First, the Mn2+ ion at the MIDAS in the αvβ3 crystal structure was replaced with Mg2+. During the preparation, all water molecules were deleted, hydrogen atoms added, and hydrogen-bonding network optimized. Finally, the positions of the hydrogen atoms were minimized. Blind Docking. The Autodock program (version 4.2) was used for the blind docking since it has previously been demonstrated to be successful in the prediction of protein binding sites.78,79 The docking area has been defined by a box large enough to comprise the entire protein and centered on the protein. Grids points of 126 × 126 × 126 with 0.375 Å spacing were calculated around the docking area for all the ligand atom types using AutoGrid4. One hundred separate docking calculations were performed. Each docking calculation consisted of 25 × 106 energy evaluations using the Lamarckian genetic algorithm local search (GALS) method. A low-frequency local search according to the method of Solis and Wets was applied to docking trials to ensure that the final solution represents a local minimum. Each docking run was performed with a population size of 150, and 300 rounds of Solis and Wets local search were applied with a probability of 0.06. A mutation rate of 0.02 and a crossover rate of 0.8 were used to generate new docking trials for subsequent generations. The docking results from each of the 100 calculations were clustered on the basis of root-mean square deviation (rmsd = 2.0 Å) between the Cartesian coordinates of the ligand atoms and were ranked on the basis of the free energy of binding. The canonical binding site and the pocket detected as putative allosteric binding site by the Autodock program were further investigated with a subsequent “focused” docking step in order to characterize compound 9’s binding modes. The “focused” docking was performed also with the aid of Glide 5.5 software67 in extra precision mode (XP), employing Glidescore for 4805



Article

ligand ranking. The receptor grids were generated using the grid generation in Glide centered around the crystallized ligand for the canonical pocket and around the L37 for the bottom pocket, using default settings. Figures 12 and 13 were rendered using PyMOL (www.pymol.org) and Chimera software package,80 respectively. Docking Calculations on Integrins. Docking to integrin receptors was performed using the standard precision (SP) mode of Glide. For the grid generation, a virtual box surrounding the ligand RGD binding cavity was created, using default settings. Per-atom van der Waals radii of residues (αv)-Y178, (β3)-Y122, (β3)-M180, and (β3)-R214 in αvβ3, and (α5)-F187 and (β1)-Y133 in α5β1, respectively, were scaled to 0.8. This partly accounted for the possible steric hindrance effects of such bulky amino acids in rigid receptor docking simulations. The OPLS3 force field81 was employed to run calculations. For each complex, the lowest energy solution able to properly recapitulate the typical RGD interaction pattern26,27 was selected for the description of the ligand binding mode. Figure 8 was rendered using PyMOL (www.pymol.org). NMR Measurements. The MDM2 protein used for NMR experiment was cloned, expressed, and purified as previously described.23 All the spectra were acquired at 298 K with a Bruker Avance NMR spectrometer operating at 700 MHz 1H Larmor frequency, equipped with a cryogenically cooled probe optimized for 13C sensitivity (TCI, S/N 1500:1, on the ASTM standard sample) as well as for 1H sensitivity. The spectra were processed with the Bruker TOPSPIN software packages and analyzed by the program Computer Aided Resonance Assignment (ETH Zurich; Keller, 2004). The protein assignment was based on the data reported in the Biological Magnetic Resonance Data Bank under the accession code 6612.82 During the NMR titration of the protein with 9, aliquots of a DMSO-d6 solution of compound 9 (25, 50, 100, 150, 200, 250, 300, 350, and 400 μM) were added to the buffered solution [50 mM KH2PO4, 50 mM Na2HPO4, pH 7.5, 150 mM NaCl] of 15N isotopically enriched MDM2 protein at the concentration of 200 μM. The competitive binding experiment was performed titrating the 15N isotopically enriched protein (200 μM) in the presence of compound 9 (400 μM) with 1, by adding increasing aliquots of the latter ligand solubilized in DMSO-d6 (25, 50, 100, 150, 200 μM) to the solution. The NH resonances of the protein were detected during the titrations through 2D 1H−15N HSQC NMR spectra.

■

Barbara Costa: 0000-0002-7598-1275 Claudia Martini: 0000-0001-9379-3027 Horst Kessler: 0000-0002-7292-9789 Ettore Novellino: 0000-0002-2181-2142 Luciana Marinelli: 0000-0002-4084-8044 Author Contributions ∇

These authors contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS The study was supported by PRIN2015 FCHJ8E. ABBREVIATIONS ANOVA, analysis of variance; CSP, chemical shift perturbation; CTC, chlorotrityl chloride; DB, database; DIEA, diisopropylethylamine; ECM, extra-cellular matrix; GBA, 3-guanidino benzoic acid; GBM, Glioblastoma Multiforme; HBTU, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate; HOAt, 1-hydroxyazabenzotriazole; HOBt, 1-hydroxybenzotriazole; MDM2, murine double minute 2; MDM4, murine double minute 4; MGMT, O6-methylguanine DNA methyltransferase; PyAOP, (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; SDL, specificity determining loop; TCGA, The Cancer Genome Atlas; TMZ, temozolomide; VS, virtual screening

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00004. Analytical data and HPLC chromatograms of compounds 6−19. Binding mode of 8 into the integrin receptors. Docking binding mode B of 9 in the canonical binding pocket. Blind docking details. Docking into the bottom pocket. Dose−response curves of active compounds toward MDM2 and/or MDM4. PDB coordinates for docking pose of 9 in the MDM2 canonical binding site. PDB coordinates for docking pose of 9 in the MDM2 bottom pocket (PDF) Molecular formula strings for compounds 6−19 (CSV)

■

REFERENCES

(1) Ostrom, Q. T.; Gittleman, H.; Farah, P.; Ondracek, A.; Chen, Y.; Wolinsky, Y.; Stroup, N. E.; Kruchko, C.; Barnholtz-Sloan, J. S. CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2006−2010. Neuro. Oncol. 2013, 15, 1−56. (2) Lacroix, M.; Abi-Said, D.; Fourney, D. R.; Gokaslan, Z. L.; Shi, W.; DeMonte, F.; Lang, F. F.; McCutcheon, I. E.; Hassenbusch, S. J.; Holland, E.; Hess, K.; Michael, C.; Miller, D.; Sawaya, R. A multivariate analysis of 416 patients with glioblastoma multiforme: prognosis, extent of resection, and survival. J. Neurosurg. 2001, 95, 190−198. (3) https://clinicaltrial.gov: (a) Autologous Cytomegalovirus (CMV)-specific cytotoxic T cells for glioblastoma (GBM) patients;. (b) Dendritic cell (DC) vaccine for malignant glioma and glioblastoma; (c) Avelumab in patients with newly diagnosed glioblastoma multiforme. Accessed Apr 13, 2018. (4) Wade, M.; Li, Y. C.; Wahl, G. M. MDM2, MDMX and p53 in oncogenesis and cancer therapy. Nat. Rev. Cancer 2013, 13, 83−96. (5) Khoo, K. H.; Verma, C. S.; Lane, D. P. Drugging the p53 pathway: understanding the route to clinical efficacy. Nat. Rev. Drug Discovery 2014, 13, 217−236. (6) Selivanova, G. Wild type p53 reactivation: from lab bench to clinic. FEBS Lett. 2014, 588, 2628−2638. (7) (a) Brown, C. J.; Lain, S.; Verma, C. S.; Fersht, A. R.; Lane, D. P. Awakening guardian angels: drugging the p53 pathway. Nat. Rev. Cancer 2009, 9, 862−873. (b) Momand, J.; Wu, H. H.; Dasgupta, G. MDM2-master regulator of the p53 tumor suppressor protein. Gene 2000, 242, 15−29. (8) Momand, J.; Jung, D.; Wilzynski, S.; Niland, J. The MDM2 gene amplification database. Nucleic Acids Res. 1998, 26, 3453−3459. (9) Zhao, Y.; Aguilar, A.; Bernard, D.; Wang, S. Small-molecule inhibitors of the MDM2-p53 protein-protein interaction (MDM2 Inhibitors) in clinical trials for cancer treatment. J. Med. Chem. 2015, 58, 1038−1052.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Francesco Merlino: 0000-0002-9607-229X Salvatore Di Maro: 0000-0002-9286-4433 Marco Fragai: 0000-0002-8440-1690 4806



Article

(10) Wade, M.; Rodewald, L. W.; Espinosa, J. M.; Wahl, G. M. BH3 activation blocks Hdmx suppression of apoptosis and co-operates with nutlin to induce cell death. Cell Cycle 2008, 7, 1973−1982. (11) Patton, J. T.; Mayo, L. D.; Singhi, A. D.; Gudkov, A. V.; Stark, G. R.; Jackson, M. W. Levels of HdmX expression dictate the sensitivity of normal and transformed cells to nutlin-3. Cancer Res. 2006, 66, 3169−3176. (12) Wade, M.; Wong, E. T.; Tang, M.; Stommel, J. M.; Wahl, G. M. Hdmx modulates the outcome of p53 activation in human tumor cells. J. Biol. Chem. 2006, 281, 33036−33044. (13) Hu, B.; Gilkes, D. M.; Farooqi, B.; Sebti, S. M.; Chen, J. MDMX overexpression prevents p53 activation by the MDM2 inhibitor nutlin. J. Biol. Chem. 2006, 281, 33030−33035. (14) Bernal, F.; Wade, M.; Godes, M.; Davis, T. N.; Whitehead, D. G.; Kung, A. L.; Wahl, G. M.; Walensky, L. D. A Stapled p53 helix overcomes HDMX-mediated suppression of p53. Cancer Cell 2010, 18, 411−422. (15) Zaytsev, A.; Dodd, B.; Magnani, M.; Ghiron, C.; Golding, B. T.; Griffin, R. J.; Liu, J.; Lu, X.; Micco, I.; Newell, D. R.; Padova, A.; Robertson, G.; Lunec, J.; Hardcastle, I. R. Searching for dual inhibitors of the MDM2-p53 and MDMX-p53 protein−protein interaction by a scaffold-hopping approach. Chem. Biol. Drug Des. 2015, 86, 180−189. (16) Neochoritis, C. G.; Wang, K.; Estrada-Ortiz, N.; Herdtweck, E.; Kubica, K.; Twarda, A.; Zak, K. M.; Holak, T. A.; Dömling, A. 2,3′Bis(1′H-indole) heterocycles: new p53/MDM2/MDMX antagonists. Bioorg. Med. Chem. Lett. 2015, 25, 5661−5666. (17) Golestanian, S.; Sharifi, A.; Popowicz, G. M.; Azizian, H.; Foroumadi, A.; Szwagierczak, A.; Holak, T. A.; Amanlou, M. Discovery of novel dual inhibitors against Mdm2 and Mdmx proteins by in silico approaches and binding assay. Life Sci. 2016, 145, 240−246. (18) Estrada-Ortiz, N.; Neochoritis, C. G.; Dömling, A. How to design a successful p53-MDM2/X interaction inhibitor: a thorough overview based on crystal structures. ChemMedChem 2016, 11, 757− 772. (19) Daniele, S.; La Pietra, V.; Barresi, E.; Di Maro, S.; Da Pozzo, E.; Robello, M.; La Motta, C.; Cosconati, S.; Taliani, S.; Marinelli, L.; Novellino, E.; Martini, C.; Da Settimo, F. Lead optimization of 2phenylindolylglyoxylyldipeptide murine double minute (MDM)2/ Translocator Protein (TSPO) dual inhibitors for the treatment of gliomas. J. Med. Chem. 2016, 59, 4526−4538. (20) Daniele, S.; Barresi, E.; Zappelli, E.; Marinelli, L.; Novellino, E.; Da Settimo, F.; Taliani, S.; Trincavelli, M. L.; Martini, C. Long lasting MDM2/Translocator protein modulator: a new strategy for irreversible apoptosis of human glioblastoma cells. Oncotarget 2016, 7, 7866−7884. (21) Daniele, S.; Costa, B.; Zappelli, E.; Da Pozzo, E.; Sestito, S.; Nesi, G.; Campiglia, P.; Marinelli, L.; Novellino, E.; Rapposelli, S.; Martini, C. Combined inhibition of AKT/mTOR and MDM2 enhances glioblastoma multiforme cell apoptosis and differentiation of cancer stem cells. Sci. Rep. 2015, 5, 9956. (22) Daniele, S.; Taliani, S.; Da Pozzo, E.; Giacomelli, C.; Costa, B.; Trincavelli, M. L.; Rossi, L.; La Pietra, V.; Barresi, E.; Carotenuto, A.; Limatola, A.; Lamberti, A.; Marinelli, L.; Novellino, E.; Da Settimo, F.; Martini, C. Apoptosis therapy in cancer: the first single-molecule coactivating p53 and the translocator protein in glioblastoma. Sci. Rep. 2015, 4, 4749. (23) Giustiniano, M.; Daniele, S.; Pelliccia, S.; La Pietra, V.; Pietrobono, D.; Brancaccio, D.; Cosconati, S.; Messere, A.; Giuntini, S.; Cerofolini, L.; Fragai, M.; Luchinat, C.; Taliani, S.; La Regina, G.; Da Settimo, F.; Silvestri, R.; Martini, C.; Novellino, E.; Marinelli, L. Computer-aided identification and lead optimization of dual murine double minute 2 and 4 binders: structure-activity relationship studies and pharmacological activity. J. Med. Chem. 2017, 60, 8115−8130. (24) Sallinen, S. L.; Sallinen, P. K.; Haapasalo, H. K.; Helin, H. J.; Helen, P. T.; Schraml, P.; Kallioniemi, O. P.; Kononen, J. Identification of differentially expressed genes in human gliomas by DNA microarray and tissue chip techniques. Cancer Res. 2000, 60, 6617−6622. (25) Colin, C.; Baeza, N.; Bartoli, C.; Fina, F.; Eudes, N.; Nanni, I.; Martin, P. M.; Ouafik, L.; Figarella-Branger, D. Identification of genes

differentially expressed in glioblastoma versus pilocytic astrocytoma using suppression subtractive hybridization. Oncogene 2006, 25, 2818− 2826. (26) Mas-Moruno, C.; Rechenmacher, F.; Kessler, H. Cilengitide: the first anti-angiogenic small molecule drug candidate design, synthesis and clinical evaluation. Anti-Cancer Agents Med. Chem. 2010, 10, 753− 768. (27) Dechantsreiter, M. A.; Planker, E.; Mathä, B.; Lohof, E.; Hölzemann, G.; Jonczyk, A.; Goodman, S. L.; Kessler, H. Nmethylated cyclic RGD peptides as highly active and selective αVβ3 integrin antagonists. J. Med. Chem. 1999, 42, 3033−3040. (28) Ray, A. M.; Schaffner, F.; Janouskova, H.; Noulet, F.; Rognan, D.; Lelong-Rebel, I.; Choulier, L.; Blandin, A. F.; Lehmann, M.; Martin, S.; Kapp, T.; Neubauer, S.; Rechenmacher, F.; Kessler, H.; Dontenwill, M. Single cell tracking assay reveals an opposite effect of selective small non-peptidic α5β1 or αvβ3/β5 integrin antagonists in U87MG glioma cells. Biochim. Biophys. Acta, Gen. Subj. 2014, 1840, 2978−2987. (29) Bredel, M.; Bredel, C.; Juric, D.; Harsh, G. R.; Vogel, H.; Recht, L. D.; Sikic, B. I. Functional network analysis reveals extended gliomagenesis pathway maps and three novel MYC-interacting genes in humangliomas. Cancer Res. 2005, 65, 8679−8689. (30) Maglott, A.; Bartik, P.; Cosgun, S.; Klotz, P.; Ronde, P.; Fuhrmann, G.; Takeda, K.; Martin, S.; Dontenwill, M. The small α5β1 integrin antagonist, SJ749, reduces proliferation and clonogenicity of human astrocytoma cells. Cancer Res. 2006, 66, 6002−6007. (31) Martin, S.; Cosset, E. C.; Terrand, J.; Maglott, A.; Takeda, K.; Dontenwill, M. Caveolin-1 regulates glioblastoma aggressiveness through the control of α5β1 integrin expression and modulates glioblastoma responsiveness to SJ749, an α5β1 integrin antagonist. Biochim. Biophys. Acta, Mol. Cell Res. 2009, 1793, 354−367. (32) Renner, G.; Janouskova, H.; Noulet, F.; Koenig, V.; Guerin, E.; Bär, S.; Nuesch, J.; Rechenmacher, F.; Neubauer, S.; Kessler, H.; Blandin, A.-F.; Choulier, L.; Etienne-Selloum, N.; Lehmann, M.; Lelong-Rebel, I.; Martin, S.; Dontenwill, M. Integrin α5β1 and p53 convergent pathways in the control of anti-apoptotic proteins PEA-15 and survivin in high-grade glioma. Cell Death Differ. 2016, 23, 640− 653. (33) Janouskova, H.; Maglott, A.; Leger, D. Y.; Bossert, C.; Noulet, F.; Guerin, E.; Guenot, D.; Pinel, S.; Chastagner, P.; Plenat, F.; EntzWerle, N.; Lehmann-Che, J.; Godet, J.; Martin, S.; Teisinger, J.; Dontenwill, M. Integrin α5β1 plays a critical role in resistance to Temozolomide by interfering with the p53 pathway in high-grade glioma. Cancer Res. 2012, 72, 3463−3470. (34) Martinkova, E.; Maglott, A.; Leger, D. Y.; Bonnet, D.; Stiborova, M.; Takeda, K.; Martin, S.; Dontenwill, M. α5β1 integrin antagonists reduce chemotherapy-induced premature senescence and facilitate apoptosis in human glioblastoma cells. Int. J. Cancer 2010, 127, 1240− 1248. (35) (a) Maltsev, O. V.; Marelli, U. K.; Kapp, T. G.; Di Leva, F. S.; Di Maro, S.; Nieberler, M.; Reuning, U.; Schwaiger, M.; Novellino, E.; Marinelli, L.; Kessler, H. Stable peptides instead of stapled peptides: highly potent αvβ6-selective integrin ligands. Angew. Chem., Int. Ed. 2016, 55, 1535−1539. (b) Di Leva, F. S.; Tomassi, S.; Di Maro, S.; Reichart, F.; Notni, J.; Dangi, A.; Marelli, U. K.; Brancaccio, D.; Merlino, F.; Wester, H.-J.; Novellino, E.; Kessler, H.; Marinelli, L. From a helix to a small cycle: metadynamics-inspired αvβ6 integrin selective ligands. Angew. Chem., Int. Ed. 2018, DOI: 10.1002/ anie.201803250. (36) Neubauer, S.; Rechenmacher, F.; Brimioulle, R.; Di Leva, F. S.; Bochen, A.; Sobahi, T. R.; Schottelius, M.; Novellino, E.; Mas-Moruno, C.; Marinelli, L.; Kessler, H. Pharmacophoric modifications lead to superpotent αvβ3 integrin ligands with suppressed α5β1 activity. J. Med. Chem. 2014, 57, 3410−3417. (37) (a) Bochen, A.; Marelli, U. K.; Otto, E.; Pallarola, D.; MasMoruno, C.; Di Leva, F. S.; Boehm, H.; Spatz, J. P.; Novellino, E.; Kessler, H.; Marinelli, L. Biselectivity of isoDGR peptides for fibronectin binding integrin subtypes α5β1 and αvβ6: conformational control through flanking amino acids. J. Med. Chem. 2013, 56, 1509− 4807



Article

cell detachment and anoikis induction. Anti-Cancer Drugs 2013, 24, 818−825. (53) (a) Lim, S. T.; Chen, X. L.; Lim, Y.; Hanson, D. A.; Vo, T. T.; Howerton, K.; Larocque, N.; Fisher, S. J.; Schlaepfer, D. D.; Ilic, D. Nuclear FAK promotes cell proliferation and survival through FERMenhanced p53 degradation. Mol. Cell 2008, 29, 9−22. (b) Golubovskaya, V. M.; Cance, W. G. FAK and p53 protein interactions. AntiCancer Agents Med. Chem. 2011, 11, 617−619. (54) (a) Russo, M. A.; Paolillo, M.; Sanchez-Hernandez, Y.; Curti, D.; Ciusani, E.; Serra, M.; Colombo, L.; Schinelli, S. A small-molecule RGD-integrin antagonist inhibits cell adhesion, cell migration and induces anoikis in glioblastoma cells. Int. J. Oncol. 2013, 42, 83−92. (b) Desgrosellier, J. S.; Cheresh, D. A. Integrins in cancer: biological implications and therapeutic opportunities. Nat. Rev. Cancer 2010, 10, 9−22. (55) Wang, C.-C.; Liao, Y.-P.; Mischel, P. S.; Iwamoto, K. S.; Cacalano, N. A.; Mcbride, W. H. HDJ-2 as a target for radiosensitization of glioblastoma multiforme cells by the farnesyltransferase inhibitor R115777 and the role of the p53/p21 pathway. Cancer Res. 2006, 66, 6756−6762. (56) (a) Verreault, M.; Schmitt, C.; Goldwirt, L.; Pelton, K.; Haidar, S.; Levasseur, C.; Guehennec, J.; Knoff, D.; Labussière, M.; Marie, Y.; Ligon, A. H.; Mokhtari, K.; Hoang-Xuan, K.; Sanson, M.; Alexander, B. M.; Wen, P. Y.; Delattre, J. Y.; Ligon, K. L.; Idbaih, A. Preclinical efficacy of the MDM2 inhibitor RG7112 in MDM2-amplified and TP53 wild-type Glioblastomas. Clin. Cancer Res. 2016, 22, 1185−1196; (b) Correction: Preclinical efficacy of the MDM2 inhibitor RG7112 in MDM2-amplified and TP53 wild-type glioblastomas. Clin. Cancer Res. 2016, 22, 2313−2313. (57) Haupt, S.; Buckley, D.; Pang, J.-M.; Panimaya, J.; Paul, P. J.; Gamell, C.; Takano, E. A.; Lee, Y. Y.; Hiddingh, S.; Rogers, T.-M.; Teunisse, A. F. A. S.; Herold, M. J.; Marine, J.-C.; Fox, S. B.; Jochemsen, A.; Haupt, Y. Targeting Mdmx to treat breast cancers with wild-type p53. Cell Death Dis. 2015, 6, e1821. (58) Geng, Q.-Q.; Dong, D.-F.; Chen, N.-Z.; Wu, Y.-Y.; Li, E.-X.; Wang, J.; Wang, S.-M. Induction of p53 expression and apoptosis by a recombinant dual-target MDM2/MDMX inhibitory protein in wildtype p53 breast cancer cells. Int. J. Oncol. 2013, 43, 1935−1942. (59) Nieberler, M.; Reuning, U.; Reichart, F.; Notni, J.; Wester, H.-J.; Schwaiger, M.; Weinmüller, M.; Räder, A.; Steiger, K.; Kessler, H. Exploring the role of RGD-recognizing integrins in cancer. Cancers 2017, 9, E116. (60) Renner, G.; Noulet, F.; Mercier, M.-C.; Choulier, L.; EtienneSelloum, N.; Gies, J.-P.; Lehmann, M.; Lelong-Rebel, I.; Martin, S.; Dontenwill, M. Expression/activation of α5β1 integrin is linked to the β-Catenin signaling pathway to drive migration in glioma cells. Oncotarget 2016, 7, 62194−62207. (61) Van Agthoven, J. F.; Xiong, J. P.; Alonso, J. L.; Rui, X.; Adair, B. D.; Goodman, S. L.; Arnaout, M. A. Structural basis for pure antagonism of integrin αvβ3 by a high-affinity form of fibronectin. Nat. Struct. Mol. Biol. 2014, 21, 383−388. (62) Xia, W.; Springer, T. A. Metal ion and ligand binding of integrin α5β1. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 17863−17868. (63) Sigurdardottir, A. G.; Winter, A.; Sobkowicz, A.; Fragai, M.; Chirgadze, D.; Ascher, D. B.; Blundell, T. L.; Gherardi, E. Exploring the chemical space of the lysine-binding pocket of the first kringle domain of hepatocyte growth factor/scatter factor (HGF/SF) yields a new class of inhibitors of HGF/SF-MET binding. Chem. Sci. 2015, 6, 6147−6157. (64) Zuiderweg, E. R. Mapping protein-protein interactions in solution by NMR spectroscopy. Biochemistry 2002, 41, 1−7. (65) Chi, S.-W.; Lee, S.-H.; Kim, D.-H.; Ahn, M.-J.; Kim, J.-S.; Woo, J.-Y.; Torizawa, T.; Kainosho, M.; Han, K.-H. Structural details on mdm2-p53 interaction. J. Biol. Chem. 2005, 280, 38795−38802. (66) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexiblity. J. Comput. Chem. 2009, 30, 2785−2791. (67) Glide, version 5.5; Schrodinger, LLC: New York, 2009.

1519. (b) Kapp, T. G.; Di Leva, F. S.; Notni, J.; Räder, A. F. B.; Fottner, M.; Reichart, F.; Reich, D.; Wurzer, A.; Steiger, K.; Novellino, E.; Marelli, U. K.; Wester, H.-J.; Marinelli, L.; Kessler, H. Nmethylation of isoDGR peptides: discovery of a selective α5β1-integrin ligand as a potent tumor imaging agent. J. Med. Chem. 2018, 61, 2490− 2499. (38) Heckmann, D.; Meyer, A.; Marinelli, L.; Zahn, G.; Stragies, R.; Kessler, H. Probing integrin selectivity: rational design of highly active and selective ligands for the α5β1 and αvβ3 integrin receptor. Angew. Chem., Int. Ed. 2007, 46, 3571−3574. (39) Marinelli, L.; Gottschalk, K. E.; Meyer, A.; Novellino, E.; Kessler, H. Human integrin αvβ5: homology modeling and ligand binding. J. Med. Chem. 2004, 47, 4166−4177. (40) Marinelli, L.; Meyer, A.; Heckmann, D.; Lavecchia, A.; Novellino, E.; Kessler, H. Ligand binding analysis for human α5β1 integrin: strategies for designing new α5β1 integrin antagonists. J. Med. Chem. 2005, 48, 4204−4207. (41) Merrifield, R. B. Solid Phase Peptide Synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149−2154. (42) Carpino, L. A.; Sadat-Aalaee, D.; Chao, H. G.; Deselms, R. H. [(9-Fluorenylmethyl)oxy]carbonyl (FMOC) amino acid fluorides. Convienient new peptide coupling reagents applicable to the FMOC/tert-butyl strategy for solution and solid-phase syntheses. J. Am. Chem. Soc. 1990, 112, 9651−9652. (43) Merlino, F.; Yousif, A. M.; Billard, É.; Dufour-Gallant, J.; Turcotte, S.; Grieco, P.; Chatenet, D.; Lubell, W. D. Urotensin II(4−11) azasulfuryl peptides: synthesis and biological activity. J. Med. Chem. 2016, 59, 4740−4752. (44) Carotenuto, A.; Merlino, F.; Cai, M.; Brancaccio, D.; Yousif, A. M.; Novellino, E.; Hruby, V. J.; Grieco, P. Discovery of novel potent and selective agonists at the melanocortin-3 receptor. J. Med. Chem. 2015, 58, 9773−9778. (45) Han, S.-Y.; Kim, Y.-A. Recent development of peptide coupling reagents in organic synthesis. Tetrahedron 2004, 60, 2447−2467. (46) Chatterjee, J.; Ovadia, O.; Zahn, G.; Marinelli, L.; Hoffman, A.; Gilon, C.; Kessler, H. Multiple N-methylation by a designed approach enhances receptor selectivity. J. Med. Chem. 2007, 50, 5878−5881. (47) (a) Mas-Moruno, C.; Beck, J. G.; Doedens, L.; Frank, A. O.; Marinelli, L.; Cosconati, S.; Novellino, E.; Kessler, H. Increasing αvβ3 selectivity of the anti-angiogenic drug cilengitide by N-methylation. Angew. Chem., Int. Ed. 2011, 50, 9496−9500. (b) Kapp, T. G.; Rechenmacher, F.; Neubauer, S.; Maltsev, O. V.; Cavalcanti-Adam, E. A.; Zarka, R.; Reuning, U.; Notni, J.; Wester, H.-J.; Mas-Moruno, C.; Spatz, J.; Geiger, B.; Kessler, H. A comprehensive evaluation of the activity and selectivity profile of ligands for RGD-binding integrins. Sci. Rep. 2017, 7, 39805. (48) Bista, M.; Smithson, D.; Pecak, A.; Salinas, G.; Pustelny, K.; Min, J.; Pirog, A.; Finch, K.; Zdzalik, M.; Waddell, B.; Wladyka, B.; Kedracka-Krok, S.; Dyer, M. A.; Dubin, G.; Guy, R. K. On the mechanism of action of SJ-172550 in inhibiting the interaction of MDM4 and p53. PLoS One 2012, 7, e37518. (49) Reed, D.; Shen, Y.; Shelat, A. A.; Arnold, L. A.; Ferreira, A. M.; Zhu, F.; Mills, N.; Smithson, D. C.; Regni, C. A.; Bashford, D.; Cicero, S. A.; Schulman, B. A.; Jochemsen, A. G.; Guy, R. K.; Dyer, M. A. Identification and characterization of the first small molecule inhibitor of MDMX. J. Biol. Chem. 2010, 285, 10786−10796. (50) Villalonga-Planells, R.; Coll-Mulet, L.; Martínez-Soler, F.; Castaño, E.; Acebes, J. J.; Giménez-Bonafé, P.; Gil, J.; Tortosa, A. Activation of p53 by nutlin-3a induces apoptosis and cellular senescence in human glioblastoma multiforme. PLoS One 2011, 6, e18588. (51) Costa, B.; Bendinelli, S.; Gabelloni, P.; Da Pozzo, E.; Daniele, S.; Scatena, F.; Vanacore, R.; Campiglia, P.; Bertamino, A.; GomezMonterrey, I.; Sorriento, D.; Del Giudice, C.; Iaccarino, G.; Novellino, E.; Martini, C. Human glioblastoma multiforme: p53 reactivation by a novel MDM2 inhibitor. PLoS One 2013, 8, e72281. (52) Leblond, P.; Dewitte, A.; Le Tinier, F.; Bal-Mahieu, C.; Baroncini, M.; Sarrazin, T.; Lartigau, E.; Lansiaux, A.; Meignan, S. Cilengitide targets pediatric glioma and neuroblastoma cells through 4808



Article

(68) Demmer, O.; Dijkgraaf, I.; Schumacher, U.; Marinelli, L.; Cosconati, S.; Gourni, E.; Wester, H.-J.; Kessler, H. Design, synthesis, and functionalization of dimeric peptides targeting chemokine receptor CXCR4. J. Med. Chem. 2011, 54, 7648−7662. (69) Lohan, S.; Cameotra, S. S.; Bisht, G. S. Antibacterial evaluation of structurally amphipathic, membrane active small cationic peptidomimetics: synthesized by incorporating 3-amino benzoic acid as peptidomimetic element. Eur. J. Med. Chem. 2014, 83, 102−115. (70) Lubell, W. D.; Blankenship, J. W.; Fridkin, G.; Kaul, R. Science of Synthesis 21.11. In Peptides; Thieme: Stuttgart, 2005; pp 713−809. (71) Gaggini, F.; Porcheddu, A.; Reginato, G.; Rodriquez, M.; Taddei, M. Colorimetric tools for solid-phase organic synthesis. J. Comb. Chem. 2004, 6, 805−810. (72) Simoni, E.; Daniele, S.; Bottegoni, G.; Pizzirani, D.; Trincavelli, M. L.; Goldoni, L.; Tarozzo, G.; Reggiani, A.; Martini, C.; Piomelli, D.; Melchiorre, C.; Rosini, M.; Cavalli, A. Combining galantamine and memantine in multitargeted, new chemical entities potentially useful in Alzheimer’s disease. J. Med. Chem. 2012, 55, 9708−9721. (73) Daniele, S.; Sestito, S.; Pietrobono, D.; Giacomelli, C.; Chiellini, G.; Di Maio, D.; Marinelli, L.; Novellino, E.; Martini, C.; Rapposelli, S. Dual inhibition of PDK1 and Aurora kinase A: an effective strategy to induce differentiation and apoptosis of human glioblastoma multiforme stem cells. ACS Chem. Neurosci. 2017, 8, 100−114. (74) Daniele, S.; Zappelli, E.; Natali, L.; Martini, C.; Trincavelli, M. L. Modulation of A1 and A2B adenosine receptor activity: a new strategy to sensitise glioblastoma stem cells to chemotherapy. Cell Death Dis. 2014, 5, e1539. (75) Daniele, S.; Giacomelli, C.; Zappelli, E.; Granchi, C.; Trincavelli, M. L.; Minutolo, F.; Martini, C. Lactate dehydrogenase-A inhibition induces human glioblastoma multiforme stem cell differentiation and death. Sci. Rep. 2015, 5, 15556. (76) Nuti, E.; Casalini, F.; Santamaria, S.; Gabelloni, P.; Bendinelli, S.; Pozzo, E. D.; Costa, B.; Marinelli, L.; La Pietra, V.; Novellino, E.; Bernardo, M. M.; Fridman, R.; Da Settimo, F.; Martini, C.; Rossello, A. Synthesis and biological evaluation in U87MG glioma cells of (ethynylthiophene)sulfonamido-based hydroxamates as matrix metalloproteinase inhibitors. Eur. J. Med. Chem. 2011, 46, 2617−2629. (77) Gabelloni, P.; Da Pozzo, E.; Bendinelli, S.; Costa, B.; Nuti, E.; Casalini, F.; Orlandini, E.; Da Settimo, F.; Rossello, A.; Martini, C. Inhibition of metalloproteinases derived from tumours: new insights in the treatment of human glioblastoma. Neuroscience 2010, 168, 514− 522. (78) Butini, S.; Gemma, S.; Brindisi, M.; Borrelli, G.; Lossani, A.; Ponte, A. M.; Torti, A.; Maga, G.; Marinelli, L.; La Pietra, V.; Fiorini, I.; Lamponi, S.; Campiani, G.; Zisterer, D. M.; Nathwani, S.-M.; Sartini, S.; La Motta, C.; Da Settimo, F.; Novellino, E.; Focher, F. Non-nucleoside inhibitors of human adenosine kinase: synthesis, molecular modeling, and biological studies. J. Med. Chem. 2011, 54, 1401−1420. (79) Milite, C.; Feoli, A.; Sasaki, K.; La Pietra, V.; Balzano, A. L.; Marinelli, L.; Mai, A.; Novellino, E.; Castellano, S.; Tosco, A.; Sbardella, G. A novel cell-permeable, selective, and noncompetitive inhibitor of KAT3 histone acetyltransferases from a combined molecular pruning/classical isosterism approach. J. Med. Chem. 2015, 58, 2779−2798. (80) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF chimera: a visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605−1612. (81) Harder, E.; Damm, W.; Maple, J.; Wu, C.; Reboul, M.; Xiang, J. Y.; Wang, L.; Lupyan, D.; Dahlgren, M. K.; Knight, J. L.; Kaus, J. W.; Cerutti, D. S.; Krilov, G.; Jorgensen, W. L.; Abel, R.; Friesner, R. A. OPLS3: a force field providing broad coverage of drug-like small molecules and proteins. J. Chem. Theory Comput. 2016, 12, 281−296. (82) Uhrinova, S.; Uhrin, D.; Powers, H.; Watt, K.; Zheleva, D.; Fischer, P.; McInnes, C.; Barlow, P. N. Structure of free MDM2 Nterminal domain reveals conformational adjustments that accompany p53-binding. J. Mol. Biol. 2005, 350, 587−598.

4809


Simultaneous Targeting of RGD-Integrins and Dual Murine Double

Recommend Documents