Simultaneous Targeting of RGD-Integrins and Dual Murine Double

Subscriber access provided by READING UNIV

Article

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 J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00004 • Publication Date (Web): 18 May 2018 Downloaded from http://pubs.acs.org on May 18, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 66

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Simultaneous Targeting of RGD-Integrins and Dual Murine Double Minute Proteins in Glioblastoma Multiforme Francesco Merlino,1,§ Simona Daniele,2,§ Valeria La Pietra,1,§ Salvatore Di Maro,3,* Francesco Saverio Di Leva,1 Diego Brancaccio,1 Stefano Tomassi,3 Stefano Giuntini,4,5 Linda Cerofolini,4,5 Marco Fragai,4,5 Claudio Luchinat,4,5 Florian Reichart,6 Chiara Cavallini,2 Barbara Costa,2 Rebecca Piccarducci,2 Sabrina Taliani,2 Federico Da Settimo,2 Claudia Martini,2,* Horst Kessler,6 Ettore Novellino,1 Luciana Marinelli1,* 1

Dipartimento di Farmacia, Università degli Studi di Napoli ‘Federico II’, via D. Montesano 49,

80131 Napoli, Italy; 2

Dipartimento di Farmacia, Università di Pisa, via Bonanno 6, 56126 Pisa, Italy;

3

DiSTABiF, Università degli Studi della Campania ‘Luigi Vanvitelli’, via Vivaldi 43, 81100

Caserta, Italy; 4

Magnetic Resonance Center (CERM) University of Florence, via L. Sacconi 6, 50019 Sesto

Fiorentino (FI), Italy; 5

Department of Chemistry ‘Ugo Schiff’, University of Florence, via della Lastruccia 3-13, 50019

Sesto Fiorentino (FI), Italy; 6

Institute for Advanced Study and Center for Integrated Protein Science, Department of Chemistry,

Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany. ACS Paragon Plus Environment

Page 2 of 66

Page 3 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 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 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 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, ACS Paragon Plus Environment


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 option, GBM has been the first cancer type 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 last 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 homolog 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; ACS Paragon Plus Environment

Page 4 of 66

Page 5 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


MDM4 IC50=6.1 nM).23 Interestingly, the lead compound 3 was originally designed to target the 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 well known 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 solely 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 aggressiveness30 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 multi-target MDM2/4-integrins ligands as potential tools for GBM treatment. In fact, a single molecule capable to hit multiple targets simultaneously, might present several advantages compared



to the co-administration 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 (Arg-Gly-Asp) 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 towards 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.


Page 6 of 66

Page 7 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Chart 1. Chemical structures of 1-7.

RESULTS AND DISCUSSION Design Strategy. We have elsewhere demonstrated that an RGD (Arg-Gly-Asp) 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 Cterminus. Compound 7 gains activity on MDM proteins 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. On the other hand, 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 towards the MDM proteins, while preserving the low nanomolar affinity observed towards 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 are 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 solid-phase 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 well-documented Fmoc/tBu orthogonal protecting group for the subsequent functionalization.43,44 As 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 FmocGly-OH and Fmoc-3-amino benzoic acid, thus providing 22. Upon Fmoc removal of 22, the resulting resin-bound aromatic primary amine was reacted with 1,3-bis(tert-butoxycarbonyl)-2methyl-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). 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-1ACS Paragon Plus Environment

Page 8 of 66

Page 9 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) and 1-hydroxyazabenzotriazole (HOAt), that 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).


Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Scheme 1. Synthetic strategy for compounds 6-19.a

a

(a) DIEA, DMF, overnight; (b) 20:80 piperidine/DMF, 1 x 5 min, 1 x 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.


Page 10 of 66

Page 11 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Biological Evaluation. Integrin Binding Assays. The affinity of compounds 6-19 towards 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/sub-nanomolar 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 benzhydryl-substituted derivatives (compare 9 vs 17). On the other hand, keeping costant the length of the linker, either the introduction of psubstituents 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.



Page 12 of 66

Table 1. Inhibition of integrin binding fibronectin (α5β1) and vitronectin (αvβ3).

IC50a α5β1 (nM)

IC50a αvβ3 (nM)

13 ± 0.4

0.21 ± 0.04

28 ± 4

2.8 ± 0.1

31 ± 5

0.79 ± 0.14

22 ± 2

1.4 ± 0.2

10

36 ± 8

4.0 ± 0.6

11

22 ± 3

4.5 ± 2.6

12

76 ± 17

2.2 ± 0.8

Cpd

6

7

8

9

Structure


Page 13 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


13

45 ± 8

1.6 ± 0.3

14

20 ± 2

0.91 ± 0.13

15

48 ± 11

1.3 ± 0.3

16

40 ± 11

1.2 ± 0.2

17

22 ± 7

0.91 ± 0.19

18

18 ± 1

1.2 ± 0.2

19

41 ± 27

1.7 ± 0.3

15.4 ± 3.4

0.54 ± 0.12

Cilengitide (5)b

a

IC50 values were derived from a competitive ELISA using immobilized ECM protein and soluble integrin; b5, c(-RGDfMeV-) was used in ELISA as an internal reference compound for αvβ3 and α5β1 assays.



Page 14 of 66

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. To this purpose, specific immune-enzymatic assays were employed, using cell lysates obtained from U87MG (for p53-MDM2) or SHSY-5Y cells (for p53MDM4).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.

Table 2. Effect of new compounds on the dissociation of human p53/MDM2 or p53/MDM4 complex.

Cpd

6

7

Structure

IC50a MDM2/p53 (nM)

IC50b MDM4/p53 (nM)

> 1000

> 1000

437 ± 41

219 ± 21


Page 15 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8

> 1000

836 ± 85

72.0 ± 6.8

77.4 ± 6.9

308 ± 31

> 1000

545 ± 53

81.9 ± 8.5

370 ± 34

57.5 ± 5.5

13

685 ± 61

12.1 ± 1.0

14

270 ± 41

> 1000

15

624 ± 63

302 ± 29

9

10

11

12



Page 16 of 66

16

720 ± 69

> 1000

17

> 1000

812 ± 83

18

333 ± 31

> 1000

19

389 ± 36

> 1000

1

108 ± 5c

> 1000c

2

>1000c

847 ± 91c

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 percentage 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.


Page 17 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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. 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 p53 pathway was assessed by evaluating p53 protein accumulation and the transcription induction of p53 target genes after 8 h and 24 h of incubation. As expected, the standard MDM2 inhibitor ACS Paragon Plus Environment


accumulated 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 2AB). Interestingly, 9 induced a high statistically significant enhancement of p53 protein levels vs control, even greater with respect to that elicited by the co-treatment of the single-target agents (Figure 2A-B). Compound 9 has not 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 multi-target compound can provide a higher efficacy with respect to formulations containing mixtures of single-target agents.


Page 18 of 66

Page 19 of 66

A nt Co

l ro 2

1

2 1+

5

5 2+

5 1+

5 2+ 9 1+

p53

GAPDH

B

^^^ ^^^ ^ ^^^ ^^^

+++ +++ ### ### §§§ §§§

*** ***

+++ OD (% vs Control)

## §§§

200

###

***

*** 150

***

**

§§§

***

100

9

2+ 5 1+ 2 +5

1+ 5

5

1+ 2

1

2

nt ro l

50

Co

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Accumulation of p53 protein. U87MG cells were treated with 9 (100 nM), or the MDM2 inhibitor 1 (100 nM), or the MDM4 inhibitor 2 (100 nM), or with 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) A representative blot is shown. Control represents cell sample in the presence of DMSO (solvent used to solubilize the tested compounds). (B) The relative quantification of optical density (OD) was evaluated by densitometric analysis. The ACS Paragon Plus Environment


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, or 1+5, or 2+5 are shown using the symbol Ʌ; Ʌ P < 0.05, ɅɅɅ P < 0.001. The comparison between the cell treatment with 9 and the combination 1+2+5 did not give statistically significant differences.

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 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 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


Page 20 of 66

Page 21 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 and 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, P98%, 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); 1H 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 (∆=1.54 ppm). (S)-4-(benzylamino)-3-(2-(3-guanidinobenzamido)acetamido)-4-oxobutanoic 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, 1090% 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 (∆=1.13 ppm). ACS Paragon Plus Environment


Page 42 of 66

(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); 1H 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); 13C 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 (∆=1.16 ppm). (S)-4-((2-(1H-indol-3-yl)ethyl)amino)-3-(2-(3-guanidinobenzamido)acetamido)-4oxobutanoic 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);

13

C 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 (∆=1.21 ppm). ACS Paragon Plus Environment

Page 43 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(S)-3-(2-(3-guanidinobenzamido)acetamido)-4-(naphthalen-1-ylamino)-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); 1H 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 (∆=2.83 ppm). (S)-3-(2-(3-guanidinobenzamido)acetamido)-4-((2-(naphthalen-2-yl)ethyl)amino)-4oxobutanoic 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, 16.9Hz); 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 (∆=2.38 ppm). (S)-3-(2-(3-guanidinobenzamido)acetamido)-4-(((R)-1-phenylethyl)amino)-4-oxobutanoic acid (13) - purity >98%, tR = 13.9 min [analytical HPLC/Gradient#1, 10-70% MeCN (0.1% TFA) ACS Paragon Plus Environment


Page 44 of 66

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);

13

C 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 (∆=1.98 ppm). (S)-3-(2-(3-guanidinobenzamido)acetamido)-4-(((S)-1-phenylethyl)amino)-4-oxobutanoic acid (14) - purity >98%, 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), 7.487.45 (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);

13

C 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 (∆=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 ACS Paragon Plus Environment

Page 45 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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), 7.71 (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);

13

C 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 (∆=1.68 ppm). (S)-3-(2-(3-guanidinobenzamido)acetamido)-4-((4-methoxybenzyl)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);

13

C 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 (∆=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), ACS Paragon Plus Environment


2.55 (1H, dd, J = 7.8, 16.9 Hz); 13C 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 (∆=1.13 ppm). (S)-4-(([1,1'-biphenyl]-3-ylmethyl)amino)-3-(2-(3-guanidinobenzamido)acetamido)-4oxobutanoic 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 tR = 18.6 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 7.76 (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 (∆=0.39 ppm). (S)-4-(([1,1'-biphenyl]-4-ylmethyl)amino)-3-(2-(3-guanidinobenzamido)acetamido)-4oxobutanoic 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), 7.447.40 (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);

13

C 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, ACS Paragon Plus Environment

Page 46 of 66

Page 47 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 (∆=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 anti-human 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 (anti-mouse IgGPOD, 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, ACS Paragon Plus Environment


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 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 (Sigma-Aldrich, Taufkirchen, Germany) in carbonate buffer, washed, and 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 anti-human 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 either MDM2/p53 or MDM4/p53 complex, quantitative sandwich immune-enzymatic assays19,20,22 were performed on cell lysates obtained from U87MG cells (for p53/MDM2) or from SHSY-5Y (for p53/MDM4). Wells were pre-coated with full-length anti-MDM2 (sc-965, Santa Cruz Biotechnology, in 0.05% Poly-L-Ornithine) or anti-MDM4 (sc74468 Santa Cruz Biotechnology, in 0.05% Poly-L-Ornithine) antibody overnight at room temperature. The compound 9 was incubated, at different concentration, with cell lysates (10 min at room temperature), and then transferred to the pre-coated wells for 90 min. After extensive washes, nonspecific sites were blocked with 1% BSA, and the wells were incubated for 90 min with an antip53 antibody (sc-6243, Santa Cruz Biotechnology, 1:250). Afterwards, 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-


Page 48 of 66

Page 49 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 (SHSY5Y) 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 5,000 cells/cm2. After 24 h, the culture medium was replaced with fresh medium containing compound solubilised 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 nM to 100 nM), or the MDM2 inhibitor 1 (100 nM), or the MDM4 inhibitor 2 (100 nM), or with 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 h 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 analysed by western blotting. In brief, U87MG cells were treated with compound 9, or compounds 1, 2 or 5, for 24 h. Then, cells were then lysed and 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 ACS Paragon Plus Environment


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 matrices 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. Non-migrating cells on the upper surface were removed with a cotton bud. Pictures of randomly picked light microscope fields were taken (5 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 post-hoc pair-wise 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 ACS Paragon Plus Environment

Page 50 of 66

Page 51 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 non-truncated X-ray structures, only those co-crystallized with an organic compound were considered, and the one with the highest resolution (1.60 Å) (PDB code: 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 code: 4MMX)61 and of α5β1 in complex with a cyclic RGD peptide (PDB code: 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 the 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 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 126x126x126 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 25x106 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 ACS Paragon Plus Environment


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 the compound 9 binding modes. The “focused” docking was performed also with the aid of Glide 5.5 software67 in extra precision mode (XP), employing Glidescore for ligand ranking. The receptor grids were generated using the grid generation in Glide centred around the crystallized ligand for the canonical pocket and around the L37 for the bottom pocket, using default settings. Figure 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

13

C 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, ACS Paragon Plus Environment

Page 52 of 66

Page 53 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


aliquots of a DMSO-d6 solution of compound 9 (25, 50, 100, 150, 200, 250, 300, 350, 400 µM) were added to the buffered solution [50 mM KH2PO4, 50 mM Na2HPO4, pH 7.5, 150 mM NaCl] of 15

N 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.

ASSOCIATED CONTENT Supporting Information 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 towards 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. Molecular formula strings for compounds 6-19.

AUTHOR INFORMATION Corresponding Authors



Page 54 of 66

Prof. Luciana Marinelli *e-mail: [email protected]; Prof. Salvatore Di Maro *e-mail: [email protected]; Prof. Claudia Martini *e-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. § These Authors contributed equally to this work. Funding Sources The study was supported by PRIN2015 FCHJ8E. Notes The authors declare no competing financial interest.

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-1yl)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.

REFERENCES


Page 55 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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) Hoe, K. K.; Verma, C. S.; Lane, D. P. Drugging the p53 pathway: understanding the route to clinical efficacy. Nat. Rev. Drug Discov. 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. 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.


Page 56 of 66

Page 57 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 2-phenylindolylglyoxylyldipeptide 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 co-activating p53 and the translocator protein in glioblastoma. Sci. Rep. 2014, 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 ACS Paragon Plus Environment


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. Anticancer 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. N-methylated 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. 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.


Page 58 of 66

Page 59 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 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.; Entz-Werle, 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. Engl. 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. ACS Paragon Plus Environment


From a helix to a small cycle: metadynamics-inspired αvβ6 integrin selective ligands. Angew. Chem. Int. Ed. Engl. 2018. [Online early access]. DOI: 10.1002/anie.201803250. Published Online: April 16, 2018. 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.; Mas-Moruno, 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, 1509–1519; (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. N-methylation 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. Engl. 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. ACS Paragon Plus Environment

Page 60 of 66

Page 61 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 Nmethylation. Angew. Chem. Int. Ed. Engl. 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.; Gomez-Monterrey, 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 cell detachment and anoikis induction. Anticancer 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 FERM-enhanced 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)


Page 62 of 66

Page 63 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 wild-type 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.; Etienne-Selloum, 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. USA. 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. 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. ACS Paragon Plus Environment

Page 64 of 66

Page 65 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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. 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

Page 66 of 66

in

U87MG

glioma

cells

of

(ethynylthiophene)sulfonamido-based hydroxamates as matrix metalloproteinase inhibitors. Eur. J. Med. Chem. 2011, 46, 2617–2629. 77) Gabelloni, P.; Pozzo, E. D.; 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.; Motta, C. L.; 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.


Page 67 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


82) Uhrinova, S.; Uhrin, D.; Powers, H.; Watt, K.; Zheleva, D.; Fischer, P.; McInnes, C.; Barlow, P. N. Structure of free MDM2 N-terminal domain reveals conformational adjustments that accompany p53-binding. J. Mol. Biol. 2005, 350, 587–598.

Table of Contents (TOC)


Simultaneous Targeting of RGD-Integrins and Dual Murine Double

Recommend Documents