Discovery of Irreversible p97 Inhibitors - Journal of Medicinal

Article Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/jmc

Discovery of Irreversible p97 Inhibitors Rui Ding,†,§ Ting Zhang,‡,§ Daniel J. Wilson,† Jiashu Xie,† Jessica Williams,† Yue Xu,‡ Yihong Ye,‡ and Liqiang Chen*,† †

Center for Drug Design, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, United States Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States

‡

J. Med. Chem. Downloaded from pubs.acs.org by WEBSTER UNIV on 03/04/19. For personal use only.

S Supporting Information *

ABSTRACT: Inhibitors of human p97 (also known as valosin-containing protein) have been actively pursued because of their potential therapeutic applications in cancer and other diseases. However, covalent and irreversible p97 inhibitors have not been well explored. Herein, we report our design, synthesis, and biological evaluation of covalent and irreversible inhibitors of p97. Among an amide and a reverse amide series we synthesized, we have identified a p97 inhibitor whose functional irreversibility has been established both in vitro and in cells. Also importantly, mass spectrometry reveals three potential cysteine residues labeled by this compound, and mutagenesis together with computer modeling suggests Cys522 as a major site, which when modified, could compromise the function of p97. Taken together, this new inhibitor may provide a template for designing more potent p97 inhibitors with covalent and irreversible characteristics.

■

for p97.9 While D1 is relatively inactive in terms of ATP hydrolysis compared to the D2 domain, the ATP binding in D1 promotes hexamer formation and overall structural integrity. The D2 domain on the other hand is believed to provide the major mechanical force.6 The energy generated by D2 ATP hydrolysis allows p97 to extract ubiquitinated proteins from membranes, cellular structures or protein complexes, acting as a “segregase” in a wide range of cellular processes, including endoplasmic reticulum-associated protein degradation (ERAD),10 mitochondria-associated degradation,11 and ribosome-associated degradation. 12 In general, proteins released by p97 are delivered to the 26S proteasome for degradation. p97 is also involved in autophagy,13 another form of protein degradation and recycling. Therefore, p97 is intimately associated with protein quality control and homeostasis. Furthermore, p97 is involved in cellular functions, such as DNA repair and cell cycle progression, through its segregase activity.14 Given that drugs targeting the 26S proteasome have been used in clinics to treat cancer and the intimate connection of p97 with the proteasome in protein homeostasis regulation, p97 has also been actively pursued as an anticancer target. Higher levels of p97 have been reported in patients with various cancer types, including colorectal cancer, pancreatic cancer, thyroid cancer, breast cancer, squamous cell carcinoma, gastric carcinoma, osteosarcoma, and lung cancer.15 In addition, elevated p97 is associated with poor clinical

INTRODUCTION Covalent chemical inhibitors targeting biological enzymes have been historically avoided in drug discovery because of potential risks of severe immune response and idiosyncratic toxicity as a result of on- or off-target covalent modifications;1 however, this view has changed recently as several covalent inhibitors have emerged as attractive drug candidates, especially in cancer therapy.2−4 Compared to noncovalent inhibitors, covalent inhibitors offer unique advantages, including superior biochemical efficiency, nonequilibrium binding that allows for effective competition with endogenous substrates of high concentration, lower dosage, and targeting of undruggable binding sites.3 Although early covalent inhibitors usually originated from natural products, de novo design of covalent inhibitors has received increasing attention recently. This exciting research area has been greatly bolstered by recent Food and Drug Administration approvals of several covalent kinase inhibitors that had been designed and developed for cancer treatment.3 The adenosinetriphosphatase (ATPase) p97, which is also known as valosin-containing protein, belongs to the AAA + (ATPase associated with various cellular activities) ATPase family.5 p97 is a homohexamer, with each subunit comprising an N-domain, two AAA ATPase domains (D1 and D2), and a short unstructured C-terminal tail.6 Upon adenosine 5′triphosphate (ATP) binding to D1, the N-domain adopts an axial position, a remarkable conformational change from the initial coplanar position.7,8 Being flexible, the N-domain can interact with a wide range of adaptor proteins that render differential subcellular localization and substrate recognition © XXXX American Chemical Society

Received: January 23, 2019

A

DOI: 10.1021/acs.jmedchem.9b00144 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Selected known p97 inhibitors.

outcomes.16 Therefore, it has been hypothesized that inhibition of p97 might be a promising anticancer therapy, especially in cancers where survival is critically dependent on cellular protein homeostasis.15 This concept is further bolstered by the fact that a p97 inhibitor has recently entered into a phase I clinical trial.17 In addition to cancer, p97 has been heavily investigated because mutations in p97 gene have been linked to pathogenesis of inclusion body myopathy, Paget’s disease of bone, and frontotemporal dementia (IBMPFD), as well as a small subset of inherited amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease).18 Recent studies have pinpointed a gain-of-function activity as a potential underlying basis for pathogenesis in IBMPFD.8,19,20 As a result, inhibition of p97 may also represent a potential strategy for therapeutic intervention in IBMPFD and potentially ALS. Since p97 is implicated in a variety of human diseases and its inhibition constitutes a promising therapeutic avenue, there has been a growing list of p97 inhibitors,16 some of which are shown in Figure 1. Eeyarestatin I (1a, EerI) was the first reported p97 inhibitor that blocked ERAD and stabilized a fluorescence-tagged proteasome reporter protein Ub-v-GFP.21 As a bifunctional compound, EerI’s nitrofuran-containing (NFC) group probably binds the D1 domain and its aromatic moiety is capable of interacting with hydrophobic membranes.22 Further efforts have led to EerI analogues with a truncated aromatic moiety22 and a derivative (1b) with improved aqueous solubility.23 In addition to EerI, several D2-targeting inhibitors have been recently discovered. N2,N4Dibenzylquinazoline-2,4-diamine (2, DBeQ) features a quinazoline core structure and mainly targets the D2 domain.24 Subsequent structure−activity relationship (SAR) studies performed on DBeQ gave rise to ML240 (3) and ML241, both of which selectively bind the D2 domain.25 More recently, inhibitors built on a pyrimidine core structure, such as 4a and 4b, have been reported.17,26 CB-5083 (4b) has been advanced into a phase I clinical trial in solid tumors, validating p97 as an anticancer target.17 Compound 5 also features a pyrimidine scaffold but a different substitution pattern.27 Photoaffinity experiments suggest that compound 5 and its analogues also target the D2 domain. Alkylsulfanyl-1,2,4triazoles as exemplified by NMS-873 (6) were identified as allosteric inhibitors as they bind into a tunnel between the D1

and D2 domains.28,29 UPCDC30245 (7) is also an allosteric inhibitor, occupying a distinct region at the interface of D1 and D2 domains as revealed by a cryo-electron microscopy (cryoEM) study.30 Upon binding, compound 7 is believed to interfere with the cross-talk between the domains and subsequent conformational changes that are required for functional p97. In addition to the p97 inhibitors shown in Figure 1, new compounds also include withaferin A analogues,31 indole amides,32 trifluoromethyl and pentafluorosulfanyl indoles,33 chlorinated analogues of dehydrocurvularin,34 and oxaspirols.35 The majority of the reported p97 inhibitors are noncovalent and reversible. Since p97 is one of the most abundant cellular proteins, covalent and irreversible inhibition under nonequilibrium conditions should provide a more effective means to neutralize p97, avoiding high doses that may cause nonspecific off-target side effects. Compared to noncovalent inhibitor counterparts designed to target the ATP-binding D1 or D2 site, irreversible blockage may also allow more effective competition with ATP. A few covalent p97 inhibitors have been suggested, including EerI and NMS-859 (8). EerI is believed to serve as a prodrug, and its anti-p97 activity requires the NFC group to be converted into a reactive metabolite in cells even though the exact nature of the active species still remains unknown.36 Compound 8 features an electrophilic α-chloroacetamide, which has been shown to selectively modify Cys522, a residue buried in the D2 active site, but the potency is low.22 In this work, we report our design, synthesis, and evaluation of covalent and irreversible p97 inhibitors based on the core structure of compounds 4a and 4b, which are among the most potent and specific reversible p97 inhibitors tested to date.17

■

RESULTS AND DISCUSSION Design of Covalent p97 Inhibitors. A general approach to designing covalent inhibitor is to attach an electrophile onto an existing reversible compound. A desirable parent compound possesses a high reversible binding affinity that is not compromised by the introduced electrophile. These two features would allow the resulting inhibitor to efficiently dock into the binding site, where the electrophile forms a covalent bond with a nearby amino acid residue. For the B



Article

Figure 2. Design of irreversible p97 inhibitors.

Table 1. Evaluation of p97 Inhibitorsa

cmpd

core

X

R

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 4a 4b

I I I I I I I I II II II II II II II II II III III

CH2 CH2 CH2 CH2 CH2 CH2 O O CH2 CH2 CH2 CH2 CH2 CH2 O O O CH2 O

CCH CN CH2CCH CH2CN CCMe Et CCH CCMe CCH CCMe CCEt CCnPr CHCH2 Et CCH CCMe CHCH2

Kiapp (nM)

GSH reactivity t1/2 (h)

GSH adduct

EC50 (μM)a

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

>24 >24

no no

>24

no

>24 >24 0.21 16.4 13.5 21.6 5.08

no no yes yes yes yes yes

0.21 16.2 4.31

yes yes yes

0.470 0.468 1.41 0.517 0.723 0.608 1.08 0.524 0.131 0.436 0.463 1.57 0.892 1.01 0.0787 0.600 0.880 0.128 0.146

30.6 28.1 24.5 24.1 37.3 40.8 18.6 42.4 37.9 33.2 43.0 26.9 31.4 20.8 32.8 32.3 26.6 28.6 26.1

3.8 3.6 3.3 3.3 4.3 4.5 2.9 4.6 4.3 4.0 4.7 3.5 3.8 3.1 3.9 3.9 3.5 3.6 7.0

a

EC50 values were determined in MIA PaCa-2 cells.

electrophile, the reactivity needs to be fine-tuned to avoid excessive activity, which can lead to nonspecific modification of protein residues and consequent off-target toxicity. Assay conditions have been developed to gauge the intrinsic reactivity of a wide range of electrophiles and the consequence of structural modifications.37−40 In short, a desired covalent inhibitor should contain structural elements that initiate tight binding to its target and subsequent efficient trapping of a target residue. To obtain covalent p97 inhibitors, we decided to use the reversible inhibitors 4a and 4b as the parent compounds because of their potency and specificity, both of which are expected to bind the D2 ATP-binding site.26 A previous computational docking study performed on compound 4a has suggested that the phenyl group projects into a narrow cavity that is normally occupied by the purine ring of ATP, while the primary amide on the indole ring is more solvent-exposed.26 More importantly, examination of the residues in proximity to compound 4a revealed three cysteine residues (Cys522, 691,

and 695), which could potentially be targeted by a properly positioned electrophile. Among these three cysteine residues, Cys522 is buried in a narrow cavity and it is the target of a known p97 inhibitor 8, in part due to its small size. In principle, Cys522 can be trapped with an electrophile appended on the phenyl ring in compound 4a. Unfortunately, previous structure−activity relationship (SAR) studies suggested that only minor structural changes can be tolerated on the phenyl ring;26 therefore, we decided to keep the ring intact. Instead, we opted to focus on the primary amide, upon which an electrophile can be introduced by minor alterations of the functionality of this compound. We devised two chemotypes derived from compounds 4a and 4b (Figure 2). In the so termed amide series, as represented by the general structure 9, the original primary amide in 4a and 4b was extended to give a secondary amide, to which an electrophile was appended. In the reverse amide series as represented by the general structure 10, the primary amide was replaced with a reverse amide equipped with an C



Article

Scheme 1. Synthesis of Compounds 11-27a

Reagents and Conditions: (a) Pd2(dba)3, XPhos, Cs2CO3, dioxane, 100 °C; (b) LiOH, H2O/MeOH/tetrahydrofuran (THF); (c) amine, N,N,N’,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), Et3N, dimethylformamide (DMF); (d) SnCl2, MeOH, 75 °C; (e) H2, Pd/C, MeOH; (f) carboxylic acid, N,N′-dicyclohexylcarbodiimide (DCC), CH2Cl2; or carboxylic acid, N-ethyl-N′-(3dimethylaminopropyl)carbodiimide (EDC), 1-hydroxybenzotriazole (HOBt), DMF; or acryloyl chloride, K2CO3, acetone/H2O. a

electrophilicity relative to propiolamide. To provide an example of a reversible inhibitor with similar structural elements, we synthesized compound 24 devoid of a Michael acceptor. Like inhibitors 17 and 18 in the amide series, we also investigated compounds 25−27, which were based on 4b and featured a propiolamide, a methyl-capped propiolamide, and an acrylamide, respectively. Chemical Synthesis. The chemical synthesis of the new p97 inhibitors is depicted in Scheme 1. To prepare the amide inhibitors based on compound 4a, known chloride 28a26 and indole 2926 underwent a palladium-mediated C−N coupling reaction to give methyl ester 30a, hydrolysis of which afforded carboxylic acid 31a. Amide-coupling reactions were performed on acid 31a, and various amines gave inhibitors 11−16. To obtain the amide inhibitors based on compound 4b, an identical synthetic sequence was performed with chloride 28b26 and indole 29 as starting materials, leading to compounds 17−18. To synthesize the reverse amide inhibitors based on compound 4a, known chloride 28a and indole 3244 were subjected to a palladium-mediated C−N coupling reaction to afford nitrate 33a. Upon reduction of 33a, the resulting aniline 34a was coupled with carboxylic acids or treated with an acyl chloride to give compounds 19−24. The reverse amide inhibitors based on compound 4b were obtained in a similar manner, starting with chloride 28b and indole 32. Biochemical Evaluation of p97 Inhibitors. We established an ATPase assay in which inorganic phosphate released by the p97-catalyzed reaction was continuously monitored using a method previously developed by Webb.45 The method relies on purine nucleoside phosphorylase (PNP), which uses

electrophile. Building on the general structures of 9 and 10, we prepared and evaluated a series of potential covalent p97 inhibitors using 4a or 4b as the reference compound (Table 1). Specifically, for the amide series built on core I, we prepared and tested compounds 11−18. In compound 11, we explored a terminal alkyne that has been shown to react with active-site cysteine residues.41,42 In compound 12, we examined a nitrile group that has been well exploited in the design of covalent cysteine protease inhibitors.43 Compounds 13 and 14 were also synthesized to investigate the effect of a longer spacer between the amide functionality and the terminal alkyne or nitrile group, respectively. In compound 15, a methyl group was appended upon the original terminal alkyne to investigate if a steric hindrance would impede the anticipated nucleophilic attack by a thiol. We also synthesized compound 16, a reversible inhibitor that contained a simple propyl secondary amide of a comparable length but without an electrophilic capacity. Compounds 11−16 were based on 4a, while inhibitors 17 and 18 were built on 4b, featuring a terminal alkyne and a methyl-capped alkyne, respectively. For the reverse amide series built on core II, we generated compounds 19−27, in which a Michael acceptor was installed. While compound 19 contained a propiolamide functionality, its terminal alkyne was further capped with alkyl groups of increasing length (namely, methyl, ethyl, and propyl), leading to compounds 20−22, respectively. These three compounds were synthesized to probe simple alkyl groups and their steric effect on the resulting inhibitors, providing a possibility to finetune the reversibility. For a similar purpose, we prepared compound 23, which had an acrylamide with attenuated D



Article

assay conditions with the possible exceptions of compounds 19 and 25. Antiproliferative Activity. To evaluate the anticancer activity of the newly identified p97 inhibitors, we tested their antiproliferative activity in MIA PaCa-2 cells, a pancreatic cancer cell line. Consistent with their biochemical anti-p97 activity, our p97 inhibitors showed high antiproliferative activity. The majority of the compounds possessed an EC50 value 24 h and no detection of GSH adducts. Lack of reactivity is probably due to the attenuated nucleophilicity of noncatalytic GSH cysteine relative to the catalytic cysteines that can be successfully captured by terminal alkynes or nitriles in properly oriented cysteine protease inhibitors. In contrast to the amide series, compounds selected from the reverse amide series showed appreciable albeit varied reactivity toward GSH. Compound 19 exhibited high intrinsic reactivity as judged by t1/2 < 30 min, a result that is in line with the reactivity reported for analogous propiolamides. This high reactivity is likely responsible for compound 19’s cytotoxicity (see below). As expected, steric capping of the terminal alkyne in compound 19 with a small alkyl group as seen in compounds 20−22 markedly reduced the intrinsic reactivity by >60-fold. When the propiolamide in 19 was replaced with an acrylamide, the resulting compound 23 possessed significantly attenuated reactivity due to the reduced electrophilicity of acrylamide. When compounds 25−27 were tested, their t1/2 values were almost identical to those determined for compounds 19, 20, and 23, respectively, indicating that an extra oxygen atom in the former three compounds had minimal effect on their intrinsic reactivity. Consistent with the observed GSH reactivity, GSH adducts were detected for compounds 19−23 and 25−27. We also determined the reactivity toward dithiothreitol (DTT), a thiol-containing reducing agent that was present in our p97 biochemical assay. Since the amide series showed no reactivity toward GSH, we focused on the reverse amide series (Table S1). The DTT reactivity generally mirrored those toward GSH. Therefore, we concluded that DTT had no significant effect on the compounds under the biochemical

Table 2. Enzymatic Activity Recovered (%) after Dialysisa WT p97 C522A p97 delta N a

E

20

4b

3

DMSO

3±1 6±1 0

19 ± 4 60 ± 21 11

82 ± 6 100 NDa

100 100 100

ND, not determined. DOI: 10.1021/acs.jmedchem.9b00144 J. Med. Chem. XXXX, XXX, XXX−XXX


Article

Figure 3. Inhibition of p97 stabilizes Ub-v-GFP. (A) Experimental scheme. (B) Whole-cell extract of Ub-v-GFP cells treated with the inhibitors as indicated (4 h) was subject to immunoblotting (IB) analysis. Anti-p97 blot serves as a loading control. (C) As in (B), except that the indicated inhibitors (2 μM) were tested.

out of a dialysis cassette. Rebinding is more likely to occur with enzymes bearing multiple binding sites for inhibitor. Indeed, because the six protomers of p97 showed positive cooperation with regard to ATP hydrolysis,49,50 reversing its activity would require complete removal of the inhibitor from all six subunits of a p97 hexamer. Consistent with our model, when 4b was tested with a p97 mutant (C522A) that bore a mutation near the inhibitor binding site, its reversibility was significantly enhanced (Table 2), presumably due to reduced affinity. Additionally, compound 3, a weaker p97 inhibitor, exhibited stronger reversibility for WT p97 than 4b with 82% activity recovered after dialysis. Likewise, this compound appeared almost completely reversible when tested with the p97 C522A mutant (Table 2, see below). Collectively, these results supported the notion that the apparent irreversibility of 4b in WT p97 was due to its high binding affinity and the intrinsic positive cooperation among p97 subunits, therefore validating our dialysis assay conditions. Under these conditions, when WT p97 was treated with compound 20, merely 3% activity was recovered after dialysis. This result indicated that 20 is an irreversible p97 inhibitor under the assay conditions. Inhibition and Recovery of p97 Activity in Cells. We next evaluated selected compounds for their p97 inhibitory activity in cells stably expressing the proteasome substrate Ubv-GFP whose degradation also requires p97 (Figure 3A).24 Inhibition of p97 ATPase activity is expected to stabilize this short-lived protein, leading to its accumulation in cells. Indeed, in cells treated with MG132 (20 μM), a proteasome inhibitor, the level of Ub-v-GFP was dramatically increased (Figure 3B, lane 2 vs 1). Likewise, cells exposed to these newly developed p97 inhibitors, even at 2 μM, accumulated Ub-v-GFP to a similar level to cells treated with 20 μM MG132 (Figure 3B,C). Among the chemicals tested, compound 19 did not

increase Ub-v-GFP as much as other compounds, but this was due to a strong toxicity unrelated to p97 inhibition, which led to reduced cell number (Figure 3B, lanes 3−5). To rule out that our compounds blocked Ub-v-GFP degradation through inhibition of the proteasome, we monitored a signal sequencedeleted T-cell receptor (TCR) variant (ΔSS TCRα-YFP), whose degradation is mediated by proteasome but does not require p97.21,22 Degradation of ΔSS TCRα-YFP was inhibited by MG132 but not by compound 20 or NMS-873 (Figure S1), excluding the possibility that our compounds inhibited Ub-vGFP degradation by blocking the proteasome. All together, these results demonstrate that the newly synthesized chemicals can inhibit p97 function in mammalian cells. To explore the reversibility of these p97 inhibitors in cells, we developed a drug washout assay (Figure 4). To this end, Ub-v-GFP cells were either treated continuously with an inhibitor (2 μM) for 4 h (treatment A) or treated for 1 h and subsequently cultured in inhibitor-free medium for three additional hours (treatment B) (Figure 4A). The total incubation time was the same for both schemes; therefore, for an irreversible inhibitor, a short period of inhibition (1 h, treatment B) should be sufficient to render complete p97 inactivation even after compound withdrawal. Consequently, the level of Ub-v-GFP accumulation between the two treatment conditions should be similar. Indeed, when cells were exposed to the irreversible proteasome inhibitor MG115, similar levels of Ub-v-GFP accumulation were observed between the two treatment conditions (Figure 4D). By contrast, the previously reported reversible p97 inhibitor NMS-87329 only caused significant Ub-v-GFP accumulation under treatment A. Thus, the assay could effectively discern the differential reversibility of inhibitors in cells. When we tested compound 4b using this assay, as predicted from its lack F



Article

Figure 4. Reversibility of newly identified p97 inhibitors in cells. (A) Experimental design. (B) Ub-v-GFP reporter cells were treated with the indicated inhibitors (2 μM) following either the A or B scheme. Cell lysates were analyzed by immunoblotting. (C) Structure of compound 20 (LC-1028). (D) Compound 20 is an irreversible inhibitor. Cells treated with the indicated inhibitors were lysed and analyzed by immunoblotting. (E) A373 cells stably expressing US11 and HA-tagged MHC class I heavy chain H2A were treated with the indicated inhibitor (2 μM) for 1 h. The cells were then incubated in inhibitor-free medium that contains cycloheximide (50 μg/mL). At the indicated time points, a portion of the cells was harvested. Cell lysates prepared were analyzed by immunoblotting. (F) Compound 20 is a potent p97 inhibitor in cells. The cells treated with the indicated compounds were analyzed by immunoblotting. G



Article

Table 3. Summary of the p97 Peptides Covalently Modified by Compound 20 As Detected by LC−MS/MS

a ModScore >20 indicates that the covalent modification by compound 20 was localized at the proposed residue with >99% certainty. C# (in red) indicates a cysteine residue that was modified by compound 20 through a conjugate addition reaction at the alkyne group of compound 20.

exhibited by compounds 20−22 could be attributed to the possibility that a longer alkyl cap impeded the necessary conformation change and subsequent covalent bond formation. In in vitro studies, compounds 4b and 20 both behaved as irreversible inhibitors; however, in cells, compound 4b was largely reversible, whereas 20 was irreversible. This discrepancy may be explained by the different biological settings in which the compounds were tested. In cells, when a reversible inhibitor like 4b is released from p97, it can be pumped out of the cells by ABC transporters, manifesting its reversible nature; however, in dialysis tubes, it may rebind p97 quickly, so it behaves like an irreversible inhibitor. Alternatively, compounds 4b upon release from p97 may be subjected to cellular metabolism, giving rise to inactive species, which would reduce its effective concentration. By contrast, for covalent inhibitor compound 20, cellular metabolism should have a minimal effect on its activity as covalent bonding with p97 may prevent metabolism. While future efforts are needed to elucidate the different cellular behavior of compounds 20 and 4b, our washout experiment corroborated that compound 20 was an irreversible inhibitor, a conclusion that was further supported by our LC−MS/MS experiment (see below). In addition to the reverse amide series, we also tested compounds 11, 15, 17, and 18, which belonged to the amide series. Consistent with their lack of intrinsic GSH reactivity, these inhibitors were essentially all reversible under the assay conditions (Figure 4B). In summary, we have identified compound 20 as a potent irreversible inhibitor of p97, which justified further investigation of its mode of action. Identification of Residues Covalently Modified by Compound 20. To experimentally identify the residue(s) modified by compound 20, recombinant p97 was treated with compound 20 with DMSO as a control. In addition, we also treated p97 with compound 4b as an additional control. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, the p97-containing bands were excised and analyzed by LC−MS/MS. The resulting tandem spectra were searched for covalent modifications that resulted from a cysteine residue’s conjugate addition to the alkyne group of compound 20. The probability of the proposed modification was scored by ModScore, a program that was based on the algorithm Ascore.52 A ModScore of >20 indicated covalent modification, a specified site with >99% certainty.52 Among the 12 cysteine residues present in p97, only three (Cys105, 522, and 535) were found to be modified (Tables 3 and S2, and Figure S2). Among them, Cys105 and Cys522 appeared more prominent as they were each identified several times (21 and 5, respectively) with ModScore >20. As expected, when p97 was treated with 4b, no covalent

of a Michael acceptor, it only induced strong accumulation of Ub-v-GFP under treatment A conditions, but not under treatment B conditions (Figure 4B). This result confirms our hypothesis that 4b is a reversible inhibitor, but its reversibility is masked by the tight affinity to p97 in vitro. When compound 19 was tested under the same experimental conditions, severe cytotoxic activity compromised a definite determination of reversibility (Figure 3B). In comparison, compound 20, which contained a methyl-capped propiolamide (Figure 4C) showed no cytotoxicity within the 4 h treatment period. More importantly, it was clearly irreversible as judged by almost the same level of Ub-v-GFP accumulation regardless of the treatment scheme (Figure 4B). To further test the irreversibility of compound 20, we tested the degradation of another p97 substrate, MHC class I heavy chain, in cells co-expressing the human cytomegalovirus protein US11. US11 is a type I membrane protein that induces the degradation of newly synthesized MHC class heavy chain.51 When these cells were treated with either NMS873 or compound 20 at 2 μM for 1 h, the MHC class I heavy chain accumulated in these cells. After drug washout and addition of cycloheximide to prevent protein synthesis, cells treated with NMS-873 degraded MHC class I heavy chain rapidly with t1/2 less than 1 h. By contrast, cells treated with compound 20 turned over MHC class I heavy chain at a much reduced rate compared to NMS-873 (Figure 4E). Collectively, these results strongly indicate that the effect of compound 20 is largely irreversible in cells. A dose titration experiment showed that treating cells with 0.5 μM compound 20 could lead to similar levels of Ub-v-GFP accumulation to cells treated with 20 μM MG132, demonstrating the potency of this compound (Figure 4F). Interestingly, compound 21, which featured an ethyl capping group, also displayed a high degree of irreversibility (Figure 4B). However, when a propyl group was attached to the terminal alkyne, the resulting compound 22 completely lost irreversibility. These findings suggest that the methyl cap in compound 20 might induce a steric effect that hinders a nucleophilic attack on its Michael acceptor. This notion is supported by a drastic increase of t1/2 observed for 20 versus 19 in the GSH reactivity assay (Table 1). Gratifyingly, the methyl cap did not lead to complete loss of irreversibility. However, a longer cap like an ethyl or propyl group had a detrimental effect on inhibition irreversibility. Since the intrinsic GSH reactivity determined for compounds 20−22 were comparable (Table 1), the difference in reversibility could not be simply explained by increased steric hindrance induced by a longer alkyl cap. Because our computational study of compound 20 and its analogues (see below) suggested that a significant conformation change was required for formation of a covalent bond between compound 20 and its potential target residue Cys522 (vide infra), the differential reversibility H



Article

Figure 5. Proposed binding modes and interactions of selected p97 inhibitors docked into the cryo-EM structure of human p97 (PDB 5FTK). (A) Binding modes of compounds 4b (magenta) and 19 (green) at the active site of the D2 domain. (B) Binding modes of compounds 20 (magenta) and 21 (green) at the active site of the D2 domain. (C) Two-dimensional (2D) representation of potential interactions between compound 20 and p97. (D) Potential binding mode of compound 20 after formation of a covalent bond with Cys522. Upon covalent bond formation, a significant change in the orientation of compound 20 was observed. The phenyl group, which was originally placed at the interface of subunits D and E, flipped and now mainly interacted with residues from subunit E.

experiments. Remarkably, the detected peptide sequence (covalently modified) was identical to the one observed for compound 20 (Table 3). Thus, compound 20 was able to label Cys522, a residue susceptible to covalent modification by small molecules. We next tested the reversibility of compound 20 using the p97 C522A mutant. Surprisingly, it remained essentially irreversible for C522A, an observation that could potentially exclude Cys522 as a main target. However, as discussed above, it is also possible that compound 20 appeared irreversible because it is a tight binder for both WT p97 and p97 C522A similarly to 4b for WT p97. Previously reported iodoacetamide and compound 8 contain chemically reactive α-iodo- and α-chloroacetamide warheads, respectively, which are functional groups undesired in drug discovery. By contrast, compound 20 features a relatively inert methyl-capped amide warhead (as judged by its low intrinsic reactivity toward GSH or DTT). Thus, it is more suitable for drug development. Additionally, Cys522 is buried within the D2 ATPase active site, indicative of a specific interaction with compound 20. Taken together, as a covalent p97 inhibitor, compound 20 is chemically inert until it binds to p97 and subsequently traps its target residue. With these desired features, compound 20 serves as an excellent template for further developing irreversible p97 inhibitors.

modification by 4b was detected. We therefore focused our study on Cys105 and C522. Cys105 is solvent-exposed on the surface of the N domain, and it does not contribute to the ATP-binding pockets (Figure S3). These features render it more accessible to compound 20, giving rise to the relatively high abundance of the identified peptide. However, previous studies have demonstrated that the N domain is dispensable for p97 ATPase activity.53 When a p97 mutant lacking the N domain was tested, compound 20 still effectively inhibited it in an irreversible manner (Table 2), further suggesting that Cys105 is unessential for compound 20-mediated p97 inhibition. Thus, although compound 20 might form a covalent interaction with Cys105, this modification could not solely account for the ATPase inhibition by compound 20. Nonetheless, because the N domain is extensively involved in interactions with a number of co-factors critical for p97’s biological functions, future investigation on the consequence of compound 20’s covalent modification of Cys105, especially in cells, is justified. The relatively abundant Cys522-containing peptides identified by our LC−MS/MS experiment together with its proximity to the D2 active site strongly suggested it as a major modification site relevant to p97 inhibition by compound 20. Cys522 has been shown to be the target residue of iodoacetamide54 and compound 828 based on MS I



Article

Noncompetitive Inhibition by Compound 20. To determine if compound 20 was competitive with ATP, we measured its Kiapp values at increasing ATP concentrations (0.312−5.00 mM) (Table S3). Under these conditions, 20 was not conclusively ATP-competitive, a finding that could be explained by several reasons. First, as a covalent and irreversible inhibitor, compound 20 has an intrinsic advantage over a noncovalent inhibitor when competing with endogenous ATP. Second, compound 20 binds p97 with a Kd value in the low nanomolar range, whereas the affinity of ATP for D1 and D2 domain is ∼2 μM (Kd).55 Therefore, ATP may not effectively compete with compound 20 even at higher concentrations. Third, there is a positive corporation with regard to ATP hydrolysis among the six p97 protomers.49,50 Hence, ATP may need to replace all of the inhibitor molecules in the p97 hexamers to restore the enzymatic capacity, posing a high barrier for ATP to compete with compound 20. Computational Study. Our studies showed that compound 20 modified Cys522, a residue that is positioned near the p97 D2 active site, where 4b and other analogous inhibitors bind. Also interestingly, this residue has been shown to be covalently modified by structurally simple iodoacetamide54 and compound 8.28 Furthermore, mutating Cys522 reduced the irreversibility of 4b and 3 but did not affect the reversibility of compound 20, suggesting that 20 might utilize a unique binding mode to engage p97 and subsequently form a covalent bond with Cys522. To obtain insight into the binding mode of compound 20 and its analogues, we performed a computational docking study (Table S4) using a cryo-EM structure of hexameric human p97 (Protein Data Bank (PDB) 5FTK) that consists of subunits A−F.30 We first focused on the D2 active site. Because the nucleotide-binding pocket is shaped by residues from two adjacent subunits, both subunits might contribute to inhibitor binding. We therefore used the combined subunits E and D as a template to generate a docking grid that encompassed the D2 active site (E subunit) and the surrounding residues, including those from subunit D. Using this docking grid, we first examined compound 4b (magenta, Figure 5A), whose phenyl group was assumed to project into a narrow cavity occupied normally by the adenine ring of adenosine 5′-diphosphate (ADP), as suggested by a previous computational modeling study.26 In this model, the primary amide of 4b was proposed to interact with Ser664 and Lys663, which were located opposite to the ADP phosphate binding site. In our study, compound 4b could be docked into the same D2 ATPase active site, but the primary amide of 4b engaged a different set of interactions with p97, forming hydrogen bonds with Lys524, Asn624, and Asp577 within the ADP phosphate binding site (Figure S4). The discrepancy is probably due to the fact that we used two adjacent subunits as the template, whereas the previous study used one isolated subunit in docking computation. When two adjacent subunits were included in the model, a relatively crowded environment created at the opposite side of the ADP-binding pocket by the intruding residues from the neighboring subunit prevented the primary amide functionality from forming hydrogen bonds. Consistent with this interpretation, when we performed computational docking using a grid generated from the isolated subunit E, compound 4b was able to adopt a docking pose (Figure S5) identical to that proposed previously, validating our computing methodology.26 In further support of our model, a recent X-ray crystallography study56 of compound 4b in complex with p97 delta N (3.77 Å) revealed

that the inhibitor adopted an orientation similar to the one predicted by our model. Next, we used the same docking method to investigate the interactions of a few selected new inhibitors with p97. Intriguingly, compound 19 (green, Figure 5A) adopted a conformation and engaged in hydrogen bonding in a manner similar to that of compound 4b (Figure S6). The terminal acetylene of compound 19 fitted into a small pocket formed by residues, including Thr623 and Ala622. By contrast, compounds 20, 21, and 22, which contained an extra methyl, ethyl, and propyl cap, respectively, exhibited a different mode of binding. Take compound 20 (magenta, Figure 5B) as an example, unlike 4b, its indole moiety instead of the phenyl ring protruded into the narrow cavity that is normally occupied by the adenine ring of ADP. As a result, the phenyl ring was sandwiched between adjacent subunits D and E, participating in mainly lipophilic interactions with the surrounding residues, including Pro636 from the subunit D. Several structural features contribute to this new binding mode. First, the small pocket formed by Thr623 and Ala622 could not accommodate the extra methyl cap of compound 20 that is absent from compound 19. Second, the linear reverse amide snugly fitted into a thin tunnel (mainly formed by lipophilic residues) at the back of the adenine-binding cavity, engaging in lipophilic interactions with the tunnel residues (Figure 5C). In addition, the amide carbonyl formed a hydrogen bond with the backbone NH of Gly480, which in 5FTK interacted with the N1 nitrogen of the adenine ring. Compounds 21 (green, Figure 5B) and 22 (Figure S7) displayed an identical binding mode to 20 because the thin tunnel was open-ended and therefore could accommodate the longer-cap groups in these two compounds. To examine whether the thin tunnel could also be accessed by other reverse amides, we also performed docking study on compounds 23 and 24, which contained a terminal double bond and an ethyl group, respectively. Intriguingly, neither compound was able to adopt the same binding mode displayed by compounds 20−22, indicative of the importance of the acetylene group and the short alkyl cap. We proposed that a straight acetylene group allowed the whole reverse amide side chain to adopt a conformation that perfectly matched the contour of the thin tunnel in p97. Moreover, the short alkyl caps were expected to interact with the lipophilic residues within the thin tunnel, enhancing binding affinity. Notably, the structural model of compound 20 in complex with p97 positioned the alkyne functional group close to Cys522 (5.7 Å between the sulfur atom of Cys522 and the terminal carbon atom of the acetylene functionality), suggesting that a nucleophilic attack by Cys522 and subsequent formation of a covalent bond was possible. This notion was further supported by our covalent docking57 using Cys522 as a nucleophile (Table S5, Figures S8 and S9), demonstrating that a covalent bond between Cys522 and 20 was indeed computationally feasible (Figure 5D). In addition to the reverse amide series, we also performed molecular docking on compounds 11 and 15 selected from the amide series. Compound 11 exhibited a binding mode similar to compounds 4b and 19 (Figure S10). Interestingly, compound 15, which contained an extra methyl cap, was also able to adopt an analogous binding conformation even though there was a minor difference in the triple-bond orientation, in contrast to the drastic change in binding mode observed for 19 versus 20. J



Article

was then diluted with water (30 mL) and extracted with dichloromethane (30 mL × 3). The combined organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography using EtOAc/ hexanes (50%) to afford compound 11 as a white solid (27.2 mg, 100%). 1H NMR (CDCl3, 600 MHz) δ 8.13 (d, J = 8.3 Hz, 1H), 7.46 (d, J = 7.5 Hz, 1H), 7.38−7.29 (m, 5H), 7.08 (t, J = 7.9 Hz, 1H), 6.77 (s, 1H), 6.31 (t, J = 5.1 Hz, 1H), 5.03 (t, J = 5.5 Hz, 1H), 4.74 (d, J = 5.5 Hz, 2H), 4.31 (dd, J1 = 5.1 Hz, J2 = 2.5 Hz, 2H), 2.82−2.78 (m, 2H), 2.62 (s, 3H), 2.41−2.38 (m, 2H), 2.28 (t, J = 2.5 Hz, 1H),1.95− 1.86 (m, 4H). High-resolution mass spectrometry (HRMS) (ESI+) calcd for C28H28N5O (M + H)+ 450.2288, found 450.2287. 1-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2-yl)-N(cyanomethyl)-2-methyl-1H-indole-4-carboxamide (12). Compound 12 was prepared from acid 31a in a fashion similar to the one described for compound 11. White solid, 27.1 mg, yield 100%. 1H NMR (CDCl3, 600 MHz) δ 8.14 (d, J = 8.2 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H), 7.38−7.29 (m, 5H), 7.08 (t, J = 7.9 Hz, 1H), 6.75 (s, 1H), 6.50 (t, J = 5.7 Hz, 1H), 5.04 (t, J = 5.5 Hz, 1H), 4.74 (d, J = 5.5 Hz, 2H), 4.40 (t, J = 5.7 Hz, 2H), 2.82−2.78 (m, 2H), 2.62 (s, 3H), 2.42−2.38 (m, 2H), 1.95−1.86 (m, 4H). HRMS (ESI+) calcd for C27H27N6O (M + H)+ 451.2241, found 451.2241. 1-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2-yl)-N(but-3-yn-1-yl)-2-methyl-1H-indole-4-carboxamide (13). Compound 13 was prepared from acid 31a in a fashion similar to the one described for compound 11. White solid, 28.7 mg, yield 100%. 1H NMR (CDCl3, 600 MHz) δ 8.11 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 7.4 Hz, 1H), 7.38−7.28 (m, 5H), 7.08 (t, J = 7.9 Hz, 1H), 6.80 (s, 1H), 6.54 (t, J = 6.0 Hz, 1H), 5.04 (t, J = 5.5 Hz, 1H), 4.75 (d, J = 5.5 Hz, 2H), 3.67 (q, J = 6.2 Hz, 2H), 2.82−2.78 (m, 2H), 2.62 (s, 3H), 2.56 (td, J1 = 6.4 Hz, J2 = 2.6 Hz, 2H), 2.42−2.37 (m, 2H), 2.05 (t, J = 2.6 Hz, 1H), 1.94−1.86 (m, 4H). HRMS (ESI+) calcd for C29H30N5O (M + H)+ 464.2445, found 464.2450. 1-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2-yl)-N-(2cyanoethyl)-2-methyl-1H-indole-4-carboxamide (14). Compound 14 was prepared from acid 31a in a fashion similar to the one described for compound 11. White solid, 27.0 mg, yield 96%. 1H NMR (CDCl3, 600 MHz) δ 8.13 (d, J = 8.2 Hz, 1H), 7.45 (d, J = 7.6 Hz, 1H), 7.38−7.28 (m, 5H), 7.08 (t, J = 7.9 Hz, 1H), 6.77 (s, 1H), 6.64 (t, J = 6.0 Hz, 1H), 5.03 (t, J = 5.5 Hz, 1H), 4.74 (d, J = 5.5 Hz, 2H), 3.74 (q, J = 6.3 Hz, 2H), 2.82−2.78 (m, 2H), 2.77 (t, J = 6.3 Hz, 2H), 2.62 (s, 3H), 2.41−2.38 (m, 2H), 1.94−1.86 (m, 4H). HRMS (ESI+) calcd for C28H29N6O (M + H)+ 465.2397, found 465.2403. 1-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2-yl)-N(but-2-yn-1-yl)-2-methyl-1H-indole-4-carboxamide (15). Compound 15 was prepared from acid 31a in a fashion similar to the one described for compound 11. White solid, 22.2 mg, yield 90%. 1H NMR (CDCl3, 600 MHz) δ 8.13 (d, J = 8.3 Hz, 1H), 7.46 (d, J = 7.4 Hz, 1H), 7.38−7.29 (m, 5H), 7.08 (t, J = 7.9 Hz, 1H), 6.78 (s, 1H), 6.24 (br s, 1H), 5.00 (br s, 1H), 4.75 (d, J = 5.3 Hz, 2H), 4.25 (q, J = 2.3 Hz, 2H), 2.83−2.78 (m, 2H), 2.63 (s, 3H), 2.42−2.38 (m, 2H), 1.95−1.87 (m, 4H), 1.84 (t, J = 2.3 Hz, 3H). HRMS (ESI+) calcd for C29H30N5O (M + H)+ 464.2445, found 464.2439. 1-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2-yl)-2methyl-N-propyl-1H-indole-4-carboxamide (16). Compound 16 was prepared from acid 31a in a fashion similar to the one described for compound 11. White solid, 13.3 mg, yield 59%. 1H NMR (CDCl3, 600 MHz) δ 8.11 (d, J = 8.3 Hz, 1H), 7.44 (d, J = 7.5 Hz, 1H), 7.38− 7.29 (m, 5H), 7.08 (t, J = 7.8 Hz, 1H), 6.72 (s, 1H), 6.16 (t, J = 5.7 Hz, 1H), 5.02 (t, J = 5.5 Hz, 1H), 4.75 (d, J = 5.5 Hz, 2H), 3.48 (q, J = 6.7 Hz, 2H), 2.82−2.78 (m, 2H), 2.62 (s, 3H), 2.41−2.37 (m, 2H), 1.94−1.86 (m, 4H), 1.71−1.65 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H). HRMS (ESI+) calcd for C28H32N5O (M + H)+ 454.2601, found 454.2600. 1-(4-(Benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-2-methyl-N-(prop-2-yn-1-yl)-1H-indole-4-carboxamide (17). Compound 17 was prepared from acid 31b in a fashion similar to the one described for compound 11. White solid, 14.9 mg, yield 68%. 1H NMR (CDCl3, 600 MHz) δ 8.18 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.40−7.30 (m, 5H), 7.11 (t, J = 7.8

In short, our docking study revealed that compound 20 was able to adopt a binding mode distinct from their parent compounds 4a and 4b. This novel binding mode allowed for covalent bond formation between compound 20 and Cys522. A covalent bond between compound 20 and p97 might account for the irreversibility of 20.

■

CONCLUSIONS We have designed and synthesized two series of p97 inhibitors based on the core structure of the known compounds 4a and 4b. While both the amide and reverse amide series exhibited largely comparable anti-p97 and antiproliferative activity, assessment of the latter group yielded compound 20 as a potential covalent and irreversible p97 inhibitor, which featured a methyl-capped acetylene electrophilic group. Evaluation of compound 20 under the dialysis conditions established it as an irreversible inhibitor. This conclusion was further supported by its functional irreversibility in cells. LC− MS/MS study revealed cysteine 522 as a candidate for covalent modification by compound 20, and our computational study yielded a plausible model that is consistent with this notion. In short, we have identified compound 20 as a potent and irreversible inhibitor that covalently modifies p97. Whether compound 20 is suitable for clinical study remains to be tested, but as an irreversible inhibitor, its pharmacokinetics and pharmacodynamics are expected to be uncoupled and its duration of action should be prolonged, leading to a less frequent dosing schedule. Such desired properties will be explored for compound 20 and its advanced analogues. Importantly, its unique binding mode and structural features provide a starting point for designing improved p97 inhibitors regardless of their mode of inhibition (covalent or noncovalent).

■

EXPERIMENTAL SECTION

Chemical Synthesis. All commercial reagents were used as provided unless otherwise indicated. An anhydrous solvent-dispensing system (J.C. Meyer) using two packed columns of neutral alumina was used for drying THF, Et2O, and CH2Cl2, whereas two packed columns of molecular sieves were used to dry DMF. Solvents were dispensed under argon. Flash chromatography was performed with Ultra Pure silica gel (SiliCycle) or with RediSep Rf silica gel columns on a Teledyne ISCO CombiFlash Rf system using the solvents as indicated. Nuclear magnetic resonance spectra were recorded on a Varian 600 MHz spectrometer with Me4Si or signals from residual solvent as the internal standard for 1H. Chemical shifts are reported in ppm, and signals are described as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br s (broad singlet), and dd (double doublet). Values given for coupling constants are of first order. Highresolution mass spectra were recorded on an Agilent TOF II TOF/ MS instrument equipped with either an electrospray ionization (ESI) or an atmospheric pressure chemical ionization interface. Analysis of sample purity was performed on an Agilent 1200 Infinity series highperformance liquid chromatography (HPLC) system with a Phenomenex Gemini C18 column (5 μm, 4.6 × 250 mm2). HPLC conditions were as follows: solvent A = water, solvent B = MeCN or MeOH, and flow rate = 2.0 mL/min. Compounds were eluted with a gradient of from 10 to 100% MeCN/water or from 10 to 100% MeOH/water in 15 min. Purity was determined by the absorbance at 254 nm. All tested compounds have a purity of ≥95%. 1-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2-yl)-2methyl-N-(prop-2-yn-1-yl)-1H-indole-4-carboxamide (11). To a solution of acid 31a (25.0 mg, 0.0606 mmol) and 2-propynylamine (3.7 mg, 0.067 mmol) in DMF (1 mL) was added HBTU (25.3 mg, 0.667 mmol) and triethylamine (36.7 mg, 0.364 mmol). The resulting mixture was allowed to stir at room temperature (rt) overnight and K



Article

Hz, 1H), 6.82 (s, 1H), 6.29 (br s, 1H), 4.76 (d, J = 5.2 Hz, 2H), 4.61 (br s, 1H), 4.58 (s, 2H), 4.35−4.30 (m, 2H), 4.07 (t, J = 5.4 Hz, 2H), 2.92 (t, J = 5.4 Hz, 2H), 2.65 (s, 3H), 2.29 (t, J = 2.4 Hz, 1H). HRMS (ESI+) calcd for C27H26N5O2 (M + H)+ 452.2081, found 452.2079. 1-(4-(Benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-N-(but-2-yn-1-yl)-2-methyl-1H-indole-4-carboxamide (18). Compound 18 was prepared from acid 31b in a fashion similar to the one described for compound 11. White solid, 15.1 mg, yield 67%. 1H NMR (CDCl3, 600 MHz) δ 8.16 (d, J = 8.3 Hz, 1H), 7.45 (d, J = 7.3 Hz, 1H), 7.38−7.29 (m, 5H), 7.09 (t, J = 7.8 Hz, 1H), 6.81 (s, 1H), 6.25 (br s, 1H), 4.77−4.70 (m, 3H), 4.57 (s, 2H), 4.24 (s, 2H), 4.06 (t, J = 5.4 Hz, 2H), 2.90 (t, J = 5.4 Hz, 2H), 2.63 (s, 3H), 1.84 (s, 3H). HRMS (ESI+) calcd for C28H28N5O2 (M + H)+ 466.2238, found 466.2234. N-(1-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2-yl)-2methyl-1H-indol-4-yl)propiolamide (19). To a solution of amine 34a (18 mg, 0.047 mmol) and propiolic acid (9.9 mg, 0.14 mmol) in CH2Cl2 (2 mL) was added DCC (29.0 mg, 0.141 mmol), and the mixture was stirred at rt for 30 min. The suspension was then filtered and the filtrate was concentrated in vacuo. The residue was purified by flash column chromatography using EtOAc/hexanes (40%) to afford compound 19 as a yellow solid (17.6 mg, 86%). 1H NMR (CDCl3, 600 MHz) δ 7.83 (d, J = 8.3 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.61 (br s, 1H), 7.39−7.29 (m, 5H), 7.05 (t, J = 8.1 Hz, 1H), 6.29 (s, 1H), 4.98 (t, J = 5.6 Hz, 1H), 4.75 (d, J = 5.6 Hz, 2H), 2.93 (s, 1H), 2.83−2.78 (m, 2H), 2.61 (s, 3H), 2.42−2.37 (m, 2H), 1.95− 1.86 (m, 4H). HRMS (ESI+) calcd for C27H26N5O (M + H)+ 436.2132, found 436.2137. N-(1-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2-yl)-2methyl-1H-indol-4-yl)but-2-ynamide (20). To a solution of amine 34a (27 mg, 0.070 mmol) and 2-butynoic acid (8.9 mg, 0.11 mmol) in DMF (1 mL) were added EDC hydrochloride (27.0 mg, 0.141 mmol) and HOBt (9.5 mg, 0.0704 mmol), and the mixture was stirred at rt overnight. The reaction mixture was then diluted with water (30 mL) and extracted with CH2Cl2 (30 mL × 3). The combined organic layer was dried over Na2SO4, and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography using EtOAc/hexanes (40%) to afford compound 20 as a white solid (24.5 mg, 77%). 1H NMR (CDCl3, 600 MHz) δ 7.80 (d, J = 8.3 Hz, 1H), 7.72 (d, J = 7.9 Hz, 1H), 7.52 (br s, 1H), 7.37−7.28 (m, 5H), 7.03 (t, J = 8.1 Hz, 1H), 6.29 (s, 1H), 5.01 (t, J = 5.6 Hz, 1H), 4.74 (d, J = 5.6 Hz, 2H), 2.82−2.77 (m, 2H), 2.60 (s, 3H), 2.40−2.35 (m, 2H), 2.01 (s, 3H), 1.93−1.85 (m, 4H). HRMS (ESI+) calcd for C28H28N5O (M + H)+ 450.2288, found 450.2283. N-(1-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2-yl)-2methyl-1H-indol-4-yl)pent-2-ynamide (21). Compound 21 was prepared from amine 34a in a fashion similar to the one described for compound 20. White solid, 15.5 mg, yield 75%. 1H NMR (CDCl3, 600 MHz) δ 7.79 (d, J = 8.2 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.54 (s, 1H), 7.38−7.27 (m, 5H), 7.03 (t, J = 8.0 Hz, 1H), 6.29 (s, 1H), 5.03 (br s, 1H), 4.73 (d, J = 5.1 Hz, 2H), 2.83−2.77 (m, 2H), 2.59 (s, 3H), 2.42−2.34 (m, 4H), 1.94−1.85 (m, 4H), 1.24 (t, J = 7.5 Hz, 3H). HRMS (ESI+) calcd for C29H30N5O (M + H)+ 464.2445, found 464.2436. N-(1-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2-yl)-2methyl-1H-indol-4-yl)hex-2-ynamide (22). Compound 22 was prepared from amine 34a in a fashion similar to the one described for compound 20. White solid, 17.3 mg, yield 82%. 1H NMR (CDCl3, 600 MHz) δ 7.79 (d, J = 8.3 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.52 (s, 1H), 7.38−7.27 (m, 5H), 7.03 (t, J = 8.0 Hz, 1H), 6.29 (s, 1H), 5.03 (br s, 1H), 4.73 (d, J = 5.2 Hz, 2H), 2.83−2.77 (m, 2H), 2.60 (s, 3H), 2.41−2.36 (m, 2H), 2.34 (t, J = 7.1 Hz, 2H), 1.94−1.84 (m, 4H), 1.69−1.61 (m, 2H), 1.05 (t, J = 7.4 Hz, 3H). HRMS (ESI+) calcd for C30H32N5O (M + H)+ 478.2601, found 478.2608. N-(1-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2-yl)-2methyl-1H-indol-4-yl)acrylamide (23). To a solution of K2CO3 (33.4 mg, 0.242 mmol) and acryloyl chloride (22 mg, 0.24 mmol) in acetone (2 mL) and water (0.5 mL) at 0 °C was added amine 34a (46 mg, 0.121 mmol), and the mixture was stirred at 0 °C for 1 h. The

reaction mixture was then diluted with water (30 mL) and extracted with CH2Cl2 (30 mL × 3). The combined organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography using EtOAc/hexanes (40%) to afford compound 23 as a yellow solid (28.8 mg, 55%). 1H NMR (CDCl3, 600 MHz) δ 7.81 (br d, J = 5.6 Hz, 2H), 7.44 (br s, 1H), 7.37−7.28 (m, 5H), 7.05 (t, J = 8.1 Hz, 1H), 6.44 (d, J = 16.9 Hz, 1H), 6.32 (dd, J1 = 16.9 Hz, J2 = 10.2 Hz, 1H), 6.28 (s, 1H), 5.75 (d, J = 9.8 Hz, 1H), 5.01 (s, 1H), 4.74 (d, J = 5.5 Hz, 2H), 2.82−2.77 (m, 2H), 2.59 (s, 3H), 2.40−2.35 (m, 2H), 1.93−1.84 (m, 4H). HRMS (ESI+) calcd for C27H28N5O (M + H)+ 438.2288, found 438.2296. N-(1-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2-yl)-2methyl-1H-indol-4-yl)propionamide (24). Compound 24 was prepared from amine 34a in a fashion similar to the one described for compound 20. White solid, 19.1 mg, yield 63%. 1H NMR (CDCl3, 600 MHz) δ 7.80 (d, J = 8.1 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.38− 7.28 (m, 5H), 7.25 (br s, 1H), 7.05 (t, J = 8.0 Hz, 1H), 6.25 (s, 1H), 4.97 (s, 1H), 4.75 (d, J = 5.3 Hz, 2H), 2.84−2.76 (m, 2H), 2.60 (s, 3H), 2.46 (q, J = 7.5 Hz, 2H), 2.42−2.35 (m, 2H), 1.95−1.85 (m, 4H), 1.30 (t, J = 7.5 Hz, 3H). HRMS (ESI+) calcd for C27H30N5O (M + H)+ 440.2445, found 440.2443. N-(1-(4-(Benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-2-methyl-1H-indol-4-yl)propiolamide (25). Compound 25 was prepared from amine 34b in a fashion similar to the one described for compound 19. White solid, 13.6 mg, yield 79%. 1 H NMR (CDCl3, 600 MHz) δ 7.85 (d, J = 8.3 Hz, 1H), 7.68 (s, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.38−7.33 (m, 2H), 7.33−7.28 (m, 3H), 7.05 (t, J = 8.0 Hz, 1H), 6.30 (s, 1H), 4.76−4.69 (m, 3H), 4.55 (s, 2H), 4.08−4.03 (m, 2H), 2.93 (s, 1H), 2.92−2.87 (m, 2H), 2.60 (s, 3H). HRMS (ESI+) calcd for C26H24N5O2 (M + H)+ 438.1925, found 438.1918. N-(1-(4-(Benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-2-methyl-1H-indol-4-yl)but-2-ynamide (26). Compound 26 was prepared from amine 34b in a fashion similar to the one described for compound 20. White solid, 15.0 mg, yield 83%. 1 H NMR (CDCl3, 600 MHz) δ 7.83 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.53 (s, 1H), 7.38−7.28 (m, 5H), 7.04 (t, J = 8.0 Hz, 1H), 6.30 (s, 1H), 4.76−4.69 (m, 3H), 4.55 (s, 2H), 4.05 (t, J = 5.2 Hz, 2H), 2.92−2.87 (m, 2H), 2.60 (s, 3H), 2.02 (s, 3H). HRMS (ESI+) calcd for C27H26N5O2 (M + H)+ 452.2081, found 452.2084. N-(1-(4-(Benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-2-methyl-1H-indol-4-yl)acrylamide (27). Compound 27 was prepared from amine 34b in a fashion similar to the one described for compound 23. White solid, 17.3 mg, yield 82%. 1H NMR (CDCl3, 600 MHz) δ 7.84 (d, J = 7.0 Hz, 1H), 7.78 (d, J = 6.5 Hz, 1H), 7.42 (br s, 1H), 7.38−7.28 (m, 5H), 7.06 (t, J = 8.1 Hz, 1H), 6.45 (d, J = 16.9 Hz, 1H), 6.37−6.30 (m, 1H), 6.29 (s, 1H), 5.76 (d, J = 9.7 Hz, 1H), 4.71 (s, 3H), 4.55 (s, 2H), 4.05 (t, J = 5.5 Hz, 2H), 2.94−2.93 (m, 2H), 2.60 (s, 3H). HRMS (ESI+) calcd for C26H26N5O2 (M + H)+ 440.2081, found 440.2082. Methyl 1-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2yl)-2-methyl-1H-indole-4-carboxylate (30a). To a solution of chloride 28a26 (323 mg, 1.18 mmol) and indole 2926 (223 mg, 1.18 mmol) in dioxane (8 mL) were added Pd2(dba)3 (108 mg, 0.118 mmol), XPhos (112 mg, 0.236 mmol), and Cs2CO3 (768 mg, 2.36 mmol) under Ar atmosphere. The resulting mixture was stirred at 100 °C overnight and allowed to cool to rt. The mixture was diluted with water (30 mL) and extracted with CH2Cl2 (30 mL × 3). The combined organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography using EtOAc/hexanes (25%) to afford compound 30a as an orange solid (466 mg, 93%). 1H NMR (CDCl3, 600 MHz) δ 8.20 (d, J = 8.2 Hz, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.37−7.28 (m, 5H), 7.08 (t, J = 7.9 Hz, 1H), 7.01 (s, 1H), 5.00 (t, J = 5.5 Hz, 1H), 4.74 (d, J = 5.5 Hz, 2H), 3.96 (s, 3H), 2.82−2.78 (m, 2H), 2.40−2.36 (m, 2H), 1.94−1.86 (m, 4H). HRMS (ESI+) calcd for C26H27N4O2 (M + H)+ 427.2129, found 427.2137. Methyl 1-(4-(Benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-2-methyl-1H-indole-4-carboxylate (30b). ComL



Article

pound 30b was prepared from intermediates 28b26 and 2926 in a fashion similar to the one described for compound 30a. White solid, 90 mg, yield 83%. 1H NMR (CDCl3, 600 MHz) δ 8.24 (d, J = 8.2 Hz, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.37−7.28 (m, 5H), 7.09 (t, J = 7.8 Hz, 1H), 7.03 (s, 1H), 4.73 (d, J = 4.9 Hz, 2H), 4.70 (t, J = 4.9 Hz, 1H), 4.55 (s, 2H), 4.05 (t, J = 5.5 Hz, 2H), 3.95 (s, 3H), 2.89 (t, J = 5.5 Hz, 2H), 2.64 (s, 3H). HRMS (ESI+) calcd for C25H25N4O3 (M + H)+ 429.1921, found 429.1923. 1-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2-yl)-2methyl-1H-indole-4-carboxylic acid (31a). To a solution of 30a (443 mg, 1.04 mmol) in water (7.5 mL), MeOH (7.5 mL), and THF (22 mL) was added LiOH (249 mg, 10.4 mmol). The resulting mixture was stirred at 80 °C for 7 h. After cooling to rt, the reaction mixture was diluted with water (30 mL), acidified with 1 N HCl to pH = 2, and then extracted with CH2Cl2 (30 mL × 3). The combined organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography using MeOH/CH2Cl2 (5%) to afford compound 31a as a white solid (288 mg, 84%). 1H NMR (CDCl3, 600 MHz) δ 8.24 (d, J = 8.2 Hz, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.38−7.29 (m, 5H), 7.13−7.09 (m, 2H), 4.99 (t, J = 5.5 Hz, 1H), 4.75 (d, J = 5.5 Hz, 2H), 2.84−2.80 (m, 2H), 2.66 (s, 3H), 2.41−2.37 (m, 2H), 1.95−1.87 (m, 4H). HRMS (ESI+) calcd for C25H25N4O2 (M + H)+ 413.1972, found 413.1979. 1-(4-(Benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-2-methyl-1H-indole-4-carboxylic acid (31b). Compound 31b was prepared from ester 30b in a fashion similar to the one described for compound 31a. White solid, 87 mg, yield 100%. 1 H NMR (CD3OD, 600 MHz) δ 7.86 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.38−7.26 (m, 5H), 7.10 (t, J = 8.0 Hz, 1H), 7.08 (s, 1H), 4.78 (s, 2H), 4.67 (s, 2H), 4.08 (t, J = 5.0 Hz, 2H), 2.89 (t, J = 5.0 Hz, 2H), 2.44 (s, 3H). HRMS (ESI+) calcd for C24H23N4O3 (M + H)+ 415.1765, found 415.1757. N-Benzyl-2-(2-methyl-4-nitro-1H-indol-1-yl)-5,6,7,8-tetrahydroquinazolin-4-amine (33a). Compound 33a was prepared from intermediates 28a26 and 3244 in a fashion similar to the one described for compound 30a. Yellow solid, 265 mg, yield 97%. 1H NMR (CDCl3, 600 MHz) δ 8.22 (d, J = 8.2 Hz, 1H), 8.07 (d, J = 8.2 Hz, 1H), 7.38−7.35 (m, 2H), 7.34−7.29 (m, 3H), 7.12 (s, 1H), 7.07 (t, J = 8.2 Hz, 1H), 5.11 (t, J = 5.6 Hz, 1H), 4.74 (d, J = 5.6 Hz, 2H), 2.83−2.78 (m, 2H), 2.65 (s, 3H), 2.43−2.39 (m, 2H), 1.96−1.87 (m, 4H). HRMS (ESI+) calcd for C24H24N5O2 (M + H)+ 414.1925, found 414.1924. N-Benzyl-2-(2-methyl-4-nitro-1H-indol-1-yl)-7,8-dihydro5H-pyrano[4,3-d]pyrimidin-4-amine (33b). Compound 33b was prepared from intermediates 28b26 and 3244 in a fashion similar to the one described for compound 30a. White solid, 90 mg, yield 83%. 1 H NMR (CDCl3, 600 MHz) δ 8.26 (d, J = 8.2 Hz, 1H), 8.09 (d, J = 8.2 Hz, 1H), 7.41−7.36 (m, 2H), 7.35−7.31 (m, 3H), 7.16 (s, 1H), 7.11 (t, J = 8.2 Hz, 1H), 4.76 (d, J = 5.1 Hz, 2H), 4.70 (t, J = 5.1 Hz, 1H), 4.60 (s, 2H), 4.10−4.07 (m, 2H), 2.94−2.90 (m, 2H), 2.68 (m, 3H). HRMS (ESI+) calcd for C23H22N5O3 (M + H)+ 416.1717, found 416.1715. 2-(4-Amino-2-methyl-1H-indol-1-yl)-N-benzyl-5,6,7,8-tetrahydroquinazolin-4-amine (34a). To a solution of 33a (220 mg, 0.533 mmol) in MeOH (10 mL) was added SnCl2 (705 mg, 3.73 mmol). The resulting mixture was stirred at 75 °C overnight. After cooling to rt, the reaction was quenched with saturated Na2CO3 (30 mL). The resulting suspension was filtered and the filtrate was extracted with CH2Cl2 (30 mL × 3). The combined organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography using MeOH/CH2Cl2 (3%) to afford compound 34a as a yellow solid (109 mg, 53%). 1H NMR (CDCl3, 600 MHz) δ 7.51 (d, J = 8.3 Hz, 1H), 7.36−7.32 (m, 4H), 7.30−7.27 (m, 1H), 6.90 (t, J = 8.0 Hz, 1H), 6.40 (d, J = 7.4 Hz, 1H), 6.24 (s, 1H), 4.90 (t, J = 5.5 Hz, 1H), 4.74 (d, J = 5.5 Hz, 2H), 3.76 (br s, 2H), 2.81−2.76 (m, 2H), 2.59 (s, 3H), 2.36−2.32 (m, 2H), 1.91−1.83 (m, 4H). HRMS (ESI+) calcd for C24H26N5 (M + H)+ 384.2183, found 384.2182.

2-(4-Amino-2-methyl-1H-indol-1-yl)-N-benzyl-7,8-dihydro5H-pyrano[4,3-d]pyrimidin-4-amine (34b). A mixture of 33b (140 mg, 0.337 mmol) and 10% Pd/C (10 mg) in MeOH (28 mL) was stirred under H2 atmosphere (balloon) at rt for 2 h. The reaction mixture was filtered, and the filtrate was concentrated in vacuo. The residue was purified by flash column chromatography using EtOAc/ hexanes (50%) to afford compound 34b as a white solid (80 mg, 61%). 1H NMR (CDCl3, 600 MHz) δ 7.54 (d, J = 8.4 Hz, 1H), 7.34− 7.31 (m, 2H), 7.30−7.325 (m, 3H), 6.90 (t, J = 7.9 Hz, 1H), 6.40 (d, J = 7.4 Hz, 1H), 6.23 (s, 1H), 4.70 (d, J = 5.4 Hz, 2H), 4.57 (t, J = 5.4 Hz, 1H), 4.47 (s, 2H), 4.03−3.98 (m, 2H), 3.76 (br s, 2H), 2.88− 2.83 (m, 2H), 2.57 (m, 3H). HRMS (ESI+) calcd for C23H24N5O (M + H)+ 386.1975, found 386.1968. Purification of p97 Protein. His-tagged p97 was purified using Ni-NTA beads (QIAGEN). Protein eluted from the Ni column was further purified by size exclusion chromatography on a Superdex 200 HR (10/30) column in 50 mM Tris−HCl, pH 8.0, 150 mM potassium chloride, 5% glycerol, 2 mM magnesium chloride, and 1 mM DTT. Purified protein was stored at −80 °C in 50 mM Tris/ HCl, pH 8.0, 150 mM potassium chloride, 2 mM magnesium chloride, and 5% glycerol. Expression and purification of p97 C522A was performed as described previously.34 p97 Biochemical Assay. Reactions were carried out in duplicate in 96-well half-area UV-transparent microplates (Corning) at a final volume of 100 μL. Assays were initiated by adding 0.028 mg/mL (approximately 300 nM) p97 enzyme to the reaction buffer (50 mM Tris−HCl pH 8.0, 150 mM KCl, 1 mM MgCl2, 15% glycerol) containing 0.2 mM ATP, 1 mM DTT, 2 U/mL PNP (Sigma-Aldrich), and 0.2 mM MesG (Berry & Associates) that contained inhibitors (0−11.1 μM). The reactions were monitored continuously at 360 nm on an i3 multimode plate reader (Molecular Devices), and the initial velocities were fit to the Morrison equation using Prism 5 (GraphPad). GSH Reactivity Assay. To assess reactivity with GSH, the test compounds (1 μM) were incubated at 37 °C with 5 mM GSH and a stable internal standard in 0.1 M potassium phosphate buffer (pH 7.4, 10% acetonitrile) in a total volume of 1 mL. The reactions were initiated upon addition of GSH, and the samples were analyzed by directly injected the mixture into LC−MS/MS immediately after the GSH addition and then at predetermined intervals within a period of 24 h. Control reactions were run in the absence of GSH. The disappearance of parent compounds was monitored as % remaining relative to time zero, and the data were fitted to first-order kinetics by plotting the natural log of % remaining as a function of time. The pseudo-first-order rate constant (k), which is the negative slope of the linear fitting, was used to calculate the reaction half-life (t1/2 = 0.693/ k). The formation of GSH conjugates was determined at the end of incubation to confirm the reactions of test compounds with GSH. Antiproliferation Assay in MIA PaCa-2 Cells. The assay was performed as described previously.58 Reversibility under the Biochemical Assay Conditions. The p97 enzyme was diluted to 0.028 μg/μL in assay buffer without glycerol (50 mM Tris, pH 8.0, 150 mM KCl, 1 mM MgCl2, and 1 mM DTT). Reactions contained 750 nM compound 20, 1610 nM compound 4b, 3200 nM 3 (10 × IC50), or DMSO (1% of final volume). The reactions were incubated at rt for 20 min to allow for binding of the inhibitors to the enzyme. After the initial incubation, the reactions were moved to hydrated D-Tube Dialyzer Mini 6−8 kDa MWCO cassettes (Millipore) and placed in separate beakers each containing 500 mL of the above assay buffer (4000 × dilution). The beakers were placed on separate stirring plates and stirred overnight (16 h) at 4 °C. After the initial dialysis, the cassettes were placed in 500 mL of fresh buffer and dialyzed as above for an additional 6 h. The enzymatic activity after dialysis was determined based on the above p97 biochemical assay. Assays were initiated by adding 50 μL of the overnight reactions to 50 μL of assay buffer (50 mM Tris, pH 8.0, 150 mM KCl, 2 mM MgCl2, 15% glycerol (w/v), 0.2 mM ATP, 1 mM DTT, 2 U/mL PNP, and 0.2 mM MesG, all given as final M



Article

concentrations in 100 μL total). Additionally, compounds 20 (750 nM), 4b (1610 nM), and 3 (3200 nM) were added to wells with the overnight DMSO reactions as negative controls. All wells were mixed by pipetting and read for 2 h in an i3 multimode microplate reader in continuous absorbance mode set to 360 nm. Inhibition and Recovery of p97 Activity Ub-v-GFP Cells. Ubv-GFP cells (3 × 105, HEK293T-based) were seeded in 12-well polylysine D-coated plates and allowed to grow overnight. p97 inhibitor (2 μM) was added to each well. Before harvesting, the control group was incubated at 37 °C for 4 h (scheme A); for the recovery group, medium containing the drug was removed carefully after 1 h incubation. The cells were washed once with PBS and incubated in drug-free medium for another 3 h. Whole-cell extract prepared in the Laemmli buffer was subject to standard SDS-PAGE and immunoblotting analysis. Identification of Covalent Modifications by Compound 20. Recombinant p97 (10 μg; final concentration, 2 μM) and 10 μM compound 20 or DMSO were incubated in 50 μL of 1× ATPase buffer (50 mM Tris/HCl, pH 8.0, 150 mM KCl, 1 mM magnesium chloride, and 15% glycerol) for 3 h at room temperature. The mixture was subjected to SDS-PAGE on a 4−12% Bis-Tris gel. After staining the gel with colloidal Coomassie Blue (Invitrogen), the p97 band was cut off and analyzed at the Taplin Mass Spectrometry Facility at Harvard Medical School to identify residues that were covalently modified by compound 20. The p97 gel band was digested using a modified in-gel trypsin digestion procedure,59 and the resulting peptides were separated by nanoscale HPLC. The eluted peptides were detected (electrospray ionization), isolated, and further fragmented to give tandem mass spectra on an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). The raw mass MS/MS images were converted into mzXML format, processed, and then searched using the SEQUEST program (version 28) with methionine oxidation (+15.9949) and covalent modification by compound 20 (+449.2216, conjugate addition the alkyne) being dynamically allowed. Sites of cysteine modification were assigned using ModScore, a variation of the Ascore algorithm.52 Molecular Modeling. The docking study was carried out using the Schrodinger modeling package (version 2015-4).60 The solved cryo-EM structure of human p97 (PDB 5FTK)30 was taken from the Protein Data Bank. The protein structure was first processed using protein preparation wizard in Maestro. During the preparation, missing hydrogens atoms were added and the missing residues were added to the cryo-EM structure using Prime. After the het states were generated, the state of the lowest energy was selected and the energy minimization was performed using OPLS 2005 force field. The receptor grid, which encompassed the active site of the D2 domain at the interface of subunits D and E, was then generated around the endogenous ligand ADP using the Receptor Grid Generation application. The p97 inhibitors were drawn in Maestro and processed using the LigPrep application to generate conformation and ionization states within pH 7 ± 2 (Epik). The p97 ligands generated by LigPrep were docked into the receptor grid in the standard Glide XP mode. Post-docking minimization was performed and up to three best poses per ligand were written out. All poses were examined manually, and the pose of the best docking score was retained unless noted otherwise. Covalent docking was performed using the CovDock57 application with Cys522 as the nucleophilic residue, which underwent a conjugate addition to an alkyne (carbonyl-activated) as preset by CovDock. Structural visualization and representation were performed with PyMOL.61

■

■

Kiapp values at increasing ATP concentrations; active site (within D2 domain) Glide XP docking scores; CovDock docking scores; degradation of ΔSS TCRα-YFP in the presence of proteasome or p97 inhibitors; examples of tandem mass spectra of peptides that were covalently modified by compound 20 at Cys105, 522, and 535; three cysteine residues (Cys105, 522, and 535) in human p97 (PDB 5FTK, subunit E) as potential targets of compound 20; 2D representation of potential interactions between compound 4b and p97; proposed binding mode and interactions of compound 4b docked into human p97 (PDB 5FTK) using subunit E as a template; 2D representation of potential interactions between compound 19 and p97; 2D representation of potential interactions between compound 22 and p97; proposed binding modes of S1−3 docked (XP mode) into human p97 (PDB 5FTK) at the active site of the D2 domain; proposed binding modes of S1−3 covalently docked into human p97 (PDB 5FTK) at the active site of the D2 domain; proposed binding modes and interactions of compounds 11 and 15 docked into human p97 (PDB 5FTK) at the active site of the D2 domain (PDF) Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 612-624-2575. Fax: 612-624-8154. ORCID

Liqiang Chen: 0000-0002-4229-863X Author Contributions §

R.D. and T.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was supported by the Center for Drug Design in the Academic Health Center of the University of Minnesota and by the intramural research program of the NIDDK at NIH. The authors thank Eli Chapman (Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona) for his generous gift of p97-C522A-pET14b construct. They thank Di Xia (NCI) for critical reading of the manuscript. The University of Minnesota Supercomputing Institute provided all of the necessary computational resources. The authors also thank Ross Tomaino of the Taplin Mass Spectrometry Facility at Harvard Medical School for his expert help with the MS experiments.

■

ABBREVIATIONS ALS, amyotrophic lateral sclerosis; DBeQ, N2,N4-dibenzylquinazoline-2,4-diamine; DCC, N,N′-dicyclohexylcarbodiimide; DTT, dithiothreitol; EDC, N-ethyl-N′-(3dimethylaminopropyl)carbodiimide; ERAD, endoplasmic reticulum-associated protein degradation; GSH, glutathione; HBTU, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; IBMPFD, inclusion body myopathy, Paget’s disease of bone, and frontotemporal dementia; MesG, 7-methyl-6-thioguanosine; NFC, nitrofuran-containing; PNP, purine nucleoside

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.9b00144. DTT reactivity of selected compounds; list of p97 peptides that were covalently modified by compound 20 N



Article

membrane proteins with a new small molecule inhibitor, eeyarestatin. Mol. Biol. Cell 2004, 15, 1635−1646. (22) Wang, Q.; Shinkre, B. A.; Lee, J. G.; Weniger, M. A.; Liu, Y.; Chen, W.; Wiestner, A.; Trenkle, W. C.; Ye, Y. The ERAD inhibitor Eeyarestatin I is a bifunctional compound with a membrane-binding domain and a p97/VCP inhibitory group. PLoS One 2010, 5, No. e15479. (23) Ding, R.; Zhang, T.; Xie, J.; Williams, J.; Ye, Y.; Chen, L. Eeyarestatin I derivatives with improved aqueous solubility. Bioorg. Med. Chem. Lett. 2016, 26, 5177−5181. (24) Chou, T. F.; Brown, S. J.; Minond, D.; Nordin, B. E.; Li, K.; Jones, A. C.; Chase, P.; Porubsky, P. R.; Stoltz, B. M.; Schoenen, F. J.; Patricelli, M. P.; Hodder, P.; Rosen, H.; Deshaies, R. J. Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 4834−4839. (25) Chou, T. F.; Li, K.; Frankowski, K. J.; Schoenen, F. J.; Deshaies, R. J. Structure-activity relationship study reveals ML240 and ML241 as potent and selective inhibitors of p97 ATPase. ChemMedChem 2013, 8, 297−312. (26) Zhou, H. J.; Wang, J.; Yao, B.; Wong, S.; Djakovic, S.; Kumar, B.; Rice, J.; Valle, E.; Soriano, F.; Menon, M. K.; Madriaga, A.; Kiss von Soly, S.; Kumar, A.; Parlati, F.; Yakes, F. M.; Shawver, L.; Le Moigne, R.; Anderson, D. J.; Rolfe, M.; Wustrow, D. Discovery of a first-in-class, potent, selective, and orally bioavailable inhibitor of the p97 AAA ATPase (CB-5083). J. Med. Chem. 2015, 58, 9480−9497. (27) Cervi, G.; Magnaghi, P.; Asa, D.; Avanzi, N.; Badari, A.; Borghi, D.; Caruso, M.; Cirla, A.; Cozzi, L.; Felder, E.; Galvani, A.; Gasparri, F.; Lomolino, A.; Magnuson, S.; Malgesini, B.; Motto, I.; Pasi, M.; Rizzi, S.; Salom, B.; Sorrentino, G.; Troiani, S.; Valsasina, B.; O’Brien, T.; Isacchi, A.; Donati, D.; D’Alessio, R. Discovery of 2(cyclohexylmethylamino)pyrimidines as a new class of reversible valosine containing protein inhibitors. J. Med. Chem. 2014, 57, 10443−10454. (28) Magnaghi, P.; D’Alessio, R.; Valsasina, B.; Avanzi, N.; Rizzi, S.; Asa, D.; Gasparri, F.; Cozzi, L.; Cucchi, U.; Orrenius, C.; Polucci, P.; Ballinari, D.; Perrera, C.; Leone, A.; Cervi, G.; Casale, E.; Xiao, Y.; Wong, C.; Anderson, D. J.; Galvani, A.; Donati, D.; O’Brien, T.; Jackson, P. K.; Isacchi, A. Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death. Nat. Chem. Biol. 2013, 9, 548−556. (29) Polucci, P.; Magnaghi, P.; Angiolini, M.; Asa, D.; Avanzi, N.; Badari, A.; Bertrand, J.; Casale, E.; Cauteruccio, S.; Cirla, A.; Cozzi, L.; Galvani, A.; Jackson, P. K.; Liu, Y.; Magnuson, S.; Malgesini, B.; Nuvoloni, S.; Orrenius, C.; Sirtori, F. R.; Riceputi, L.; Rizzi, S.; Trucchi, B.; O’Brien, T.; Isacchi, A.; Donati, D.; D’Alessio, R. Alkylsulfanyl-1,2,4-triazoles, a new class of allosteric valosine containing protein inhibitors. Synthesis and structure-activity relationships. J. Med. Chem. 2013, 56, 437−450. (30) Banerjee, S.; Bartesaghi, A.; Merk, A.; Rao, P.; Bulfer, S. L.; Yan, Y.; Green, N.; Mroczkowski, B.; Neitz, R. J.; Wipf, P.; Falconieri, V.; Deshaies, R. J.; Milne, J. L.; Huryn, D.; Arkin, M.; Subramaniam, S. 2.3 A resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition. Science 2016, 351, 871−875. (31) Tao, S.; Tillotson, J.; Wijeratne, E. M.; Xu, Y. M.; Kang, M.; Wu, T.; Lau, E. C.; Mesa, C.; Mason, D. J.; Brown, R. V.; La Clair, J. J.; Gunatilaka, A. A.; Zhang, D. D.; Chapman, E. Withaferin A analogs that target the AAA + chaperone p97. ACS Chem. Biol. 2015, 10, 1916−1924. (32) Alverez, C.; Bulfer, S. L.; Chakrasali, R.; Chimenti, M. S.; Deshaies, R. J.; Green, N.; Kelly, M.; LaPorte, M. G.; Lewis, T. S.; Liang, M.; Moore, W. J.; Neitz, R. J.; Peshkov, V. A.; Walters, M. A.; Zhang, F.; Arkin, M. R.; Wipf, P.; Huryn, D. M. Allosteric indole amide inhibitors of p97: identification of a novel probe of the ubiquitin pathway. ACS Med. Chem. Lett. 2016, 7, 182−187. (33) Alverez, C.; Arkin, M. R.; Bulfer, S. L.; Colombo, R.; Kovaliov, M.; LaPorte, M. G.; Lim, C.; Liang, M.; Moore, W. J.; Neitz, R. J.; Yan, Y.; Yue, Z.; Huryn, D. M.; Wipf, P. Structure-activity study of

phosphorylase; SAR, structure−activity relationship; TCR, Tcell receptor

■

REFERENCES

(1) Nakayama, S.; Atsumi, R.; Takakusa, H.; Kobayashi, Y.; Kurihara, A.; Nagai, Y.; Nakai, D.; Okazaki, O. A zone classification system for risk assessment of idiosyncratic drug toxicity using daily dose and covalent binding. Drug Metab. Dispos. 2009, 37, 1970−1977. (2) Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discovery 2011, 10, 307−317. (3) Bauer, R. A. Covalent inhibitors in drug discovery: from accidental discoveries to avoided liabilities and designed therapies. Drug Discovery Today 2015, 20, 1061−1073. (4) Jackson, P. A.; Widen, J. C.; Harki, D. A.; Brummond, K. M. Covalent modifiers: a chemical perspective on the reactivity of alpha,beta-unsaturated carbonyls with thiols via hetero-Michael addition reactions. J. Med. Chem. 2017, 60, 839−885. (5) Ye, Y.; Tang, W. K.; Zhang, T.; Xia, D. A mighty “protein extractor” of the cell: structure and function of the p97/CDC48 ATPase. Front. Mol. Biosci. 2017, 4, 39. (6) Xia, D.; Tang, W. K.; Ye, Y. Structure and function of the AAA + ATPase p97/Cdc48p. Gene 2016, 583, 64−77. (7) Tang, W. K.; Li, D.; Li, C. C.; Esser, L.; Dai, R.; Guo, L.; Xia, D. A novel ATP-dependent conformation in p97 N-D1 fragment revealed by crystal structures of disease-related mutants. EMBO J. 2010, 29, 2217−2229. (8) Tang, W. K.; Xia, D. Altered intersubunit communication is the molecular basis for functional defects of pathogenic p97 mutants. J. Biol. Chem. 2013, 288, 36624−36635. (9) Buchberger, A.; Schindelin, H.; Hanzelmann, P. Control of p97 function by cofactor binding. FEBS Lett. 2015, 589, 2578−2589. (10) Zhang, T.; Ye, Y. The final moments of misfolded proteins en route to the proteasome. DNA Cell Biol. 2014, 33, 477−483. (11) Xu, S.; Peng, G.; Wang, Y.; Fang, S.; Karbowski, M. The AAAATPase p97 is essential for outer mitochondrial membrane protein turnover. Mol. Biol. Cell 2011, 22, 291−300. (12) Meyer, H.; Bug, M.; Bremer, S. Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system. Nat. Cell Biol. 2012, 14, 117−123. (13) Bug, M.; Meyer, H. Expanding into new markets–VCP/p97 in endocytosis and autophagy. J. Struct. Biol. 2012, 179, 78−82. (14) Dantuma, N. P.; Acs, K.; Luijsterburg, M. S. Should I stay or should I go: VCP/p97-mediated chromatin extraction in the DNA damage response. Exp. Cell Res. 2014, 329, 9−17. (15) Fessart, D.; Marza, E.; Taouji, S.; Delom, F.; Chevet, E. P97/ CDC-48: proteostasis control in tumor cell biology. Cancer Lett. 2013, 337, 26−34. (16) Chapman, E.; Maksim, N.; de la Cruz, F.; La Clair, J. J. Inhibitors of the AAA + chaperone p97. Molecules 2015, 20, 3027− 3049. (17) Anderson, D. J.; Le Moigne, R.; Djakovic, S.; Kumar, B.; Rice, J.; Wong, S.; Wang, J.; Yao, B.; Valle, E.; Kiss von Soly, S.; Madriaga, A.; Soriano, F.; Menon, M. K.; Wu, Z. Y.; Kampmann, M.; Chen, Y.; Weissman, J. S.; Aftab, B. T.; Yakes, F. M.; Shawver, L.; Zhou, H. J.; Wustrow, D.; Rolfe, M. Targeting the AAA ATPase p97 as an approach to treat cancer through disruption of protein homeostasis. Cancer Cell 2015, 28, 653−665. (18) Tang, W. K.; Xia, D. Mutations in the human AAA + chaperone p97 and related diseases. Front. Mol. Biosci. 2016, 3, 79. (19) Zhang, T.; Mishra, P.; Hay, B. A.; Chan, D.; Guo, M. Valosincontaining protein (VCP/p97) inhibitors relieve Mitofusin-dependent mitochondrial defects due to VCP disease mutants. Elife 2017, 6, No. e17834. (20) Blythe, E. E.; Olson, K. C.; Chau, V.; Deshaies, R. J. Ubiquitinand ATP-dependent unfoldase activity of P97/VCP*NPLOC4*UFD1L is enhanced by a mutation that causes multisystem proteinopathy. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, E4380−E4388. (21) Fiebiger, E.; Hirsch, C.; Vyas, J. M.; Gordon, E.; Ploegh, H. L.; Tortorella, D. Dissection of the dislocation pathway for type I O



Article

bioisosteric trifluoromethyl and pentafluorosulfanyl indole inhibitors of the AAA ATPase p97. ACS Med. Chem. Lett. 2015, 6, 1225−1230. (34) Tillotson, J.; Bashyal, B. P.; Kang, M.; Shi, T.; De La Cruz, F.; Gunatilaka, A. A.; Chapman, E. Selective inhibition of p97 by chlorinated analogues of dehydrocurvularin. Org. Biomol. Chem. 2016, 14, 5918−5921. (35) Wijeratne, E. M.; Gunaherath, G. M.; Chapla, V. M.; Tillotson, J.; de la Cruz, F.; Kang, M.; U’Ren, J. M.; Araujo, A. R.; Arnold, A. E.; Chapman, E.; Gunatilaka, A. A. Oxaspirol B with p97 inhibitory activity and other oxaspirols from Lecythophora sp. FL1375 and FL1031, endolichenic fungi inhabiting Parmotrema tinctorum and Cladonia evansii. J. Nat. Prod. 2016, 79, 340−352. (36) Wang, Q.; Li, L.; Ye, Y. Inhibition of p97-dependent protein degradation by Eeyarestatin I. J. Biol. Chem. 2008, 283, 7445−7454. (37) Schwartz, P. A.; Kuzmic, P.; Solowiej, J.; Bergqvist, S.; Bolanos, B.; Almaden, C.; Nagata, A.; Ryan, K.; Feng, J.; Dalvie, D.; Kath, J. C.; Xu, M.; Wani, R.; Murray, B. W. Covalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistance. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 173−178. (38) Flanagan, M. E.; Abramite, J. A.; Anderson, D. P.; Aulabaugh, A.; Dahal, U. P.; Gilbert, A. M.; Li, C.; Montgomery, J.; Oppenheimer, S. R.; Ryder, T.; Schuff, B. P.; Uccello, D. P.; Walker, G. S.; Wu, Y.; Brown, M. F.; Chen, J. M.; Hayward, M. M.; Noe, M. C.; Obach, R. S.; Philippe, L.; Shanmugasundaram, V.; Shapiro, M. J.; Starr, J.; Stroh, J.; Che, Y. Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors. J. Med. Chem. 2014, 57, 10072−10079. (39) Jöst, C.; Nitsche, C.; Scholz, T.; Roux, L.; Klein, C. D. Promiscuity and selectivity in covalent enzyme inhibition: a systematic study of electrophilic fragments. J. Med. Chem. 2014, 57, 7590−7599. (40) Cee, V. J.; Volak, L. P.; Chen, Y.; Bartberger, M. D.; Tegley, C.; Arvedson, T.; McCarter, J.; Tasker, A. S.; Fotsch, C. Systematic study of the glutathione (GSH) reactivity of N-arylacrylamides: 1. effects of aryl substitution. J. Med. Chem. 2015, 58, 9171−9178. (41) Sommer, S.; Weikart, N. D.; Linne, U.; Mootz, H. D. Covalent inhibition of SUMO and ubiquitin-specific cysteine proteases by an in situ thiol-alkyne addition. Bioorg. Med. Chem. 2013, 21, 2511−2517. (42) Ekkebus, R.; van Kasteren, S. I.; Kulathu, Y.; Scholten, A.; Berlin, I.; Geurink, P. P.; de Jong, A.; Goerdayal, S.; Neefjes, J.; Heck, A. J.; Komander, D.; Ovaa, H. On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. J. Am. Chem. Soc. 2013, 135, 2867−2870. (43) Oballa, R. M.; Truchon, J. F.; Bayly, C. I.; Chauret, N.; Day, S.; Crane, S.; Berthelette, C. A generally applicable method for assessing the electrophilicity and reactivity of diverse nitrile-containing compounds. Bioorg. Med. Chem. Lett. 2007, 17, 998−1002. (44) Moskalev, N.; Makosza, M. A novel method of indole ring system construction: one-pot synthesis of 4-and 6-nitroindole derivatives via base promoted reaction between 3-nitroaniline and ketones. Tetrahedron Lett. 1999, 40, 5395−5398. (45) Webb, M. R. A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 4884−4887. (46) Morrison, J. F. Kinetics of the reversible inhibition of enzymecatalysed reactions by tight-binding inhibitors. Biochim. Biophys. Acta, Enzymol. 1969, 185, 269−286. (47) Witte, J. F.; Bray, K. E.; Thornburg, C. K.; McClard, R. W. ‘Irreversible’ slow-onset inhibition of orotate phosphoribosyltransferase by an amidrazone phosphate transition-state mimic. Bioorg. Med. Chem. Lett. 2006, 16, 6112−6115. (48) McClerren, A. L.; Endsley, S.; Bowman, J. L.; Andersen, N. H.; Guan, Z.; Rudolph, J.; Raetz, C. R. A slow, tight-binding inhibitor of the zinc-dependent deacetylase LpxC of lipid A biosynthesis with antibiotic activity comparable to ciprofloxacin. Biochemistry 2005, 44, 16574−16583.

(49) Nishikori, S.; Esaki, M.; Yamanaka, K.; Sugimoto, S.; Ogura, T. Positive cooperativity of the p97 AAA ATPase is critical for essential functions. J. Biol. Chem. 2011, 286, 15815−15820. (50) Hänzelmann, P.; Schindelin, H. Structural basis of ATP hydrolysis and intersubunit signaling in the AAA + ATPase p97. Structure 2016, 24, 127−139. (51) Wiertz, E. J.; Jones, T. R.; Sun, L.; Bogyo, M.; Geuze, H. J.; Ploegh, H. L. The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell 1996, 84, 769−779. (52) Beausoleil, S. A.; Villen, J.; Gerber, S. A.; Rush, J.; Gygi, S. P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 2006, 24, 1285− 1292. (53) Ye, Y.; Meyer, H. H.; Rapoport, T. A. Function of the p97Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains. J. Cell Biol. 2003, 162, 71−84. (54) Noguchi, M.; Takata, T.; Kimura, Y.; Manno, A.; Murakami, K.; Koike, M.; Ohizumi, H.; Hori, S.; Kakizuka, A. ATPase activity of p97/valosin-containing protein is regulated by oxidative modification of the evolutionally conserved cysteine 522 residue in Walker A motif. J. Biol. Chem. 2005, 280, 41332−41341. (55) Briggs, L. C.; Baldwin, G. S.; Miyata, N.; Kondo, H.; Zhang, X.; Freemont, P. S. Analysis of nucleotide binding to P97 reveals the properties of a tandem AAA hexameric ATPase. J. Biol. Chem. 2008, 283, 13745−13752. (56) Tang, W. K.; Odzorig, T.; Jin, W.; Xia, D. Structural basis of p97 inhibition by the site-selective anti-cancer compound CB-5083. Mol. Pharmacol. 2018, 286−293. (57) Zhu, K.; Borrelli, K. W.; Greenwood, J. R.; Day, T.; Abel, R.; Farid, R. S.; Harder, E. Docking covalent inhibitors: a parameter free approach to pose prediction and scoring. J. Chem. Inf. Model. 2014, 54, 1932−1940. (58) Ai, T.; Willett, R.; Williams, J.; Ding, R.; Wilson, D. J.; Xie, J.; Kim, D. H.; Puertollano, R.; Chen, L. N-(1-Benzyl-3,5-dimethyl-1Hpyrazol-4-yl)benzamides: antiproliferative activity and effects on mTORC1 and autophagy. ACS Med. Chem. Lett. 2017, 8, 90−95. (59) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 1996, 68, 850−858. (60) Schrödinger Release 2015-4; Schrödinger, LLC: New York, NY, 2015. (61) The PyMOL Molecular Graphics System, version 1.8; Schrödinger, LLC.

P


Discovery of Irreversible p97 Inhibitors - Journal of Medicinal

Recommend Documents