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Structural Basis of p97 Inhibition by the Site-Selective Anticancer Compound CB-5083.

ABSTRACT: Inhibition of p97, a key player in the ubiquitin-proteasome degradation pathway, has been proposed as a treatment of cancer. This concept was nearly realized recently when a potent p97 inhibitor, 1-[4-(benzylamino)-5H,7H,8H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl-1H-indole-4-carboxamide (CB-5083), was developed and demonstrated broad antitumor activity in various tumor models. CB-5083 functions as a competitive inhibitor that binds selectively to the ATP-binding site of the D2 domain, although both the D1 and D2 ATPase sites of p97 are highly similar. Despite its promising anticancer activity, CB-5083 failed its phase I clinical trials due to an unexpected off-target effect, which necessitates further improvement of the inhibitor. In this study, we determined the crystal structure of N-terminal domain-truncated p97 in complex with CB-5083. It provides a structural basis for the specificity of CB-5083 toward the D2 domain, offers an explanation in atomic detail for the mutations that confer resistance to CB-5083, and establishes a foundation for future structure-guided efforts to develop the next generation of p97 inhibitors.

SUBMITTER: Tang WK

PROVIDER: S-EPMC6355941 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Structural Basis of p97 Inhibition by the Site-Selective Anticancer Compound CB-5083.

Tang Wai Kwan WK Odzorig Taivan T Jin Whitney W Xia Di D

Molecular pharmacology 20181227 3

Inhibition of p97, a key player in the ubiquitin-proteasome degradation pathway, has been proposed as a treatment of cancer. This concept was nearly realized recently when a potent p97 inhibitor, 1-[4-(benzylamino)-5H,7H,8H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl-1H-indole-4-carboxamide (CB-5083), was developed and demonstrated broad antitumor activity in various tumor models. CB-5083 functions as a competitive inhibitor that binds selectively to the ATP-binding site of the D2 domain, although bot ...[more]

PMID: 30591537

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Project description:BackgroundPathogenic gain of function variants in Valosin-containing protein (VCP) cause a unique disease characterized by inclusion body myopathy with early-onset Paget disease of bone and frontotemporal dementia (also known as Multisystem proteinopathy (MSP)). Previous studies in drosophila models of VCP disease indicate treatment with VCP inhibitors mitigates disease pathology. Earlier-generation VCP inhibitors display off-target effects and relatively low therapeutic potency. New generation of VCP inhibitors needs to be evaluated in a mouse model of VCP disease. In this study, we tested the safety and efficacy of a novel and potent VCP inhibitor, CB-5083 using VCP patient-derived myoblast cells and an animal model of VCP disease.MethodsFirst, we analyzed the effect of CB-5083 in patient-derived myoblasts on the typical disease autophagy and TDP-43 profile by Western blot. Next, we determined the maximum tolerated dosage of CB-5083 in mice and treated the 2-month-old VCPR155H/R155H mice for 5 months with 15 mg/kg CB-5083. We analyzed motor function monthly by Rotarod; and we assessed the end-point blood toxicology, and the muscle and brain pathology, including autophagy and TDP-43 profile, using Western blot and immunohistochemistry. We also treated 12-month-old VCPR155H/+ mice for 6 months and performed similar analysis. Finally, we assessed the potential side effects of CB-5083 on retinal function, using electroretinography in chronically treated VCPR155H/155H mice.ResultsIn vitro analyses using patient-derived myoblasts confirmed that CB-5083 can modulate expression of the proteins in the autophagy pathways. We found that chronic CB-5083 treatment is well tolerated in the homozygous mice harboring patient-specific VCP variant, R155H, and can ameliorate the muscle pathology characteristic of the disease. VCP-associated pathology biomarkers, such as elevated TDP-43 and p62 levels, were significantly reduced. Finally, to address the potential adverse effect of CB-5083 on visual function observed in a previous oncology clinical trial, we analyzed retinal function in mice treated with moderate doses of CB-5083 for 5 months and documented the absence of permanent ocular toxicity.ConclusionsAltogether, these findings suggest that long-term use of CB-5083 by moderate doses is safe and can improve VCP disease-associated muscle pathology. Our results provide translationally relevant evidence that VCP inhibitors could be beneficial in the treatment of VCP disease.

Project description:Small-molecule inhibitors of p97 are useful tools to study p97 function. Human p97 is an important AAA ATPase due to its diverse cellular functions and implication in mediating the turnover of proteins involved in tumorigenesis and virus infections. Multiple p97 inhibitors identified from previous high-throughput screening studies are thiol-reactive compounds targeting Cys522 in the D2 ATP-binding domain. Thus, these findings suggest a potential strategy to develop covalent p97 inhibitors. We first used purified p97 to assay several known covalent kinase inhibitors to determine if they can inhibit ATPase activity. We evaluated their selectivity using our dual reporter cells that can distinguish p97 dependent and independent degradation. We selected a β-nitrostyrene scaffold to further study the structure-activity relationship. In addition, we used p97 structures to design and synthesize analogues of pyrazolo[3,4-d]pyrimidine (PP). We incorporated electrophiles into a PP-like compound 17 (4-amino-1-tert-butyl-3-phenyl pyrazolo[3,4-d]pyrimidine) to generate eight compounds. A selective compound 18 (N-(1-(tert-butyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)acrylamide, PPA) exhibited excellent selectivity in an in vitro ATPase activity assay: IC50 of 0.6 μM, 300 μM, and 100 μM for wild type p97, yeast Cdc48, and N-ethylmaleimide sensitive factor (NSF), respectively. To further examine the importance of Cys522 on the active site pocket during PPA inhibition, C522A and C522T mutants of p97 were purified and shown to increase IC50 values by 100-fold, whereas replacement of Thr532 of yeast Cdc48 with Cysteine decreased the IC50 by 10-fold. The molecular modeling suggested the hydrogen bonds and hydrophobic interactions in addition to the covalent bonding at Cys522 between WT-p97 and PPA. Furthermore, tandem mass spectrometry confirmed formation of a covalent bond between Cys522 and PPA. An anti-proliferation assay indicated that the proliferation of HCT116, HeLa, and RPMI8226 was inhibited by PPA with IC50 of 2.7 μM, 6.1 μM, and 3.4 μM, respectively. In addition, PPA is able to inhibit proliferation of two HCT116 cell lines that are resistant to CB-5083 and NMS-873, respectively. Proteomic analysis of PPA-treated HCT116 revealed Gene Ontology enrichment of known p97 functional pathways such as the protein ubiquitination and the ER to Golgi transport vesicle membrane. In conclusion, we have identified and characterized PPA as a selective covalent p97 inhibitor, which will allow future exploration to improve the potency of p97 inhibitors with different mechanisms of action.

Project description:CB-5083 is an inhibitor of p97/valosin-containing protein (VCP), for which phase I trials for cancer were terminated because of adverse effects on vision, such as photophobia and dyschromatopsia. Lower dose CB-5083 could combat inclusion body myopathy with early-onset Paget disease and frontotemporal dementia or multisystem proteinopathy caused by gain-of-function mutations in VCP. We hypothesized that the visual impairment in the cancer trial was due to CB-5083's inhibition of phosphodiesterase (PDE)-6, which mediates signal transduction in photoreceptors. To test our hypothesis, we used in vivo and ex vivo electroretinography (ERG) in mice and a PDE6 activity assay of bovine rod outer segment (ROS) extracts. Additionally, histology and optical coherence tomography were used to assess CB-5083's long-term ocular toxicity. A single administration of CB-5083 led to robust ERG signal deterioration, specifically in photoresponse kinetics. Similar recordings with known PDE inhibitors sildenafil, tadalafil, vardenafil, and zaprinast showed that only vardenafil had as strong an effect on the ERG signal in vivo as did CB-5083. In the biochemical assay, CB-5083 inhibited PDE6 activity with a potency higher than sildenafil but lower than that of vardenafil. Ex vivo ERG revealed a PDE6 inhibition constant of 80 nM for CB-5083, which is 7-fold smaller than that for sildenafil. Finally, we showed that the inhibitory effect of CB-5083 on visual function is reversible, and its chronic administration does not cause permanent retinal anomalies in aged VCP-disease model mice. Our results warrant re-evaluation of CB-5083 as a clinical therapeutic agent. We recommend preclinical ERG recordings as a routine drug safety screen. SIGNIFICANCE STATEMENT: This report supports the use of a valosin-containing protein (VCP) inhibitor drug, CB-5083, for the treatment of neuromuscular VCP disease despite CB-5083's initial clinical failure for cancer treatment due to side effects on vision. The data show that CB-5083 displays a dose-dependent but reversible inhibitory action on phosphodiesterase-6, an essential enzyme in retinal photoreceptor function, but no long-term consequences on retinal function or structure.

Project description:Nitric oxide synthase (NOS) converts l-arginine into l-citrulline and releases the important signaling molecule nitric oxide (NO). In the cardiovascular system, NO produced by endothelial NOS (eNOS) relaxes smooth muscle which controls vascular tone and blood pressure. Neuronal NOS (nNOS) produces NO in the brain, where it influences a variety of neural functions such as neural transmitter release. NO can also support the immune system, serving as a cytotoxic agent during infections. Even with all of these important functions, NO is a free radical and, when overproduced, it can cause tissue damage. This mechanism can operate in many neurodegenerative diseases, and as a result the development of drugs targeting nNOS is a desirable therapeutic goal. However, the active sites of all three human isoforms are very similar, and designing inhibitors specific for nNOS is a challenging problem. It is critically important, for example, not to inhibit eNOS owing to its central role in controlling blood pressure. In this Account, we summarize our efforts in collaboration with Rick Silverman at Northwestern University to develop drug candidates that specifically target NOS using crystallography, computational chemistry, and organic synthesis. As a result, we have developed aminopyridine compounds that are 3800-fold more selective for nNOS than eNOS, some of which show excellent neuroprotective effects in animal models. Our group has solved approximately 130 NOS-inhibitor crystal structures which have provided the structural basis for our design efforts. Initial crystal structures of nNOS and eNOS bound to selective dipeptide inhibitors showed that a single amino acid difference (Asp in nNOS and Asn in eNOS) results in much tighter binding to nNOS. The NOS active site is open and rigid, which produces few large structural changes when inhibitors bind. However, we have found that relatively small changes in the active site and inhibitor chirality can account for large differences in isoform-selectivity. For example, we expected that the aminopyridine group on our inhibitors would form a hydrogen bond with a conserved Glu inside the NOS active site. Instead, in one group of inhibitors, the aminopyridine group extends outside of the active site where it interacts with a heme propionate. For this orientation to occur, a conserved Tyr side chain must swing out of the way. This unanticipated observation taught us about the importance of inhibitor chirality and active site dynamics. We also successfully used computational methods to gain insights into the contribution of the state of protonation of the inhibitors to their selectivity. Employing the lessons learned from the aminopyridine inhibitors, the Silverman lab designed and synthesized symmetric double-headed inhibitors with an aminopyridine at each end, taking advantage of their ability to make contacts both inside and outside of the active site. Crystal structures provided yet another unexpected surprise. Two of the double-headed inhibitor molecules bound to each enzyme subunit, and one molecule participated in the generation of a novel Zn(2+) site that required some side chains to adopt alternate conformations. Therefore, in addition to achieving our specific goal, the development of nNOS selective compounds, we have learned how subtle differences in dynamics and structure can control protein-ligand interactions and often in unexpected ways.

Dataset Information

Structural Basis of p97 Inhibition by the Site-Selective Anticancer Compound CB-5083.

Publications

Structural Basis of p97 Inhibition by the Site-Selective Anticancer Compound CB-5083.

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