Comparative Analysis of Drug-like EP300/CREBBP Acetyltransferase Inhibitors.

The human acetyltransferase paralogues EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potencies of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize the binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of the relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

Targeted Investigational Oncology Agents in the NCI-60: A Phenotypic Systems-based Resource.

The NCI-60 human tumor cell line panel has proved to be a useful tool for the global cancer research community in the search for novel chemotherapeutics. The publicly available cell line characterization and compound screening data from the NCI-60 assay have significantly contributed to the understanding of cellular mechanisms targeted by new oncology agents. Signature sensitivity/resistance patterns generated for a given chemotherapeutic agent against the NCI-60 panel have long served as fingerprint presentations that encompass target information and the mechanism of action associated with the tested agent. We report the establishment of a new public NCI-60 resource based on the cell line screening of a large and growing set of 175 FDA-approved oncology drugs (AOD) plus >825 clinical and investigational oncology agents (IOA), representing a diverse set (>250) of therapeutic targets and mechanisms. This data resource is available to the public (https://ioa.cancer.gov) and includes the raw data from the screening of the IOA and AOD collection along with an extensive set of visualization and analysis tools to allow for comparative study of individual test compounds and multiple compound sets.

Multicellular Complex Tumor Spheroid Response to DNA Repair Inhibitors in Combination with DNA-damaging Drugs.

Multicellular spheroids comprised of malignant cells, endothelial cells, and mesenchymal stem cells served as an in vitro model of human solid tumors to investigate the potentiation of DNA-damaging drugs by pharmacologic modulation of DNA repair pathways. The DNA-damaging drugs, topotecan, trabectedin, and temozolomide were combined with varied inhibitors of DNA damage response enzymes including PARP (olaparib or talazoparib), ATM (ataxia telangiectasia mutated; AZD-1390), ATR (ataxia telangiectasia and Rad3-related protein; berzosertib or elimusertib), and DNA-PK (DNA-dependent protein kinase; nedisertib or VX-984). A range of clinically achievable concentrations were tested up to the clinical Cmax, if known. Mechanistically, the types of DNA damage induced by temozolomide, topotecan, and trabectedin are distinct, which was apparent from the response of spheroids to combinations with various DNA repair inhibitors. Although most combinations resulted in additive cytotoxicity, synergistic activity was observed for temozolomide combined with PARP inhibitors as well as combinations of the ATM inhibitor AZD-1390 with either topotecan or trabectedin. These findings might provide guidance for the selection of anticancer agent combinations worthy of further investigation. Significance: Clinical efficacy of DNA-damaging anticancer drugs can be influenced by the DNA damage response in tumor cells. The potentiation of DNA-damaging drugs by pharmacologic modulation of DNA repair pathways was assessed in multicellular tumor spheroids. Although most combinations demonstrated additive cytotoxicity, synergistic cytotoxicity was observed for several drug combinations.

Comparative analysis of drug-like EP300/CREBBP acetyltransferase inhibitors.

The human acetyltransferase paralogs EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potency of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

Discovery of Novel Small-Molecule Scaffolds for the Inhibition and Activation of WIP1 Phosphatase from a RapidFire Mass Spectrometry High-Throughput Screen.

Wild-type P53-induced phosphatase 1 (WIP1), also known as PPM1D or PP2Cδ, is a serine/threonine protein phosphatase induced by P53 after genotoxic stress. WIP1 inhibition has been proposed as a therapeutic strategy for P53 wild-type cancers in which it is overexpressed, but this approach would be ineffective in P53-negative cancers. Furthermore, there are several cancers with mutated P53 where WIP1 acts as a tumor suppressor. Therefore, activating WIP1 phosphatase might also be a therapeutic strategy, depending on the P53 status. To date, no specific, potent WIP1 inhibitors with appropriate pharmacokinetic properties have been reported, nor have WIP1-specific activators. Here, we report the discovery of new WIP1 modulators from a high-throughput screen (HTS) using previously described orthogonal biochemical assays suitable for identifying both inhibitors and activators. The primary HTS was performed against a library of 102 277 compounds at a single concentration using a RapidFire mass spectrometry assay. Hits were further evaluated over a range of 11 concentrations with both the RapidFire MS assay and an orthogonal fluorescence-based assay. Further biophysical, biochemical, and cell-based studies of confirmed hits revealed a WIP1 activator and two inhibitors, one competitive and one uncompetitive. These new scaffolds are prime candidates for optimization which might enable inhibitors with improved pharmacokinetics and a first-in-class WIP1 activator.

Discovery of Small-Molecule VapC1 Nuclease Inhibitors by Virtual Screening and Scaffold Hopping from an Atomic Structure Revealing Protein-Protein Interactions with a Native VapB1 Inhibitor.

Nontypeable Haemophilus influenzae (NTHi) are clinically important Gram-negative bacteria that are responsible for various human mucosal diseases, including otitis media (OM). Recurrent OM caused by NTHi is common, and infections that recur less than 2 weeks following antimicrobial therapy are largely attributable to the recurrence of the same strain of bacteria. Toxin-antitoxin (TA) modules encoded by bacteria enable rapid responses to environmental stresses and are thought to facilitate growth arrest, persistence, and tolerance to antibiotics. The vapBC-1 locus of NTHi encodes a type II TA system, comprising the ribonuclease toxin VapC1 and its cognate antitoxin VapB1. The activity of VapC1 has been linked to the survival of NTHi during antibiotic treatment both in vivo and ex vivo. Therefore, inhibitors of VapC1 might serve as adjuvants to antibiotics, preventing NTHi from entering growth arrest and surviving; however, none have been reported to date. A truncated VapB1 peptide from a crystal structure of the VapBC-1 complex was used to generate pharmacophore queries to facilitate a scaffold hopping approach for the identification of small-molecule VapC1 inhibitors. The National Center for Advancing Translational Sciences small-molecule library was virtually screened using the shape-based method rapid overlay of chemical structures (ROCS), and the top-ranking hits were docked into the VapB1 binding pocket of VapC1. Two hundred virtual screening hits with the best docking scores were selected and tested in a biochemical VapC1 activity assay, which confirmed eight compounds as VapC1 inhibitors. An additional 60 compounds were selected with structural similarities to the confirmed VapC1 inhibitors, of which 20 inhibited VapC1 activity. Intracellular target engagement of five inhibitors was indicated by the destabilization of VapC1 within bacterial cells from a cellular thermal shift assay; however, no impact on bacterial growth was observed. Thus, this virtual screening and scaffold hopping approach enabled the discovery of VapC1 ribonuclease inhibitors that might serve as starting points for preclinical development.

Complex Tumor Spheroids, a Tissue-Mimicking Tumor Model, for Drug Discovery and Precision Medicine.

Malignant tumors are complex tissues composed of malignant cells, vascular cells, structural mesenchymal cells including pericytes and carcinoma-associated fibroblasts, infiltrating immune cells, and others, collectively called the tumor stroma. The number of stromal cells in a tumor is often much greater than the number of malignant cells. The physical associations among all these cell types are critical to tumor growth, survival, and response to therapy. Most cell-based screens for cancer drug discovery and precision medicine validation use malignant cells in isolation as monolayers, embedded in a matrix, or as spheroids in suspension. Medium- and high-throughput screening with multiple cell lines requires a scalable, reproducible, robust cell-based assay. Complex spheroids include malignant cells and two normal cell types, human umbilical vein endothelial cells and highly plastic mesenchymal stem cells, which rapidly adapt to the malignant cell microenvironment. The patient-derived pancreatic adenocarcinoma cell line, K24384-001-R, was used to explore complex spheroid structure and response to anticancer agents in a 96-well format. We describe the development of the complex spheroid assay as well as the growth and structure of complex spheroids over time. Subsequently, we demonstrate successful assay miniaturization to a 384-well format and robust performance in a high-throughput screen. Implementation of the complex spheroid assay was further demonstrated with 10 well-established pancreatic cell lines. By incorporating both human stromal and tumor components, complex spheroids might provide an improved model for tumor response in vivo.

Addressing Compound Reactivity and Aggregation Assay Interferences: Case Studies of Biochemical High-Throughput Screening Campaigns Benefiting from the National Institutes of Health Assay Guidance Manual Guidelines.

Compound-dependent assay interferences represent a continued burden in drug and chemical probe discovery. The open-source National Institutes of Health/National Center for Advancing Translational Sciences (NIH/NCATS) Assay Guidance Manual (AGM) established an "Assay Artifacts and Interferences" section to address different sources of artifacts and interferences in biological assays. In addition to the frequent introduction of new chapters in this important topic area, older chapters are periodically updated by experts from academia, industry, and government to include new technologies and practices. Section chapters describe many best practices for mitigating and identifying compound-dependent assay interferences. Using two previously reported biochemical high-throughput screening campaigns for small-molecule inhibitors of the epigenetic targets Rtt109 and NSD2, the authors review best practices and direct readers to high-yield resources in the AGM and elsewhere for the mitigation and identification of compound-dependent reactivity and aggregation assay interferences.

Interaction of the N terminus of ADP-ribosylation factor with the PH domain of the GTPase-activating protein ASAP1 requires phosphatidylinositol 4,5-bisphosphate.

Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). The mechanistic basis of PIP2-stimulated GAP activity is incompletely understood. Here, we investigated whether PIP2 controls binding of the N-terminal extension of ARF1 to ASAP1's PH domain and thereby regulates its GAP activity. Using [Δ17]ARF1, lacking the N terminus, we found that PIP2 has little effect on ASAP1's activity. A soluble PIP2 analog, dioctanoyl-PIP2 (diC8PIP2), stimulated GAP activity on an N terminus-containing variant, [L8K]ARF1, but only marginally affected activity on [Δ17]ARF1. A peptide comprising residues 2-17 of ARF1 ([2-17]ARF1) inhibited GAP activity, and PIP2-dependently bound to a protein containing the PH domain and a 17-amino acid-long interdomain linker immediately N-terminal to the first ß-strand of the PH domain. Point mutations in either the linker or the C-terminal α-helix of the PH domain decreased [2-17]ARF1 binding and GAP activity. Mutations that reduced ARF1 N-terminal binding to the PH domain also reduced the effect of ASAP1 on cellular actin remodeling. Mutations in the ARF N terminus that reduced binding also reduced GAP activity. We conclude that PIP2 regulates binding of ASAP1's PH domain to the ARF1 N terminus, which may partially regulate GAP activity.

Physiologically relevant orthogonal assays for the discovery of small-molecule modulators of WIP1 phosphatase in high-throughput screens.

WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.

The Opioid Crisis and the Future of Addiction and Pain Therapeutics.

Opioid misuse and addiction are a public health crisis resulting in debilitation, deaths, and significant social and economic impact. Curbing this crisis requires collaboration among academic, government, and industrial partners toward the development of effective nonaddictive pain medications, interventions for opioid overdose, and addiction treatments. A 2-day meeting, The Opioid Crisis and the Future of Addiction and Pain Therapeutics: Opportunities, Tools, and Technologies Symposium, was held at the National Institutes of Health (NIH) to address these concerns and to chart a collaborative path forward. The meeting was supported by the NIH Helping to End Addiction Long-TermSM (HEAL) Initiative, an aggressive, trans-agency effort to speed scientific solutions to stem the national opioid crisis. The event was unique in bringing together two research disciplines, addiction and pain, in order to create a forum for crosscommunication and collaboration. The output from the symposium will be considered by the HEAL Initiative; this article summarizes the scientific presentations and key takeaways. Improved understanding of the etiology of acute and chronic pain will enable the discovery of novel targets and regulatable pain circuits for safe and effective therapeutics, as well as relevant biomarkers to ensure adequate testing in clinical trials. Applications of improved technologies including reagents, assays, model systems, and validated probe compounds will likely increase the delivery of testable hypotheses and therapeutics to enable better health outcomes for patients. The symposium goals were achieved by increasing interdisciplinary collaboration to accelerate solutions for this pressing public health challenge and provide a framework for focused efforts within the research community. SIGNIFICANCE STATEMENT: This article summarizes key messages and discussions resulting from a 2-day symposium focused on challenges and opportunities in developing addiction- and pain-related medications. Speakers and attendees came from 40 states in the United States and 15 countries, bringing perspectives from academia, industry, government, and healthcare by researchers, clinicians, regulatory experts, and patient advocates.

Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection.

Toxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into various types based upon the characteristics of their antitoxins, with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal (PIN) domain containing conserved amino acids considered essential for activity. The vapBC (virulence-associated protein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable Haemophilus influenzae (NTHi). In this study, the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20 Å resolution. Based on this structure, aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of the VapC-1 toxin were constructed and the effects of the mutations on protein-protein interactions, growth of Escherichia coli, and pathogenesis ex vivo were tested. Finally, a novel model system was designed and utilized that consists of an NTHi ΔvapBC-1 strain complemented in cis with the TA module containing a mutated or wild-type toxin at an ectopic site on the chromosome. This enabled the analysis of the effect of PIN domain toxin mutants in tandem with their wild-type antitoxin under the control of the vapBC-1 native promoter and in single copy. This is the first report of a system facilitating the study of TA mutant operons in the background of NTHi during infections of primary human tissues ex vivoIMPORTANCE Herein the crystal structure of the VapBC-1 complex from nontypeable Haemophilus influenzae (NTHi) is described. Our results show that some of the mutations in the PIN domain of the VapC-1 toxin were associated with decreased toxicity in E. coli, but the mutants retained the ability to homodimerize and to heterodimerize with the wild-type cognate antitoxin, VapB-1. A new system was designed and constructed to quantify the effects of these mutations on NTHi survival during infections of primary human tissues ex vivo Any mutation to a conserved amino acid in the PIN domain significantly decreased the number of survivors compared to that of the in cis wild-type toxin under the same conditions.

Assay Guidance Manual: Quantitative Biology and Pharmacology in Preclinical Drug Discovery.

The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a "testing funnel" of assays to identify genuine hits using high-throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists.

A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase.

Assessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment constitutes a major challenge in the pre-clinical drug discovery space. The Cellular Thermal Shift Assay (CETSA) enables such an assessment by quantifying the changes in the thermal stability of proteins upon ligand binding in intact cells. Here, we present the development and validation of a homogeneous, standardized, target-independent, and high-throughput (384- and 1536-well formats) CETSA platform that uses a split Nano Luciferase approach (SplitLuc CETSA). The broad applicability of the assay was demonstrated for diverse targets, and its performance was compared with independent biochemical and cell-based readouts using a set of well-characterized inhibitors. Moreover, we investigated the utility of the platform as a primary assay for high-throughput screening. The SplitLuc CETSA presented here enables target engagement studies for medium and high-throughput applications. Additionally, it provides a rapid assay development and screening platform for targets where phenotypic or other cell-based assays are not readily available.

High-throughput screening with nucleosome substrate identifies small-molecule inhibitors of the human histone lysine methyltransferase NSD2.

The histone lysine methyltransferase nuclear receptor-binding SET domain protein 2 (NSD2, also known as WHSC1/MMSET) is an epigenetic modifier and is thought to play a driving role in oncogenesis. Both NSD2 overexpression and point mutations that increase its catalytic activity are associated with several human cancers. Although NSD2 is an attractive therapeutic target, no potent, selective, and bioactive small molecule inhibitors of NSD2 have been reported to date, possibly due to the challenges of developing high-throughput assays for NSD2. Here, to establish a platform for the discovery and development of selective NSD2 inhibitors, we optimized and implemented multiple assays. We performed quantitative high-throughput screening with full-length WT NSD2 and a nucleosome substrate against a diverse collection of bioactive small molecules comprising 16,251 compounds. We further interrogated 174 inhibitory compounds identified in the primary screen with orthogonal and counter assays and with activity assays based on the clinically relevant NSD2 variants E1099K and T1150A. We selected five confirmed inhibitors for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. We found that all five NSD2 inhibitors bind the catalytic SET domain and one exhibited apparent activity in cells, validating the workflow and providing a template for identifying selective NSD2 inhibitors. In summary, we have established a robust discovery pipeline for identifying potent NSD2 inhibitors from small-molecule libraries.

Irreversible inhibition of cytosolic thioredoxin reductase 1 as a mechanistic basis for anticancer therapy.

Cancer cells adapt to their inherently increased oxidative stress through activation of the glutathione (GSH) and thioredoxin (TXN) systems. Inhibition of both of these systems effectively kills cancer cells, but such broad inhibition of antioxidant activity also kills normal cells, which is highly unwanted in a clinical setting. We therefore evaluated targeting of the TXN pathway alone and, more specifically, selective inhibition of the cytosolic selenocysteine-containing enzyme TXN reductase 1 (TXNRD1). TXNRD1 inhibitors were discovered in a large screening effort and displayed increased specificity compared to pan-TXNRD inhibitors, such as auranofin, that also inhibit the mitochondrial enzyme TXNRD2 and additional targets. For our lead compounds, TXNRD1 inhibition correlated with cancer cell cytotoxicity, and inhibitor-triggered conversion of TXNRD1 from an antioxidant to a pro-oxidant enzyme correlated with corresponding increases in cellular production of H2O2 In mice, the most specific TXNRD1 inhibitor, here described as TXNRD1 inhibitor 1 (TRi-1), impaired growth and viability of human tumor xenografts and syngeneic mouse tumors while having little mitochondrial toxicity and being better tolerated than auranofin. These results display the therapeutic anticancer potential of irreversibly targeting cytosolic TXNRD1 using small molecules and present potent and selective TXNRD1 inhibitors. Given the pronounced up-regulation of TXNRD1 in several metastatic malignancies, it seems worthwhile to further explore the potential benefit of specific irreversible TXNRD1 inhibitors for anticancer therapy.