Points For Drug Discovery Programs Research Articles

X-ray Screening of an Electrophilic Fragment Library and Application toward the Development of a Novel ERK 1/2 Covalent Inhibitor.

Fragment-based drug discovery (FBDD) has become an established method for the identification of efficient starting points for drug discovery programs. In recent years, electrophilic fragment screening has garnered increased attention from both academia and industry to identify novel covalent hits for tool compound or drug development against challenging drug targets. Herein, we describe the design and characterization of an acrylamide-focused electrophilic fragment library and screening campaign against extracellular signal-regulated kinase 2 (ERK2) using high-throughput protein crystallography as the primary hit-finding technology. Several fragments were found to have covalently modified the adenosine triphosphate (ATP) binding pocket Cys166 residue. From these hits, 22, a covalent ATP-competitive inhibitor with improved potency (ERK2 IC50 = 7.8 μM), was developed.

Abstract 1816: Identification of novel VPS4A inhibitors for the treatment of VPS4B-deleted cancers

Abstract Synthetic lethality occurs when a single gene alteration is compatible with cell viability, but an additional co-occurring genetic alteration leads to cell death. In the context of cancer therapy, synthetic lethality can occur through the inhibition of a target that is selectively essential to tumors harboring a specific genetic alteration. Gene paralog pairs represent one promising class of synthetic lethal cancer targets, wherein the function of one paralog is lost in tumor cells, rendering them dependent on the remaining paralog to carry out an essential cellular process. To identify essential gene paralog pairs as starting points for drug discovery programs, we mined publicly available CRISPR genetic loss-of-function data and associated molecular datasets collected across a diverse panel of cancer cell lines. We first identified pairs of gene paralogs where one paralog was essential in a subset of cell lines, and then filtered these genes based on function, known literature, enrichment in specific lineages and integration of external datasets. These efforts identified VPS4A as a synthetic lethal target in cancers harboring copy number loss of VPS4B. VPS4A and VPS4B are highly homologous AAA ATPases that carry out multiple essential cellular processes including nuclear membrane remodeling and endosomal membrane biogenesis. VPS4B loss occurs as a passenger deletion during loss of the tumor suppressors SMAD2 and SMAD4. Loss of VPS4B creates a genetic dependency on VPS4A to drive essential VPS4-dependent processes. VPS4B deletion occurs at a frequency of up to 3% in multiple solid tumor types including esophageal, head and neck, pancreatic and colorectal cancers. To further explore the potential of VPS4A as a therapeutic target in VPS4B-deleted tumors, we first validated the synthetic lethal relationship between VPS4A/B using isogenic cell line pairs. HCT116 cells with an engineered homozygous loss of VPS4B, but not wild-type HCT116 cells, showed profound cell kill in response to genetic silencing of VPS4A. Moreover, simultaneous siRNA-mediated knockdown of VPS4A and VPS4B resulted in cell death across a panel of cancer cell lines (e.g. H1975, Panc0403), while knockdown of either gene alone was compatible with cell viability. Encouraged by these results, we profiled several previously reported small-molecule inhibitors of VPS4A (e.g. DBeQ and MSC1094308) in a suite of biochemical assays. Notably, these molecules were inactive against VPS4A. We have discovered a novel series of VPS4A inhibitors and are advancing this inhibitor series through lead optimization. Potent, selective, and pharmacologically active VPS4A inhibitors are expected to be well tolerated and have strong single-agent activity in tumors bearing VPS4B homozygous deletions. Citation Format: Meredith Kuo, Jason Chen, Sacha Holland, Eugene Lurie, An-Angela Ngoc Van, Francesco Parlati, Tayna Santos, Eric Sjogren, Natalija Sotirovska, Susanne Steggerda, Andrew MacKinnon. Identification of novel VPS4A inhibitors for the treatment of VPS4B-deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1816.

Guided by Evolution: Biology-Oriented Synthesis of Bioactive Compound Classes

Biology-oriented-synthesis (BIOS), is a chemocentric approach to identifying structurally novel molecules as tools for chemical biology and medicinal chemistry research. The vast chemical space cannot be exhaustively covered by synthetic chemistry. Thus, methods which reveal biologically relevant portions of chemical space are of high value. Guided by structural conservation in the evolution of both proteins and natural products, BIOS classifies bioactive compound classes in a hierarchical manner based on molecular architecture and bioactivity. Biologically relevant scaffolds inspire and guide the synthesis of BIOS libraries, which calls for the development of suitable synthetic methodologies. These compound collections have enriched biological relevance, leading to the discovery of bioactive small molecules. These potent and selective modulators allow the study of complex biological pathways and may serve as starting points for drug discovery programs. Thus, BIOS can also be regarded as a hypothesis-generating tool, guiding the design and preparation of novel, bioactive molecular scaffolds. This review elaborates the principles of BIOS and highlights selected examples of their application, which have in turn created future opportunities for the expansion of BIOS and its combination with fragment-based compound discovery for the identification of biologically relevant small molecules with unprecedented molecular scaffolds.1 Introduction2 Structural Classification of Natural Products3 Implications and Opportunities for Biology-Oriented Synthesis4 Applications of Biology-Oriented Synthesis4.1 Chemical Structure and Bioactivity Guided Approaches4.2 Natural-Product-Derived Fragment-Based Approaches5 Conclusions and Outlook

Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease.

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

BioAssay Ontology Annotations Facilitate Cross-Analysis of Diverse High-Throughput Screening Data Sets

High-throughput screening data repositories, such as PubChem, represent valuable resources for the development of small-molecule chemical probes and can serve as entry points for drug discovery programs. Although the loose data format offered by PubChem allows for great flexibility, important annotations, such as the assay format and technologies employed, are not explicitly indexed. The authors have previously developed a BioAssay Ontology (BAO) and curated more than 350 assays with standardized BAO terms. Here they describe the use of BAO annotations to analyze a large set of assays that employ luciferase- and β-lactamase-based technologies. They identified promiscuous chemotypes pertaining to different subcategories of assays and specific mechanisms by which these chemotypes interfere in reporter gene assays. Results show that the data in PubChem can be used to identify promiscuous compounds that interfere nonspecifically with particular technologies. Furthermore, they show that BAO is a valuable toolset for the identification of related assays and for the systematic generation of insights that are beyond the scope of individual assays or screening campaigns.

Fragment-Based Screening by Biochemical Assays: Systematic Feasibility Studies with Trypsin and MMP12

Fragment-based screening (FBS) has gained acceptance in the pharmaceutical industry as an attractive approach for the identification of new chemical starting points for drug discovery programs in addition to classical strategies such as high-throughput screening. There is the concern that screening of fragments at high µM concentrations in biochemical assays results in increased false-positive and false-negative rates. Here the authors systematically compare the data quality of FBS obtained by enzyme activity-based fluorescence intensity, fluorescence lifetime, and mobility shift assays with the data quality from surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) methods. The serine protease trypsin and the matrix metalloprotease MMP12 were selected as model systems. For both studies, 352 fragments were selected each. From the data generated, all 3 biochemical protease assay methods can be used for screening of fragments with low false-negative and low false-positive rates, comparable to those achieved with the SPR-based assays. It can also be concluded that only fragments with a solubility higher than the screening concentration determined by means of NMR should be used for FBS purposes. Extrapolated to 10,000 fragments, the biochemical assays speed up the primary FBS process by approximately a factor of 10 and reduce the protease consumption by approximately 10,000-fold compared to NMR protein observation experiments.

From Fragment Screening to Potent Binders: Strategies for Fragment-to-Lead Evolution

To be an effective medicine a drug has to possess many attributes to ensure target potency and specificity, lack of toxicity, bioavailability and duration of action. Discovering a compound with these properties is invariably an evolutionary process. Fragment based drug discovery sets out to identify a starting compound by screening a library of small molecules representing fragments which cover the chemical space of drug like matter. Fragment based screening is increasingly used in the pharmaceutical industry in the early stages of lead identification and optimization. We will provide an introduction into this approach and discuss a number of examples which show how fragment based drug discovery has been used in the discovery of starting points for drug discovery programs and in their optimization.

Development of a Method for the High‐Throughput Quantification of Cellular Proteins

The quantification of cellular proteins is essential for the study of many different biological processes. This study describes an assay for the detection of the intracellular mutant huntingtin, the causative agent of Huntington's disease, with a method that may be generally applicable to other cellular proteins. A small recombinant protein tag that is recognized by a pair of readily available, high-affinity monoclonal antibodies was designed. This tag was then added to an inducible fragment of the mutant huntingtin protein by genetic engineering. We show that it is possible to use time-resolved FRET to detect low intracellular levels of huntingtin by a simple lysis and detection procedure. This assay was then adapted into a homogeneous, miniaturized format suitable for screening in 1536-well plates. The use of time-resolved FRET also permits the assay to be multiplexed with a standard readout of cell toxicity, thus allowing the identification of conditions causing reduction of protein levels simply due to cytotoxicity. The screening results demonstrated that the assay is able to identify compounds that modulate the levels of huntingtin both positively and negatively and that represent valuable starting points for drug discovery programs.

An Integrated Approach to Fragment-Based Lead Generation:Philosophy, Strategy and Case Studies from AstraZenecas Drug Discovery Programmes

Fragment-based lead generation (FBLG) has recently emerged as an alternative to traditional high throughput screening (HTS) to identify initial chemistry starting points for drug discovery programs. In comparison to HTS screening libraries, the screening sets for FBLG tend to contain orders of magnitude fewer compounds, and the compounds themselves are less structurally complex and have lower molecular weight. This report summarises the advent of FBLG within the industry and then describes the FBLG experience at AstraZeneca. We discuss (1) optimising the design of screening libraries, (2) hit detection methodologies, (3) evaluation of hit quality and use of ligand efficiency calculations, and (4) approaches to evolve fragment-based, low complexity hits towards drug-like leads. Furthermore, we exemplify our use of FBLG with case studies in the following drug discovery areas: antibacterial enzyme targets, GPCRs (melanocortin 4 receptor modulators), prostaglandin D2 synthase inhibitors, phosphatase inhibitors (protein tyrosine phosphotase 1B), and protease inhibitors (b-secretase).

Deconstructing fragment-based inhibitor discovery

Fragment-based screens test multiple low-molecular weight molecules for binding to a target. Fragments often bind with low affinities but typically have better ligand efficiencies (DeltaG(bind)/heavy atom count) than traditional screening hits. This efficiency, combined with accompanying atomic-resolution structures, has made fragments popular starting points for drug discovery programs. Fragment-based design adopts a constructive strategy: affinity is enhanced either by cycles of functional-group addition or by joining two independent fragments together. The final inhibitor is expected to adopt the same geometry as the original fragment hit. Here we consider whether the inverse, deconstructive logic also applies--can one always parse a higher-affinity inhibitor into fragments that recapitulate the binding geometry of the larger molecule? Cocrystal structures of fragments deconstructed from a known beta-lactamase inhibitor suggest that this is not always the case.