Mechanistic safety assessment via multi-omic characterisation of systemic pathway perturbations following in vivo MAT2A inhibition.

The tumour suppressor p16/CDKN2A and the metabolic gene, methyl-thio-adenosine phosphorylase (MTAP), are frequently co-deleted in some of the most aggressive and currently untreatable cancers. Cells with MTAP deletion are vulnerable to inhibition of the metabolic enzyme, methionine-adenosyl transferase 2A (MAT2A), and the protein arginine methyl transferase (PRMT5). This synthetic lethality has paved the way for the rapid development of drugs targeting the MAT2A/PRMT5 axis. MAT2A and its liver- and pancreas-specific isoform, MAT1A, generate the universal methyl donor S-adenosylmethionine (SAM) from ATP and methionine. Given the pleiotropic role SAM plays in methylation of diverse substrates, characterising the extent of SAM depletion and downstream perturbations following MAT2A/MAT1A inhibition (MATi) is critical for safety assessment. We have assessed in vivo target engagement and the resultant systemic phenotype using multi-omic tools to characterise response to a MAT2A inhibitor (AZ'9567). We observed significant SAM depletion and extensive methionine accumulation in the plasma, liver, brain and heart of treated rats, providing the first assessment of both global SAM depletion and evidence of hepatic MAT1A target engagement. An integrative analysis of multi-omic data from liver tissue identified broad perturbations in pathways covering one-carbon metabolism, trans-sulfuration and lipid metabolism. We infer that these pathway-wide perturbations represent adaptive responses to SAM depletion and confer a risk of oxidative stress, hepatic steatosis and an associated disturbance in plasma and cellular lipid homeostasis. The alterations also explain the dramatic increase in plasma and tissue methionine, which could be used as a safety and PD biomarker going forward to the clinic.

Development of a Series of Pyrrolopyridone MAT2A Inhibitors.

The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 (21), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.

A patent review of MAT2a inhibitors (2018-2021).

INTRODUCTION: In methylthioadenosine phosphorylase (MTAP)-deficient tumor cells, reduced S-adenosylmethionine (SAM) levels in the context of elevated methylthioadenosine (MTA) has been hypothesized to lead to inhibition of protein arginine methyltransferase 5 (PRMT5) and tumor growth inhibition. Inhibitors of methionine adenosyltransferase 2A (MAT2a) prevent the synthesis of SAM from methionine and have therefore attracted increasing attention as potential chemotherapeutic agents in cancers characterized by MTAP-loss. AREAS COVERED: This review covers patent applications between January 2018 and December 2021. 18 patent applications from 5 different applicants are evaluated. EXPERT OPINION: Recent advances in the field show a significant interest in the MAT2a therapeutic hypothesis. Agios and Ideaya in particular have capitalized on an allosteric binding mode first published by Pfizer in at least two of the filings during this time period, leading to potent, selective inhibitors. They have advanced MAT2a inhibitors to phase I clinical studies to explore their benefit to patients suffering with MTAP-deficient solid tumors or lymphoma. Whilst the other patent disclosures during this time frame have not led to disclosed candidates, the trials initiated by Agios and Ideaya studies will clearly inform on the potential for such inhibitors as viable therapeutic agents either as single agent or in combination.

Design, Synthesis, and Characterization of I-BET567, a Pan-Bromodomain and Extra Terminal (BET) Bromodomain Oral Candidate.

Through regulation of the epigenome, the bromodomain and extra terminal (BET) family of proteins represent important therapeutic targets for the treatment of human disease. Through mimicking the endogenous N-acetyl-lysine group and disrupting the protein-protein interaction between histone tails and the bromodomain, several small molecule pan-BET inhibitors have progressed to oncology clinical trials. This work describes the medicinal chemistry strategy and execution to deliver an orally bioavailable tetrahydroquinoline (THQ) pan-BET candidate. Critical to the success of this endeavor was a potency agnostic analysis of a data set of 1999 THQ BET inhibitors within the GSK collection which enabled identification of appropriate lipophilicity space to deliver compounds with a higher probability of desired oral candidate quality properties. SAR knowledge was leveraged via Free-Wilson analysis within this design space to identify a small group of targets which ultimately delivered I-BET567 (27), a pan-BET candidate inhibitor that demonstrated efficacy in mouse models of oncology and inflammation.

Fragment-based Scaffold Hopping: Identification of Potent, Selective, and Highly Soluble Bromo and Extra Terminal Domain (BET) Second Bromodomain (BD2) Inhibitors.

The profound efficacy of pan-BET inhibitors is well documented, but these epigenetic agents have shown pharmacology-driven toxicity in oncology clinical trials. The opportunity to identify inhibitors with an improved safety profile by selective targeting of a subset of the eight bromodomains of the BET family has triggered extensive medicinal chemistry efforts. In this article, we disclose the identification of potent and selective drug-like pan-BD2 inhibitors such as pyrazole 23 (GSK809) and furan 24 (GSK743) that were derived from the pyrrole fragment 6. We transpose the key learnings from a previous pyridone series (GSK620 2 as a representative example) to this novel class of inhibitors, which are characterized by significantly improved solubility relative to our previous research.

GSK973 Is an Inhibitor of the Second Bromodomains (BD2s) of the Bromodomain and Extra-Terminal (BET) Family.

Pan-BET inhibitors have shown profound efficacy in a number of in vivo preclinical models and have entered the clinic in oncology trials where adverse events have been reported. These inhibitors interact equipotently with the eight bromodomains of the BET family of proteins. To better understand the contribution of each domain to their efficacy and to improve from their safety profile, selective inhibitors are required. This Letter discloses the profile of GSK973, a highly selective inhibitor of the second bromodomains of the BET proteins that has undergone extensive preclinical in vitro and in vivo characterization.

Selective targeting of BD1 and BD2 of the BET proteins in cancer and immunoinflammation.

The two tandem bromodomains of the BET (bromodomain and extraterminal domain) proteins enable chromatin binding to facilitate transcription. Drugs that inhibit both bromodomains equally have shown efficacy in certain malignant and inflammatory conditions. To explore the individual functional contributions of the first (BD1) and second (BD2) bromodomains in biology and therapy, we developed selective BD1 and BD2 inhibitors. We found that steady-state gene expression primarily requires BD1, whereas the rapid increase of gene expression induced by inflammatory stimuli requires both BD1 and BD2 of all BET proteins. BD1 inhibitors phenocopied the effects of pan-BET inhibitors in cancer models, whereas BD2 inhibitors were predominantly effective in models of inflammatory and autoimmune disease. These insights into the differential requirement of BD1 and BD2 for the maintenance and induction of gene expression may guide future BET-targeted therapies.

Discovery of Tetrahydroquinoxalines as Bromodomain and Extra-Terminal Domain (BET) Inhibitors with Selectivity for the Second Bromodomain.

The bromodomain and extra-terminal domain (BET) family of proteins bind acetylated lysine residues on histone proteins. The four BET bromodomains-BRD2, BRD3, BRD4, and BRDT-each contain two bromodomain modules. BET bromodomain inhibition is a potential therapy for various cancers and immunoinflammatory diseases, but few reported inhibitors show selectivity within the BET family. Inhibitors with selectivity for the first or second bromodomain are desired to aid investigation of the biological function of these domains. Focused library screening identified a series of tetrahydroquinoxalines with selectivity for the second bromodomains of the BET family (BD2). Structure-guided optimization of the template improved potency, selectivity, and physicochemical properties, culminating in potent BET inhibitors with BD2 selectivity.

Potent and Selective Inhibitors of Histone Deacetylase-3 Containing Chiral Oxazoline Capping Groups and a N-(2-Aminophenyl)-benzamide Binding Unit.

A novel series of potent chiral inhibitors of histone deacetylase (HDAC) is described that contains an oxazoline capping group and a N-(2-aminophenyl)-benzamide unit. Among several new inhibitors of this type exhibiting Class I selectivity and potent inhibition of HDAC3-NCoR2, in vitro assays for the inhibition of HDAC1, HDAC2, and HDAC3-NCoR2 by N-(2-aminophenyl)-benzamide 15k gave respective IC50 values of 80, 110, and 6 nM. Weak inhibition of all other HDAC isoforms (HDAC4, 5, 6, 7, and 9: IC50 > 100 000 nM; HDAC8: IC50 = 25 000 nM; HDAC10: IC50 > 4000 nM; HDAC11: IC50 > 2000 nM) confirmed the Class I selectivity of 15k. 2-Aminoimidazolinyl, 2-thioimidazolinyl, and 2-aminooxazolinyl units were shown to be effective replacements for the pyrimidine ring present in many other 2-(aminophenyl)-benzamides previously reported, but the 2-aminooxazolinyl unit was the most potent in inhibiting HDAC3-NCoR2. Many of the new HDAC inhibitors showed higher solubilities and lower binding to human serum albumin than that of Mocetinostat. Increases in histone H3K9 acetylation in the human cell lines U937 and PC-3 was observed for all three oxazolinyl inhibitors evaluated; those HDAC inhibitors also lowered cyclin E expression in U937 cells but not in PC-3 cells, indicating underlying differences in the mechanisms of action of the inhibitors on those two cell lines.

Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation.

PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.

Discovery of potent, isoform-selective inhibitors of histone deacetylase containing chiral heterocyclic capping groups and a N-(2-aminophenyl)benzamide binding unit.

The synthesis of a novel series of potent chiral inhibitors of histone deacetylase (HDAC) is described that contain a heterocyclic capping group and a N-(2-aminophenyl)benzamide unit that binds in the active site. In vitro assays for the inhibition of HDAC1, HDAC2, HDAC3-NCoR1, and HDAC8 by the N-(2-aminophenyl)benzamide 24a gave respective IC50 values of 930, 85, 12, and 4100 nM, exhibiting class I selectivity and potent inhibition of HDAC3-NCoR1. Both imidazolinone and thiazoline rings are shown to be effective replacements for the pyrimidine ring present in many other 2-(aminophenyl)benzamides previously reported, an example of each ring system at 1 µM causing an increase in histone H3K9 acetylation in the human cell lines Jurkat and HeLa and an increase in cell death consistent with induction of apoptosis. Inhibition of the growth of MCF-7, A549, DU145, and HCT116 cell lines by 24a was observed, with respective IC50 values of 5.4, 5.8, 6.4, and 2.2 mM.

Total synthesis of aplyronine C.

A highly stereocontrolled total synthesis of the cytotoxic marine macrolide aplyronine C is described. The route exploits aldol methodology to install the requisite stereochemistry and features a crucial boron-mediated aldol coupling of an N-vinylformamide-bearing methyl ketone with a macrocyclic aldehyde to introduce the full side chain. The synthesis of two novel C21-C34 side chain analogs is also reported.