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Osimertinib is a covalent, third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) approved for treating non-small cell lung cancer patients with activating EGFR mutations (Exon19del or L858R) or with the T790M resistance mutation following disease progression on first- or second-generation EGFR TKIs. The aim of this work is to rationalize and understand how osimertinib achieves mutant EGFR selectivity over the wild-type (WT) by evaluating its kinetic mechanism of action. In doing so, we developed methodologies combining steady-state and pre-steady-state kinetics to determine the covalent inactivation rates (kinact) and reversible binding affinities (Ki) for osimertinib against WT, L858R, and L858R/T790M EGFR and compared these data to the inhibition kinetics of earlier generations of EGFR TKIs. The kinact/KI values indicate osimertinib inactivates L858R and L858R/T790M with 20- and 50-fold higher overall efficiencies, respectively, compared to that for WT. The Ki and kinact values reveal that osimertinib binds 3-fold tighter to and reacts 3-fold faster with L858R than WT EGFR and binds 17-fold tighter to and reacts 3-fold faster with L858R/T790M than with the WT EGFR. We conclude that osimertinib overcomes the T790M mutation through improved affinities from stronger hydrophobic interactions with Met790 versus Thr790 and an improved rate of covalent bond formation via better positioning of the acrylamide warhead, while osimertinib targets the L858R mutation through better affinities and reactivities with the mutant in the context of differential binding affinities of the competing substrate ATP. This work highlights the importance of optimizing both reversible drug-target interactions and inactivation rates for covalent inhibitors to achieve selectivity in targeting mutant EGFRs.

Methionine adenosyltransferase (MAT) catalyzes the adenosine 5'-triphosphate (ATP) and l-methionine (l-Met) dependent formation of S-adenosyl-l-methionine (SAM), the principal methyl donor of most biological transmethylation reactions. We carried out in-depth kinetic studies to further understand its mechanism and interaction with a potential regulator, Mat2B. The initial velocity pattern and results of product inhibition by SAM, phosphate, and pyrophosphate, and dead-end inhibition by the l-Met analog cycloleucine (l-cLeu) suggest that Mat2A follows a strictly ordered kinetic mechanism where ATP binds before l-Met and with SAM released prior to random release of phosphate and pyrophosphate. Isothermal titration calorimetry (ITC) showed binding of ATP to Mat2A with a Kd of 80 ± 30 μM, which is close to the Km(ATP) of 50 ± 10 μM. In contrast, l-Met or l-cLeu showed no binding to Mat2A in the absence of ATP; however, binding to l-cLeu was observed in the presence of ATP. The ITC results are fully consistent with the product and dead-inhibition results obtained. We also carried out kinetic studies in the presence of the physiological regulator Mat2B. Under conditions where all Mat2A is found in complex with Mat2B, no significant change in the kinetic parameters was observed despite confirmation of a very high binding affinity of Mat2A to Mat2B (Kd of 6 ± 1 nM). Finally, we found that while Mat2A is unstable at low concentrations (

Tuberculosis is the leading cause of death worldwide from infectious diseases. With the development of drug-resistant strains of Mycobacterium tuberculosis, there is an acute need for new medicines with novel modes of action. Herein, we report the discovery and profiling of a novel hydantoin-based family of antimycobacterial inhibitors of the decaprenylphospho-β-d-ribofuranose 2-oxidase (DprE1). In this study, we have prepared a library of more than a 100 compounds and evaluated them for their biological and physicochemical properties. The series is characterized by high enzymatic and whole-cell activity, low cytotoxicity, and a good overall physicochemical profile. In addition, we show that the series acts via reversible inhibition of the DprE1 enzyme. Overall, the novel compound family forms an attractive base for progression to further stages of optimization and may provide a promising drug candidate in the future.

The biosynthetic pathway of peptidoglycan is essential for Mycobacterium tuberculosis. We report here the acetyltransferase substrate specificity and catalytic mechanism of the bifunctional N-acetyltransferase/uridylyltransferase from M. tuberculosis (GlmU). This enzyme is responsible for the final two steps of the synthesis of UDP- N-acetylglucosamine, which is an essential precursor of peptidoglycan, from glucosamine 1-phosphate, acetyl-coenzyme A, and uridine 5'-triphosphate. GlmU utilizes ternary complex formation to transfer an acetyl from acetyl-coenzyme A to glucosamine 1-phosphate to form N-acetylglucosamine 1-phosphate. Steady-state kinetic studies and equilibrium binding experiments indicate that GlmU follows a steady-state ordered kinetic mechanism, with acetyl-coenzyme A binding first, which triggers a conformational change in GlmU, followed by glucosamine 1-phosphate binding. Coenzyme A is the last product to dissociate. Chemistry is partially rate-limiting as indicated by pH-rate studies and solvent kinetic isotope effects. A novel crystal structure of a mimic of the Michaelis complex, with glucose 1-phosphate and acetyl-coenzyme A, helps us to propose the residues involved in deprotonation of glucosamine 1-phosphate and the loop movement that likely generates the active site required for glucosamine 1-phosphate to bind. Together, these results pave the way for the rational discovery of improved inhibitors against M. tuberculosis GlmU, some of which might become candidates for antibiotic discovery programs.

We have targeted the Mycobacterium tuberculosis decaprenylphosphoryl-β-d-ribose oxidase (Mt-DprE1) for potential chemotherapeutic intervention of tuberculosis. A multicopy suppression strategy that overexpressed Mt-DprE1 in M. bovis BCG was used to profile the publically available GlaxoSmithKline antimycobacterial compound set, and one compound (GSK710) was identified that showed an 8-fold higher minimum inhibitory concentration relative to the control strain. Analogues of GSK710 show a clear relationship between whole cell potency and in vitro activity using an enzymatic assay employing recombinant Mt-DprE1, with binding affinity measured by fluorescence quenching of the flavin cofactor of the enzyme. M. bovis BCG spontaneous resistant mutants to GSK710 and a closely related analogue were isolated and sequencing of ten such mutants revealed a single point mutation at two sites, E221Q or G248S within DprE1, providing further evidence that DprE1 is the main target of these compounds. Finally, time-lapse microscopy experiments showed that exposure of M. tuberculosis to a compound of this series arrests bacterial growth rapidly followed by a slower cytolysis phase.

Expression of mutant Huntingtin (HTT) protein is central to the pathophysiology of Huntington's Disease (HD). The E3 ubiquitin ligase MID1 appears to have a key role in facilitating translation of the mutant HTT mRNA suggesting that interference with the function of this complex could be an attractive therapeutic approach. Here we describe a peptide that is able to disrupt the interaction between MID1 and the α4 protein, a regulatory subunit of protein phosphatase 2A (PP2A). By fusing this peptide to a sequence from the HIV-TAT protein we demonstrate that the peptide can disrupt the interaction within cells and show that this results in a decrease in levels of ribosomal S6 phosphorylation and HTT expression in cultures of cerebellar granule neurones derived from HdhQ111/Q7 mice. This data serves to validate this pathway and paves the way for the discovery of small molecule inhibitors of this interaction as potential therapies for HD.

Summary Deregulation of the PRC2 complex, comprised of the core subunits EZH2, SUZ12, and EED, drives aberrant hypermethylation of H3K27 and tumorigenicity of many cancers. Although inhibitors of EZH2 have shown promising clinical activity, preclinical data suggest that resistance can be acquired through secondary mutations in EZH2 that abrogate drug target engagement. To address these limitations, we have designed several hetero-bifunctional PROTACs (proteolysis-targeting chimera) to efficiently target EED for elimination. Our PROTACs bind to EED (pKD ∼ 9.0) and promote ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC50 ∼ 8.1) and induce rapid degradation of not only EED but also EZH2 and SUZ12 within the PRC2 complex. Furthermore, the PROTACs selectively inhibit proliferation of PRC2-dependent cancer cells (half maximal growth inhibition [GI50] = 49–58 nM). In summary, our data demonstrate a therapeutic modality to target PRC2-dependent cancer through a PROTAC-mediated degradation mechanism.

Deregulation of the PRC2 components EZH2, SUZ12, and EED plays critical roles in driving aberrant hypermethylation of H3K27 and tumorigenicity of many solid and hematological malignancies. Although SAM competitive small molecule inhibitors of EZH2 show promising clinical activity in PRC2-dependent cancers, preclinical data suggests that resistance can be acquired through secondary mutations in EZH2 that abrogate drug target engagement. To address these limitations, we have designed several hetero-bifunctional PROTACs (Proteolysis Targeting Chimera) to efficiently target EED for elimination. Our EED-targeting PROTACs bind to EED (pKD= 9.02-9.27) and promote stable ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC50= 8.11-8.17) that results in a decrease in H3K27me3 levels in cells. Interestingly, EED-targeting PROTACs induce rapid degradation of EED, as well as its associated proteins, including EZH2 and SUZ12 in the PRC2 complex. Inhibition of the ubiquitin proteasome pathway abrogates PROTAC-mediated degradation of EED and its associated proteins. Furthermore, the EED targeting PROTACs selectively inhibit proliferation and survival of PRC2-dependent cancer cells (GI50= 49-58 nM). In summary, our data demonstrate a novel therapeutic modality in treating PRC2 dependent cancer through PROTAC mediated platform. Citation Format: Jessie Hao-Ru Hsu, Timothy Rasmusson, James Robinson, Fiona Pachl, Jon Read, Sameer Kawatkar, Daniel H O'Donovan, Sharan Bagal, Erin Code, Philip Rawlins, Argyrides Argyrou, Ronald Tomlinson, Ning Gao, Xiahui Zhu, Elisabetta Chiarparin, Kelly Jacques, Minhui Shen, Haley Woods, Emma Bednarski, David M. Wilson, Lisa Drew, M. Paola Castaldi, Stephen Fawell, Andrew Bloecher. EED targeted PROTACs degrade EED, EZH2, and SUZ12 in the PRC2 complex [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A09. doi:10.1158/1535-7163.TARG-19-LB-A09

Significance Better understanding of the mechanisms used by bacteria to counter antibacterial agents is essential to cope with the rising prevalence of antimicrobial resistance. Here, we identified the mechanism of resistance of Mycobacterium tuberculosis to an antimycobacterial cyano-substituted fused pyrido-benzimidazole. Clones bearing mutations in a transcription factor, Rv2887, markedly up-regulated the expression of rv0560c , a putative methyltransferase. Rv0560c N -methylated the pyrido-benzimidazole in vitro and in Mycobacterium tuberculosis , abrogating its bactericidal activity. Resistant mutants selected in the absence of rv0560c led to the identification of the target of the compound, the essential oxidoreductase, decaprenylphosphoryl-β- d -ribose 2-oxidase (DprE1). Methylation of an antibacterial compound is a previously uncharacterized mode of antimicrobial resistance.

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis., Screens for bactericidal compounds have resulted in promising anti-tubercular hits. Here, the authors analyse in detail the target of an indazole sulfonamide (GSK3011724A), and find that it has a different mode of inhibition compared to other Kas inhibitors of fatty acid biosynthesis in bacteria.