The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-β-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.

Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (S N 2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MSbased method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-L-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13 C KIE of 1.04, an inverse intrinsic α-secondary CD 3 KIE of 0.90, and a small but statistically significant inverse CD 3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical S N 2 transition state with the C-N and C-S distances of 2.35-2.40 Å and 2.00-2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steadystate kinetics with the SAM analog Se-adenosyl-L-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.S tepwise progression of an enzyme-catalyzed chemical reaction is accompanied by changes of bond orders and vibrational modes involved with specific atoms of the reactant(s) (1, 2). Such changes can be traced experimentally by measuring the ratios of turnover rates [kinetic isotope effects (KIEs)] or binding affinities [binding isotope effects (BIEs)] of the reactant(s) when the relevant atoms are replaced by heavy isotopes (3, 4). KIEs and BIEs are thus useful parameters for elucidating transition-state (TS) structures and catalytic mechanisms, which sometimes cannot be elucidated readily through sole measurement of steady-state kinetics (5-9). A sufficient set of KIEs and BIEs at the positions involved with bond motions can afford electrostatic and geometric constraints, when combined with computational modeling, to define an enzymatic TS (10-12). This information provides not only the atomic resolution of the transient structure at the highest energy summit along the reaction path, but also structural guidance for designing tight-binding TS analog inhibitors (13...

Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the ␤-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acylenzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices ␣5 and ␣6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100 -200 g/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton.

Blocking the activity of the programmed cell death protein 1 (PD-1) inhibitory receptor with therapeutic antibodies against either the ligand (PD-L1) or PD-1 itself has proven to be an effective treatment modality for multiple cancers. Contrasting with antibodies, small molecules could demonstrate increased tissue penetration, distinct pharmacology and potentially enhanced antitumor activity. Here, we describe the identification and characterization of INCB086550, a novel, oral, small-molecule PD-L1 inhibitor. In vitro, INCB086550 selectively and potently blocked the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, and induced stimulation-dependent cytokine production in primary human immune cells. In vivo, INCB086550 reduced tumor growth in CD34+ humanized mice and induced T-cell activation gene signatures, consistent with PD-L1/PD-1 pathway blockade. Preliminary data from an ongoing phase 1 study confirmed PD-L1/PD-1 blockade in peripheral blood cells, with increased immune activation and tumor growth control. These data support continued clinical evaluation of INCB086550 as an alternative to antibody-based therapies.

Drug-target kinetics enable time-dependent changes in target engagement to be quantified as a function of drug concentration. When coupled to drug pharmacokinetics (PK), drug-target kinetics can thus be used to predict in vivo pharmacodynamics (PD). Previously we described a mechanistic PK/PD model that successfully predicted the antibacterial activity of an LpxC inhibitor in a model of Pseudomonas aeruginosa infection. In the present work we demonstrate that the same approach can be used to predict the in vivo activity of an enoyl-ACP reductase (FabI) inhibitor in a model of methicillin-resistant Staphylococcus aureus (MRSA) infection. This is significant because the LpxC inhibitors are cidal, whereas the FabI inhibitors are static. In addition P. aeruginosa is a Gram-negative organism whereas MRSA is Gram-positive. Thus this study supports the general applicability of our modeling approach across antibacterial space.

Salmonellae are pathogenic bacteria that induce immunosuppression by mechanisms that remain largely unknown. Previously, we showed that a putative type II l-asparaginase produced by Salmonella Typhimurium inhibits T cell responses and mediates virulence in a murine model of infection. Here, we report that this putative L-asparaginase exhibits L-asparagine hydrolase activity required for Salmonella Typhimurium to inhibit T cells. We show that L-asparagine is a nutrient important for T cell activation and that L-asparagine deprivation, such as that mediated by the Salmonella Typhimurium L-asparaginase, causes suppression of activation-induced mammalian target of rapamycin signaling, autophagy, Myc expression, and L-lactate secretion. We also show that L-asparagine deprivation mediated by the Salmonella Typhimurium L-asparaginase causes suppression of cellular processes and pathways involved in protein synthesis, metabolism, and immune response. Our results advance knowledge of a mechanism used by Salmonella Typhimurium to inhibit T cell responses and mediate virulence, and provide new insights into the prerequisites of T cell activation. We propose a model in which l-asparagine deprivation inhibits T cell exit from quiescence by causing suppression of activation-induced metabolic reprogramming.

No abstract

Resveratrol is a natural polyphenol with plethora of biological activities. Resveratrol has previously shown to decrease DNA methyltransferase (DNMT) enzymes expression and to reactivate silenced tumor suppressor genes. Currently, it seems that no resveratrol analogues have been developed as DNMT inhibitors. Recently, we reported the synthesis of resveratrol-salicylate derivatives and by examining the chemical structure of these analogues, we proposed that these compounds could exhibit DNMT inhibition especially that they resembled NSC 14778, a compound we previously identified as DNMT inhibitor by virtual screening. Indeed, using in vitro DNMT inhibition assay, some of the resveratrol-salicylate analogues we screened in this work showed selective inhibition against DNMT3 enzymes which was greater than resveratrol. A molecular docking study revealed key binding interactions with DNMT3A and DNMT3B enzymes. Additionally, the most active analogues, 10 showed considerable cytotoxicity against three human cancer cells; HT-29, HepG2 and SK-BR-3 which was greater than resveratrol. Further studies are needed to understand the anticancer mechanisms of these derivatives.