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A hallmark of Mycobacterium tuberculosis (M. tb), the aetiologic agent of tuberculosis, is its ability to metabolise host-derived lipids. However, the enzymes and mechanisms underlying such metabolism are still largely unknown. We previously reported that the Cyclophostin & Cyclipostins (CyC) analogues, a new family of potent antimycobacterial molecules, react specifically and covalently with (Ser/Cys)-based enzymes mostly involved in bacterial lipid metabolism. Here, we report the synthesis of new CyC alkyne-containing inhibitors (CyC yne ) and their use for the direct fishing of target proteins in M. tb culture via bio-orthogonal click-chemistry activity-based protein profiling (CC-ABPP). This approach led to the capture and identification of a variety of enzymes, and many of them involved in lipid or steroid metabolisms. One of the captured enzymes, HsaD (Rv3569c), is required for the survival of M. tb within macrophages and is thus a potential therapeutic target. This prompted us to further explore and validate, through a combination of biochemical and structural approaches, the specificity of HsaD inhibition by the CyC analogues. We confirmed that the CyC bind covalently to the catalytic Ser 114 residue, leading to a total loss of enzyme activity. These data were supported by the X-ray structures of four HsaD-CyC complexes, obtained at resolutions between 1.6 and 2.6 Å. The identification of mycobacterial enzymes directly captured by the CyC yne probes through CC-ABPP paves the way to better understand and potentially target key players at crucial stages of the bacilli life cycle., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Patients with cystic fibrosis (CF) have a significantly higher risk of acquiring nontuberculous mycobacteria infections, predominantly due to Mycobacterium abscessus , than the healthy population. Because M. abscessus infections are a major cause of clinical decline and morbidity in CF patients, improving treatment and the detection of this mycobacterium in the context of a polymicrobial culture represents a critical component to better manage patient care. We report here the synthesis of fluorescent Dansyl derivatives of four active cyclipostins and cyclophostin analogues ( CyCs ) and provide new insights regarding the CyC 's lack of activity against Gram-negative and Gram-positive bacteria, and above all into their mode of action against intramacrophagic M. abscessus cells. Our results pointed out that the intracellularly active CyC accumulate in acidic compartments within macrophage cells, that this accumulation appears to be essential for their delivery to mycobacteria-containing phagosomes, and consequently, for their antimicrobial effect against intracellular replicating M. abscessus , and that modification of such intracellular localization via disruption of endolysosomal pH strongly affects the CyC accumulation and efficacy. Moreover, we discovered that these fluorescent compounds could become efficient probes to specifically label mycobacterial species with high sensitivity, including M. abscessus in the presence several other pathogens like Pseudomonas aeruginosa and Staphylococcus aureus . Collectively, all present and previous data emphasized the therapeutic potential of unlabeled CyC s and the attractiveness of the fluorescent CyC as a potential new efficient diagnostic tool to be exploited in future diagnostic developments against mycobacterial-related infections, especially against M. abscessus .

Compound VBT-5445 was identified as an inhibitor to block the association of Pim and the protein Enhancer of Decapping 3 (EDC3), a Pim substrate, which normally functions to enhance the decapping of messenger RNA (mRNA). It was also shown to inhibit both the Pim and mTORC protein kinases. The activity of this compound class can be fine-tuned by structural modification. A series of VBT analogs were designed, synthesized, and evaluated. These compounds decrease the growth of multiple cancer types, including pancreas, prostate, breast, lung, and leukemia. Notably, 6-methyl (GRG-1-31, 6d ), 4-Bromo (GRG-1-34, 6e ), 4-Chloro (GRG-1-35, 6f ), and phenylthio substituted (GRG-1-104, 6n ) derivatives are highly potent at inhibiting tumor growth. The ability of these compounds to block cancer growth in vitro is highly correlated with their activity as mTORC inhibitors. The toxicity of GRG 1-34 is low in mice treated with twice-daily gavage for 30 days and did not induce weight loss. Pharmacokinetics of a single oral dose demonstrated a peak concentration at 0.5 hours after gavage. In summary, further development of this compound class has the potential to inhibit important signaling pathways and impact cancer treatment., Competing Interests: Conflict of interest – The authors declare that they have no conflict of interest.

We investigate the gas-phase structures and fragmentation pathways of model compounds of anthracene derivatives of the general formula C c H h N 1 utilizing tandem mass spectrometry and computational methods. We vary the substituent alkyl chain length, composition, and degree of branching. We find substantial experimental and theoretical differences between the linear and branched congeners in terms of fragmentation thresholds, available pathways, and distribution of products. Our calculations predict that the linear substituents initially isomerize to form lower energy branched isomers prior to loss of the alkyl substituents as alkenes. The rate-determining chemistry underlying these related processes is dominated by the ability to stabilize the alkene loss transition structures. This task is more effectively undertaken by branched substituents. Consequently, analyte lability systematically increased with degree of branching (linear < secondary < tertiary). The resulting anthracen-9-ylmethaniminium ion generated from these alkene loss reactions undergoes rate-limiting proton transfer to enable expulsion of either hydrogen cyanide or CNH. The combination of the differences in primary fragmentation thresholds and degree of radical-based fragmentation processes provide a potential means of distinguishing compounds that contain branched alkyl chain substituents from those with linear ones.

The reaction of epoxy aldehydes with tetraethyl methylenediphosphonate gave γ,δ-epoxy vinyl phosphonates. The palladium-catalyzed addition of primary alcohols gave the monoprotected diols as single diastereoisomers. The trans- and cis-epoxides lead to opposite ( syn and anti) diastereoisomers of the addition products. The alkene of the vinyl phosphonates was subjected to hydrogenation, and the resulting saturated phosphonates underwent base-catalyzed cyclization to give phostones with a very high diastereoselectivity in the formation of the new chiral center at the phosphorus atom.

An increasing prevalence of cases of drug-resistant tuberculosis requires the development of more efficacious chemotherapies. We previously reported the discovery of a new class of cyclipostins and cyclophostin (CyC) analogs exhibiting potent activity against Mycobacterium tuberculosis both in vitro and in infected macrophages. Competitive labeling/enrichment assays combined with MS have identified several serine or cysteine enzymes in lipid and cell wall metabolism as putative targets of these CyC compounds. These targets included members of the antigen 85 (Ag85) complex ( i.e. Ag85A, Ag85B, and Ag85C), responsible for biosynthesis of trehalose dimycolate and mycolylation of arabinogalactan. Herein, we used biochemical and structural approaches to validate the Ag85 complex as a pharmacological target of the CyC analogs. We found that CyC 7β , CyC 8β , and CyC 17 bind covalently to the catalytic Ser 124 residue in Ag85C; inhibit mycolyltransferase activity ( i.e. the transfer of a fatty acid molecule onto trehalose); and reduce triacylglycerol synthase activity, a property previously attributed to Ag85A. Supporting these results, an X-ray structure of Ag85C in complex with CyC 8β disclosed that this inhibitor occupies Ag85C's substrate-binding pocket. Importantly, metabolic labeling of M. tuberculosis cultures revealed that the CyC compounds impair both trehalose dimycolate synthesis and mycolylation of arabinogalactan. Overall, our study provides compelling evidence that CyC analogs can inhibit the activity of the Ag85 complex in vitro and in mycobacteria, opening the door to a new strategy for inhibiting Ag85. The high-resolution crystal structure obtained will further guide the rational optimization of new CyC scaffolds with greater specificity and potency against M. tuberculosis ., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

A new class of Cyclophostin and Cyclipostins (CyC) analogs have been investigated against Mycobacterium tuberculosis H37Rv (M. tb) grown either in broth medium or inside macrophages. Our compounds displayed a diversity of action by acting either on extracellular M. tb bacterial growth only, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth with very low toxicity towards host macrophages. Among the eight potential CyCs identified, CyC 17 exhibited the best extracellular antitubercular activity (MIC 50  = 500 nM). This compound was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 23 potential candidates, most of them being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA and HsaD, have previously been reported as essential for in vitro growth of M. tb and/or survival and persistence in macrophages. Overall, our findings support the assumption that CyC 17 may thus represent a novel class of multi-target inhibitor leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes participating in important physiological processes.