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(10, 11) In the proposed mechanism, the serine hydroxyl group from the bacterial SPase attacks the peptide substrate from the si-face rather than the re-face as seen in eukaryotic SPase. Eukaryotic SPases utilize a catalytic triad composed for Ser-His-Asp residues, whereas bacterial SPases I use a unique Ser-Lys catalytic dyad mechanism. (4-8) Inhibition of LepB would also interfere with the translocation of proteins critical for various cellular processes and could ultimately lead to cell death.īacterial SPases are membrane-bound endopeptidases belonging to the serine protease family S26 (9) and are structurally and mechanistically distinct from their eukaryotic counterparts. (2) Inhibiting LepB would prevent cleavage of the signal peptide from the preprotein consequently, the proteins destined to be secreted would remain membrane bound.
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(3) M. tuberculosis has a single LepB homologue, which is essential for cell viability. LepB catalyzes the cleavage of the N-terminal signal peptide from preproteins during or shortly after translocation, releasing the mature protein into the extracellular space. (3) The Sec pathway is highly conserved in bacteria and is the primary route involved in the export of proteins across the cytoplasmic membrane. (2) Approximately 20% of all bacterial proteins synthesized are secreted, and they play vital roles in numerous processes, including nutrient uptake, pathogenicity, environmental response, resuscitation, cell wall biogenesis, and respiration. The type I signal peptidase (SPase I), also known as the leader peptidase (LepB), is a key enzyme involved in protein secretion via the general secretion (Sec) pathway and is a potential drug target for tuberculosis. Ideally, new drugs should target essential pathways in M. tuberculosis that are not currently targeted by first- and second-line drugs. Consequently, there is an urgent need for the development of new antitubercular therapies that are effective against resistant as well as persistent forms of tuberculosis. (1) Resistant strains are not susceptible to the standard drugs, and although MDR-TB is treatable using second-line drugs, such treatments are costly, toxic, and/or not readily available.
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According to the World Health Organization (WHO), approximately 480,000 people developed MDR-TB in 2014 and the cure rate of those patients was only 50%. (1) Although the mortality rate has dropped by 47% since the 1990s, the emergence of multidrug resistant (MDR-TB) and extremely drug resistant (XDR-TB) strains has complicated our ability to control the disease. In 2014, 9.6 million patients were diagnosed with TB infection and ∼1.5 million people died. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), has plagued mankind for centuries and is one of the world’s deadliest infectious diseases. Compounds did not increase cell permeability, dissipate membrane potential, or inhibit an unrelated mycobacterial enzyme, suggesting a specific mode of action related to the LepB secretory mechanism. Inhibition of LepB activity was observed for a number of compounds in a biochemical assay using cell membrane fraction derived from M. tuberculosis. A number of chemical modifications around the hydrazone moiety resulted in improved potency. We conducted a limited structure–activity relationship determination around a representative PHY compound with differential activity (MICs of 3.0 μM against the LepB-UE strain and 18 μM against the wt) several analogues were less potent against the LepB overexpressing strain.
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We identified the phenylhydrazone (PHY) series as having higher activity against the LepB-UE strain. We screened 72,000 compounds against both the Lep-UE and wild-type (wt) strains. We developed a target-based whole cell assay to screen for potential inhibitors of LepB, the sole signal peptidase in Mycobacterium tuberculosis, using a strain engineered to underexpress LepB (LepB-UE). During protein export, the signal peptidase LepB catalyzes the cleavage of the signal peptide and subsequent release of mature proteins into the extracellular space. The general secretion (Sec) pathway is a conserved essential pathway in bacteria and is the primary route of protein export across the cytoplasmic membrane.