RESUMO
As a growing number of clinical isolates of Mycobacterium abscessus are resistant to most antibiotics, new treatment options that are effective against these drug-resistant strains are desperately needed. The majority of the linkages in the cell wall peptidoglycan of M. abscessus are synthesized by nonclassical transpeptidases, namely, the l,d-transpeptidases. Emerging evidence suggests that these enzymes represent a new molecular vulnerability in this pathogen. Recent studies have demonstrated that inhibition of these enzymes by the carbapenem class of ß-lactams determines their activity against Mycobacterium tuberculosis Here, we studied the interactions of ß-lactams with two l,d-transpeptidases in M. abscessus, namely, LdtMab1 and LdtMab2, and found that both the carbapenem and cephalosporin, but not penicillin, subclasses of ß-lactams inhibit these enzymes. Contrary to the commonly held belief that combination therapy with ß-lactams is redundant, doripenem and cefdinir exhibit synergy against both pansusceptible M. abscessus and clinical isolates that are resistant to most antibiotics, which suggests that dual-ß-lactam therapy has potential for the treatment of M. abscessus Finally, we solved the first crystal structure of an M. abscessus l,d-transpeptidase, LdtMab2, and using substitutions of critical amino acids in the catalytic site and computational simulations, we describe the key molecular interactions between this enzyme and ß-lactams, which provide an insight into the molecular basis for the relative efficacy of different ß-lactams against M. abscessus.
Assuntos
Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Cefalosporinas/farmacologia , Mycobacterium abscessus/efeitos dos fármacos , Penicilinas/farmacologia , Peptidoglicano/biossíntese , Peptidil Transferases/antagonistas & inibidores , Parede Celular/metabolismo , Cristalografia por Raios X , Sinergismo Farmacológico , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium abscessus/isolamento & purificação , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: In this report, we outline our approach to implementing a hybrid in-person and virtual clinic model at a student-run free clinic (SRFC) during the COVID-19 pandemic. Individuals of low socioeconomic status (SES) are at an increased risk for COVID-19 infection and severe clinical outcomes. It is unclear if telehealth is a viable continuity of care enabler for the underserved. METHODS: The Weill Cornell Community Clinic (WCCC) implemented a novel telehealth clinic model to serve uninsured patients in May 2020. A phone survey of was conducted to assess WCCC patients access to technology needed for telehealth visits (eg, personal computers, smartphones). Patient no-show rates were retrospectively assessed for both in-person (pre-pandemic) and hybrid continuity of care models. RESULTS: The phone survey found that 90% of WCCC patients had access to technology needed for telehealth visits. In the 8 months following implementation of the hybrid model, telehealth and in-person no-show rates were 11% (14/128) and 15% (10/67) respectively; the combined hybrid no-show rate was 12% (24/195). For comparison, the in-person 2019 no-show rate was 23% (84/367). This study aligns with previous reports that telehealth improves patient attendance. CONCLUSION: Literature on the transition of SRFCs from in-person to telehealth care delivery models is limited. At the WCCC, the reduction in no-show rates supports the feasibility and benefits of adopting telehealth for the delivery of care to underserved patient populations. We believe the hybrid telehealth model described here is a viable model for other student run free clinics to increase access to care in low SES communities.
Assuntos
COVID-19 , Clínica Dirigida por Estudantes , Estudantes de Medicina , Telemedicina , Humanos , Pandemias , Atenção Primária à Saúde , Estudos RetrospectivosRESUMO
The Clp protease system is a promising, noncanonical drug target against Mycobacterium tuberculosis (Mtb). Unlike in Escherichia coli, the Mtb Clp protease consists of two distinct proteolytic subunits, ClpP1 and ClpP2, which hydrolyze substrates delivered by the chaperones ClpX and ClpC1. While biochemical approaches uncovered unique aspects of Mtb Clp enzymology, its essentiality complicates in vivo studies. To address this gap, we leveraged new genetic tools to mechanistically interrogate the in vivo essentiality of the Mtb Clp protease. While validating some aspects of the biochemical model, we unexpectedly found that only the proteolytic activity of ClpP1, but not of ClpP2, is essential for substrate degradation and Mtb growth and infection. Our observations not only support a revised model of Mtb Clp biology, where ClpP2 scaffolds chaperone binding while ClpP1 provides the essential proteolytic activity of the complex; they also have important implications for the ongoing development of inhibitors toward this emerging therapeutic target.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Proteínas de Bactérias/metabolismo , Endopeptidase Clp/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Serina Endopeptidases/químicaRESUMO
Phosphopantetheinyl hydrolase, PptH (Rv2795c), is a recently discovered enzyme from Mycobacterium tuberculosis that removes 4'-phosphopantetheine (Ppt) from holo-carrier proteins (CPs) and thereby opposes the action of phosphopantetheinyl transferases (PPTases). PptH is the first structurally characterized enzyme of the phosphopantetheinyl hydrolase family. However, conditions for optimal activity of PptH have not been defined, and only one substrate has been identified. Here, we provide biochemical characterization of PptH and demonstrate that the enzyme hydrolyzes Ppt in vitro from more than one M. tuberculosis holo-CP as well as holo-CPs from other organisms. PptH provided the only detectable activity in mycobacterial lysates that dephosphopantetheinylated acyl carrier protein M (AcpM), suggesting that PptH is the main Ppt hydrolase in M. tuberculosis. We could not detect a role for PptH in coenzyme A (CoA) salvage, and PptH was not required for virulence of M. tuberculosis during infection of mice. It remains to be determined why mycobacteria conserve a broadly acting phosphohydrolase that removes the Ppt prosthetic group from essential CPs. We speculate that the enzyme is critical for aspects of the life cycle of M. tuberculosis that are not routinely modeled. IMPORTANCE Tuberculosis (TB), caused by Mycobacterium tuberculosis, was the leading cause of death from an infectious disease before COVID, yet the in vivo essentiality and function of many of the protein-encoding genes expressed by M. tuberculosis are not known. We biochemically characterize M. tuberculosis's phosphopantetheinyl hydrolase, PptH, a protein unique to mycobacteria that removes an essential posttranslational modification on proteins involved in synthesis of lipids important for the bacterium's cell wall and virulence. We demonstrate that the enzyme has broad substrate specificity, but it does not appear to have a role in coenzyme A (CoA) salvage or virulence in a mouse model of TB.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Panteteína/análogos & derivados , Diester Fosfórico Hidrolases/metabolismo , Animais , Parede Celular/metabolismo , Feminino , Humanos , Lipídeos/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Panteteína/metabolismo , Processamento de Proteína Pós-Traducional , Tuberculose/patologia , Virulência/fisiologiaRESUMO
Diphenylphosphonates (DPPs) have been used for 50 years to inactivate serine hydrolases, creating adducts representative of tetrahedral intermediates of this important class of enzymes. Failure to react at active site serine residues, however, can thwart their usefulness. Here, we describe a facile route and allied mechanistic studies to highly reactive, structurally complex organofluorophosphonates (FPs) by direct fluorinative hydrolysis of DPPs. Advantages over current preparations of FPs are exemplified by the synthesis of a ß-lactam-containing peptide substrate analog capable of modifying the C-terminal, dual-function thioesterase involved in nocardicin A biosynthesis. Although this serine hydrolase was found to resist modification by classic DPP inhibitors, active site selective phosphonylation by the corresponding FP occurs rapidly to form a stable adduct. This simple, late-stage method enables the ready preparation of FP probes that retain important structural motifs of native substrates, thus promoting efforts in mechanistic enzymology by accessing biologically relevant enzyme-inhibitor co-structures.
Assuntos
Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Hidrolases/metabolismo , Organofosfonatos/metabolismo , Serina/metabolismo , Corantes Fluorescentes/química , Humanos , Hidrolases/química , Hidrólise , Estrutura Molecular , Organofosfonatos/química , Serina/químicaRESUMO
Nonribosomal peptide synthetases (NRPSs) underlie the biosynthesis of many natural products that have important medicinal utility. Protection of the NRPS peptide products from proteolysis is critical to these pathways and is often achieved by structural modification, principally the introduction of D-amino acid residues into the elongating peptide. These amino acids are generally formed in situ from their L-stereoisomers by epimerization domains or dual-function condensation/epimerization domains. In singular contrast, the thioesterase domain of nocardicin biosynthesis mediates both the effectively complete L- to D-epimerization of its C-terminal amino acid residue (≥100:1) and hydrolytic product release. We report herein high-resolution crystal structures of the nocardicin thioesterase domain in ligand-free form and reacted with a structurally precise fluorophosphonate substrate mimic that identify the complete peptide binding pocket to accommodate both stereoisomers. These structures combined with additional functional studies provide detailed mechanistic insight into this unique dual-function NRPS domain.
Assuntos
Isomerases de Aminoácido/metabolismo , Proteínas de Bactérias/metabolismo , Hidrolases/metabolismo , Lactamas/metabolismo , Peptídeo Sintases/metabolismo , Isomerases de Aminoácido/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X , Hidrolases/ultraestrutura , Modelos Moleculares , Nocardia/enzimologia , Organofosfonatos/metabolismo , Peptídeo Sintases/ultraestrutura , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Estereoisomerismo , Especificidade por SubstratoRESUMO
An efficient route to substituted N-fused aromatic heterocycles, including indolizines, imidazo[1,2-a]pyridines, and imidazo[1,5-a]pyridines from azole aldehydes, is reported. Wittig olefination of the aldehydes with fumaronitrile and triethylphosphine affords predominantly E-alkenes that undergo rapid cyclization upon treatment with a mild base. Substituent control of the 1-, 2-, and 3-positions of the resulting heteroaromatic bicycles is shown. Alternatively, the isolable E-alkene undergoes selective alkylation with electrophiles, followed by in situ annulation to indolizines additionally substituted at the 6-position.
Assuntos
Indolizinas/síntese química , Piridinas/síntese química , Indolizinas/química , Estrutura Molecular , Piridinas/química , EstereoisomerismoRESUMO
The surface of nanoporous gold (np-Au) monoliths was modified via a flow method with the lectin Concanavalin A (Con A) to develop a substrate for separation and extraction of glycoproteins. Self-assembled monolayers (SAMs) of α-lipoic acid (LA) on the np-Au monoliths were prepared followed by activation of the terminal carboxyl groups to create amine reactive esters that were utilized in the immobilization of Con A. Thermogravimetric analysis (TGA) was used to determine the surface coverages of LA and Con A on np-Au monoliths which were found to be 1.31×10(18) and 1.85×10(15)moleculesm(-2), respectively. An in situ solution depletion method was developed that enabled surface coverage characterization without damaging the substrate and suggesting the possibility of regeneration. Using this method, the surface coverages of LA and Con A were found to be 0.989×10(18) and 1.32×10(15)moleculesm(-2), respectively. The selectivity of the Con A-modified np-Au monolith for the high mannose-containing glycoprotein ovalbumin (OVA) versus negative control non-glycosylated bovine serum albumin (BSA) was demonstrated by the difference in the ratio of the captured molecules to the immobilized Con A molecules, with OVA:Con A=2.3 and BSA:Con A=0.33. Extraction of OVA from a 1:3 mole ratio mixture with BSA was demonstrated by the greater amount of depletion of OVA concentration during the circulation with the developed substrate. A significant amount of captured OVA was eluted using α-methyl mannopyranoside as a competitive ligand. This work is motivated by the need to develop new materials for chromatographic separation and extraction substrates for use in preparative and analytical procedures in glycomics.