RESUMO
Infections by Staphylococcus aureus have been treated historically with ß-lactam antibiotics. However, these antibiotics have become obsolete in methicillin-resistant S. aureus by acquisition of the bla and mec operons. The presence of the ß-lactam antibiotic is detected by the sensor domains of BlaR and/or MecR, and the information is transmitted to the cytoplasm, resulting in derepression of the antibiotic-resistance genes. We hypothesized that inhibition of the sensor domain would shut down this response system, and ß-lactam susceptibility would be restored. An in silico search of 11 million compounds led to a benzimidazole-based hit and, ultimately, to the boronate 4. The X-ray structure of 4 is covalently engaged with the active-site serine of BlaR. Compound 4 potentiates by 16- to 4,096-fold the activities of oxacillin and of meropenem against methicillin-resistant S. aureus strains. The combination of 4 with oxacillin or meropenem shows efficacy in infected mice, validating the strategy.
RESUMO
Clostridioides difficile infection (CDI) is the most lethal of the five CDC urgent public health treats, resulting in 12,800 annual deaths in the United States alone [Antibiotic Resistance Threats in the United States, 2019 (2019), www.cdc.gov/DrugResistance/Biggest-Threats.html]. The high recurrence rate and the inability of antibiotics to treat such infections mandate discovery of new therapeutics. A major challenge with CDI is the production of spores, leading to multiple recurrences of infection in 25% of patients [C. P. Kelly, J. T. LaMont, N. Engl. J. Med. 359, 1932-1940 (2008)], with potentially lethal consequence. Herein, we describe the discovery of an oxadiazole as a bactericidal anti-C. difficile agent that inhibits both cell-wall peptidoglycan biosynthesis and spore germination. We document that the oxadiazole binds to the lytic transglycosylase SleC and the pseudoprotease CspC for prevention of spore germination. SleC degrades the cortex peptidoglycan, a critical step in the initiation of spore germination. CspC senses germinants and cogerminants. Binding to SleC is with higher affinity than that to CspC. Prevention of spore germination breaks the nefarious cycles of CDI recurrence in the face of the antibiotic challenge, which is a primary cause of therapeutic failure. The oxadiazole exhibits efficacy in a mouse model of recurrent CDI and holds promise in clinical treatment of CDI.
Assuntos
Clostridioides difficile , Clostridioides , Animais , Camundongos , Clostridioides/metabolismo , Clostridioides difficile/metabolismo , Peptidoglicano/metabolismo , Esporos Bacterianos/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Clostridioides difficile is a spore-forming human pathogen responsible for significant morbidity and mortality. Infections by this pathogen ensue dysbiosis of the intestinal tract, which leads to germination of the spores. The process of spore formation requires a transition for the cell-wall peptidoglycan of the vegetative C. difficile to that of spores, which entails the formation of muramyl-δ-lactam. We describe a set of reactions for three recombinant C. difficile proteins, GerS, CwlD, and PdaA1, with the use of four synthetic peptidoglycan analogs. CwlD and PdaA1 excise the peptidoglycan stem peptide and the acetyl moiety of N-acetyl muramate, respectively. The reaction of CwlD is accelerated in the presence of GerS. With the use of a suitable substrate, we document that PdaA1 catalyzes a novel zinc-dependent transamidation/transpeptidation reaction, an unusual reaction that requires excision of the stem peptide as a pre-requisite.
Assuntos
Clostridioides difficile , Clostridioides , Humanos , Clostridioides/metabolismo , Esporos Bacterianos/metabolismo , Peptidoglicano/metabolismo , Lactamas/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Diabetic foot ulcers (DFUs) are chronic wounds that develop in 30% of diabetic patients. In DFUs, the normal wound healing process consisting of inflammation, angiogenesis, and extracellular matrix (ECM) remodeling is dysregulated and stalled. Upon injury, neutrophils and monocytes arrive at the wound and secrete matrix metalloproteinase (MMP)-8 and reactive oxygen species (ROS). ROS activates nuclear factor kappa beta (NF-κB), which upregulates MMP-9. Monocytes become macrophages, secreting tumor growth factor (TGF)-ß1 and vascular endothelial growth factor (VEGF) for angiogenesis, resulting in remodeling of the ECM. MMP-9 cleaves laminin for keratinocyte migration. MMP-8 is beneficial for remodeling the ECM and healing the wound. In DFUs, the excess unregulated MMP-9 is detrimental, destroying the ECM and preventing the wound from healing. DFUs are typically infected, many with biofilm-producing bacteria that are resistant to antibiotics. Infection increases the time for wound healing and the likelihood for a lower-limb amputation. Despite the use of antibiotics, amputations occur in 24.5% of patients with DFUs. Clearly, new strategies for treatment of DFUs are needed. With the use of an affinity resin that binds exclusively to the active forms of MMPs and proteomics, we identified two proteinases, MMP-8 and MMP-9, in wounds of diabetic mice and diabetic humans. With the use of selective inhibitors, gene ablation of MMP-9, and exogenous application of MMP-8, we demonstrated that MMP-8 is beneficial to wound repair and that MMP-9 prevents the diabetic wound from healing. Our research has shown that infection increases active MMP-9, increasing inflammation and decreasing angiogenesis. As a result, infected diabetic wounds take a longer time to heal than uninfected ones. We found that active MMP-9 and NF-κB increased in human DFUs with wound severity and infection. The best strategy for treatment of DFUs is to selectively inhibit the detrimental proteinase MMP-9 without affecting the beneficial MMP-8 so that the body can repair the wound. Lead optimization of the thiirane class of inhibitors led to the discovery of (R)-ND-336, a potent (19 nM) and selective (450-fold) MMP-9 inhibitor. (R)-ND-336 accelerated wound healing in diabetic mice by decreasing ROS and NF-κB, lowering inflammation, and increasing angiogenesis. (R)-ND-336 in combination with the antibiotic linezolid improved wound healing in infected diabetic mice by inhibiting MMP-9, which mitigated macrophage infiltration and increased angiogenesis, thereby restoring the normal wound healing process.
Assuntos
Antibacterianos/farmacologia , Pé Diabético/tratamento farmacológico , Inibidores de Metaloproteinases de Matriz/farmacologia , Animais , Pé Diabético/metabolismo , Pé Diabético/microbiologia , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
The need for new classes of antibacterials is genuine in light of the dearth of clinical options for the treatment of bacterial infections. The prodigious discoveries of antibiotics during the 1940s to 1970s, a period wistfully referred to as the Golden Age of Antibiotics, have not kept up in the face of emergence of resistant bacteria in the past few decades. There has been a renewed interest in old drugs, the repurposing of the existing antibiotics and pairing of synergistic antibiotics or of an antibiotic with an adjuvant. Notwithstanding, discoveries of novel classes of these life-saving drugs have become increasingly difficult, calling for new paradigms. We describe, herein, three strategies from our laboratories toward discoveries of new antibacterials and adjuvants using computational and multidisciplinary experimental methods. One approach targets penicillin-binding proteins (PBPs), biosynthetic enzymes of cell-wall peptidoglycan, for discoveries of non-ß-lactam inhibitors. Oxadiazoles and quinazolinones emerged as two structural classes out of these efforts. Several hundred analogs of these two classes of antibiotics have been synthesized and fully characterized in our laboratories. A second approach ventures into inhibition of allosteric regulation of cell-wall biosynthesis. The mechanistic details of allosteric regulation of PBP2a of Staphylococcus aureus, discovered in our laboratories, is outlined. The allosteric site in this protein is at 60 Å distance to the active site, whereby ligand binding at the former makes access to the latter by the substrate possible. We have documented that both quinazolinones and ceftaroline, a fifth-generation cephalosporin, bind to the allosteric site in manifestation of the antibacterial activity. Attempts at inhibition of the regulatory phosphorylation events identified three classes of antibacterial adjuvants and one class of antibacterials, the picolinamides. The chemical structures for these hits went through diversification by synthesis of hundreds of analogs. These analogs were characterized in various assays for identification of leads with adjuvant and antibacterial activities. Furthermore, we revisited the mechanism of bulgecins, a class of adjuvants discovered and abandoned in the 1980s. These compounds potentiate the activities of ß-lactam antibiotics by the formation of bulges at the sites of septum formation during bacterial replication, which are points of structural weakness in the envelope. These bulges experience rupture, which leads to bacterial death. Bulgecin A inhibits the lytic transglycosylase Slt of Pseudomonas aeruginosa as a likely transition-state mimetic for its turnover of the cell-wall peptidoglycan. Once damage to cell wall is inflicted by a ß-lactam antibiotic, the function of Slt is to repair the damage. When Slt is inhibited by bulgecin A, the organism cannot cope with it and would undergo rapid lysis. Bulgecin A is an effective adjuvant of ß-lactam antibiotics. These discoveries of small-molecule classes of antibacterials or of adjuvants to antibacterials hold promise in strategies for treatment of bacterial infections.
Assuntos
Adjuvantes Imunológicos/química , Antibacterianos/química , Sítio Alostérico , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Descoberta de Drogas , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Simulação de Dinâmica Molecular , Proteínas de Ligação às Penicilinas/química , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/antagonistas & inibidores , Peptidoglicano Glicosiltransferase/metabolismo , Pseudomonas aeruginosa/enzimologia , Quinazolinonas/química , Quinazolinonas/metabolismo , Staphylococcus aureus/metabolismoRESUMO
Diabetic foot ulcers are characterized by hypoxia. For many patients, hyperbaric oxygen (HBO) therapy is the last recourse for saving the limb from amputation, for which the molecular basis is not understood. We previously identified the active form of matrix metalloproteinase-9 (MMP-9) as responsible for diabetic foot ulcer's recalcitrance to healing. Transcription of mmp-9 to the inactive zymogen is upregulated during hypoxia. Activation of the zymogen is promoted by proteases and reactive oxygen species (ROS). We hypothesized that the dynamics of these two events might lead to a lowering of active MMP-9 levels in the wounded tissue. We employed the full-thickness excisional db/db mouse model to study wound healing, and treated the mice to 3.0 atm of molecular oxygen for 90 minutes, 5 days per week for 10 days in an HBO research chamber. Treatment with HBO accelerated diabetic wound healing compared to untreated mice, with more completed and extended reepithelialization. We imaged the wounds for ROS in vivo with a luminol-based probe and found that HBO treatment actually decreases ROS levels. The levels of superoxide dismutase, catalase, and glutathione peroxidase-enzymes that turn over ROS-increased after HBO treatment, hence the observation of decreased ROS. Since ROS levels are lowered, we explored the effect that this would have on activation of MMP-9. Quantitative analysis with an affinity resin that binds and pulls down the active MMPs exclusively, coupled with proteomics, revealed that HBO treatment indeed reduces the active MMP-9 levels. This work for the first time demonstrates that diminution of active MMP-9 is a contributing factor and a mechanism for enhancement of diabetic wound healing by HBO therapy.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Pé Diabético/metabolismo , Oxigenoterapia Hiperbárica , Metaloproteinase 9 da Matriz/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Cicatrização , Animais , Catalase/metabolismo , Modelos Animais de Doenças , Precursores Enzimáticos/metabolismo , Glutationa Peroxidase/metabolismo , Camundongos , Receptores para Leptina/genética , Superóxido Dismutase/metabolismoRESUMO
The in vitro activities of five quinazolinone antibacterials, compounds Q1 to Q5, were tested against 210 strains of methicillin-resistant Staphylococcus aureus (MRSA). The MIC50/MIC90 values (in µg/ml) were as follows: Q1, 0.5/2; Q2, 1/4; Q3, 2/4; Q4, 0.06/0.25; and Q5, 0.125/0.5. Several strains with high MIC values (from 8 to >32 µg/ml) for some of these compounds exhibited amino acid changes in the penicillin-binding proteins, which are targeted by these antibacterials.
Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Quinazolinonas/farmacologia , Substituição de Aminoácidos , Antibacterianos/química , Humanos , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Quinazolinonas/química , Espanha , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologiaRESUMO
The quinazolinones are a new class of antibacterials with in vivo efficacy against methicillin-resistant Staphylococcus aureus (MRSA). The quinazolinones target cell wall biosynthesis and have a unique mechanism of action by binding to the allosteric site of penicillin-binding protein 2a (PBP 2a). We investigated the potential for synergism of a lead quinazolinone with several antibiotics of different classes using checkerboard and time-kill assays. The quinazolinone synergized with ß-lactam antibiotics. The combination of the quinazolinone with commercial piperacillin-tazobactam showed bactericidal synergy at sub-MICs of all three drugs. We demonstrated the efficacy of the triple-drug combination in a mouse MRSA neutropenic thigh infection model. The proposed mechanism for the synergistic activity in MRSA involves inhibition of the ß-lactamase by tazobactam, which protects piperacillin from hydrolysis, which can then inhibit its target, PBP 2. Furthermore, the quinazolinone binds to the allosteric site of PBP 2a, triggering the allosteric response. This leads to the opening of the active site, which, in turn, binds another molecule of piperacillin. In other words, PBP 2a, which is not normally inhibited by piperacillin, becomes vulnerable to inhibition in the presence of the quinazolinone. The collective effect is the impairment of cell wall biosynthesis, with bactericidal consequence. Two crystal structures for complexes of the antibiotics with PBP 2a provide support for the proposed mechanism of action.
Assuntos
Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Piperacilina/farmacologia , Quinazolinonas/farmacologia , Tazobactam/farmacologia , Antibacterianos/farmacologia , Sinergismo Farmacológico , Testes de Sensibilidade MicrobianaRESUMO
The focus of this article is to highlight novel inhibitors and current examples where the use of selective small-molecule inhibitors has been critical in defining the roles of matrix metalloproteinases (MMPs) in disease. Selective small-molecule inhibitors are surgical chemical tools that can inhibit the targeted enzyme; they are the method of choice to ascertain the roles of MMPs and complement studies with knockout animals. This strategy can identify targets for therapeutic development as exemplified by the use of selective small-molecule MMP inhibitors in diabetic wound healing, spinal cord injury, stroke, traumatic brain injury, cancer metastasis, and viral infection. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.
Assuntos
Animais Geneticamente Modificados , Lesões Encefálicas Traumáticas/tratamento farmacológico , Metaloproteinases da Matriz/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Traumatismos da Medula Espinal/tratamento farmacológico , Acidente Vascular Cerebral/tratamento farmacológico , Viroses/tratamento farmacológico , Animais , Lesões Encefálicas Traumáticas/enzimologia , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/patologia , Humanos , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/uso terapêutico , Metaloproteinases da Matriz/genética , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Traumatismos da Medula Espinal/enzimologia , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia , Acidente Vascular Cerebral/enzimologia , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia , Viroses/enzimologia , Viroses/genética , Viroses/patologiaRESUMO
The activities of four oxadiazoles were investigated with 210 methicillin-resistant Staphylococcus aureus (MRSA) strains. MIC50 and MIC90 values of 1 to 2 and 4 µg/ml, respectively, were observed. We also evaluated the activity of oxadiazole ND-421 against other staphylococci and enterococci and in the presence of oxacillin for selected MRSA strains. The MIC for ND-421 is lowered severalfold in combination with oxacillin, as they synergize. The MIC90 of ND-421 against vancomycin-resistant enterococci is ≤1 µg/ml.
Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxidiazóis/farmacologia , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Antibacterianos/química , Cefalosporinas/farmacologia , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Oxacilina/farmacologia , Oxidiazóis/química , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/crescimento & desenvolvimento , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/crescimento & desenvolvimento , Staphylococcus saprophyticus/efeitos dos fármacos , Staphylococcus saprophyticus/crescimento & desenvolvimento , Relação Estrutura-Atividade , Enterococos Resistentes à Vancomicina/crescimento & desenvolvimento , CeftarolinaRESUMO
Nonhealing chronic wounds are major complications of diabetes resulting in >70,000 annual lower-limb amputations in the United States alone. The reasons the diabetic wound is recalcitrant to healing are not fully understood, and there are limited therapeutic agents that could accelerate or facilitate its repair. We previously identified two active forms of matrix metalloproteinases (MMPs), MMP-8 and MMP-9, in the wounds of db/db mice. We argued that the former might play a role in the body's response to wound healing and that the latter is the pathological consequence of the disease with detrimental effects. Here we demonstrate that the use of compound ND-336, a novel highly selective inhibitor of gelatinases (MMP-2 and MMP-9) and MMP-14, accelerates diabetic wound healing by lowering inflammation and by enhancing angiogenesis and re-epithelialization of the wound, thereby reversing the pathological condition. The detrimental role of MMP-9 in the pathology of diabetic wounds was confirmed further by the study of diabetic MMP-9-knockout mice, which exhibited wounds more prone to healing. Furthermore, topical administration of active recombinant MMP-8 also accelerated diabetic wound healing as a consequence of complete re-epithelialization, diminished inflammation, and enhanced angiogenesis. The combined topical application of ND-336 (a small molecule) and the active recombinant MMP-8 (an enzyme) enhanced healing even more, in a strategy that holds considerable promise in healing of diabetic wounds.
Assuntos
Complicações do Diabetes , Inibidores de Proteases/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Metaloproteinase 8 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Ferimentos e Lesões/enzimologiaRESUMO
The mechanism of the ß-lactam antibacterials is the functionally irreversible acylation of the enzymes that catalyze the cross-linking steps in the biosynthesis of their peptidoglycan cell wall. The Gram-positive pathogen Staphylococcus aureus uses one primary resistance mechanism. An enzyme, called penicillin-binding protein 2a (PBP2a), is brought into this biosynthetic pathway to complete the cross-linking. PBP2a effectively discriminates against the ß-lactam antibiotics as potential inhibitors, and in favor of the peptidoglycan substrate. The basis for this discrimination is an allosteric site, distal from the active site, that when properly occupied concomitantly opens the gatekeeper residues within the active site and realigns the conformation of key residues to permit catalysis. We address the molecular basis of this regulation using crystallographic studies augmented by computational analyses. The crystal structures of three ß-lactams (oxacillin, cefepime, ceftazidime) complexes with PBP2a-each with the ß-lactam in the allosteric site-defined (with preceding PBP2a structures) as the "open" or "partially open" PBP2a states. A particular loop motion adjacent to the active site is identified as the driving force for the active-site conformational change that accompanies active-site opening. Correlation of this loop motion to effector binding at the allosteric site, in order to identify the signaling pathway, was accomplished computationally in reference to the known "closed" apo-PBP2a X-ray crystal structure state. This correlation enabled the computational simulation of the structures coinciding with initial peptidoglycan substrate binding to PBP2a, acyl enzyme formation, and acyl transfer to a second peptidoglycan substrate to attain cross-linking. These studies offer important insights into the structural bases for allosteric site-to-active site communication and for ß-lactam mimicry of the peptidoglycan substrates, as foundational to the mechanistic understanding of emerging PBP2a resistance mutations.
Assuntos
Proteínas de Bactérias/metabolismo , Staphylococcus aureus Resistente à Meticilina/química , Proteínas de Ligação às Penicilinas/metabolismo , Termodinâmica , Regulação Alostérica , Proteínas de Bactérias/química , Biocatálise , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Proteínas de Ligação às Penicilinas/química , Conformação ProteicaRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent multidrug-resistant pathogens worldwide, exhibiting increasing resistance to the latest antibiotic therapies. Here we show that the triple ß-lactam combination meropenem-piperacillin-tazobactam (ME/PI/TZ) acts synergistically and is bactericidal against MRSA subspecies N315 and 72 other clinical MRSA isolates in vitro and clears MRSA N315 infection in a mouse model. ME/PI/TZ suppresses evolution of resistance in MRSA via reciprocal collateral sensitivity of its constituents. We demonstrate that these activities also extend to other carbapenem-penicillin-ß-lactamase inhibitor combinations. ME/PI/TZ circumvents the tight regulation of the mec and bla operons in MRSA, the basis for inducible resistance to ß-lactam antibiotics. Furthermore, ME/PI/TZ subverts the function of penicillin-binding protein-2a (PBP2a) via allostery, which we propose as the mechanism for both synergy and collateral sensitivity. Showing in vivo activity similar to that of linezolid, ME/PI/TZ demonstrates that combinations of older ß-lactam antibiotics could be effective against MRSA infections in humans.
Assuntos
Antibacterianos/farmacologia , Resistência a Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Inibidores de beta-Lactamases/farmacologia , Regulação Alostérica , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Expressão Gênica , Humanos , Linezolida/farmacologia , Meropeném , Resistência a Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/química , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana , Óperon , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/farmacologia , Proteínas de Ligação às Penicilinas , Piperacilina/farmacologia , Infecções Estafilocócicas/microbiologia , Tazobactam , Tienamicinas/farmacologia , beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
The oxadiazole antibacterials target the bacterial cell wall and are bactericidal. We investigated the synergism of ND-421 with the commonly used ß-lactams and non-ß-lactam antibiotics by the checkerboard method and by time-kill assays. ND-421 synergizes well with ß-lactam antibiotics, and it also exhibits a long postantibiotic effect (4.7 h). We also evaluated the in vivo efficacy of ND-421 in a murine neutropenic thigh infection model alone and in combination with oxacillin. ND-421 has in vivo efficacy by itself in a clinically relevant infection model (1.49 log10 bacterial reduction for ND-321 versus 0.36 log10 for linezolid with NRS119) and acts synergistically with ß-lactam antibiotics in vitro and in vivo, and the combination of ND-421 with oxacillin is efficacious in a mouse neutropenic thigh methicillin-resistant Staphylococcus aureus (MRSA) infection model (1.60 log10 bacterial reduction). The activity of oxacillin was potentiated in the presence of ND-421, as the strain would have been resistant to oxacillin otherwise.
Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxidiazóis/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , beta-Lactamas/farmacologia , Animais , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana , Oxacilina/farmacologiaRESUMO
Diabetic foot ulcers are a complication of diabetes for which treatment options are limited and not effective, resulting in 73,000 lower-limb amputations in the United States every year. Wound healing is a complex process with a highly orchestrated cascade of events, in which the extracellular matrix (ECM) interacts with growth factors and cells. Matrix metalloproteinases (MMPs) are involved in all wound healing events, in particular MMP-8 and MMP-9, whose physiological functions are to degrade damaged collagen type I and to facilitate keratinocyte migration and re-epithelialization, respectively. MMP substrate redundancy permits another MMP to substitute for MMP-9 during normal wound healing. Under the hypoxic and inflammatory environment of diabetic wounds, increased reactive oxygen species (ROS) and upregulation of MMP-9 results in wounds that are recalcitrant to healing. We have determined that MMP-8 plays a role in the body's response to wound healing and that MMP-9 is the pathological consequence of the disease with detrimental effects. Thus, selective inhibition of MMP-9, while leaving MMP-8 activity unaffected, is desirable. ND-336 has such inhibitory profile and is a promising strategy for treatment of diabetic foot ulcers.
Assuntos
Complicações do Diabetes/tratamento farmacológico , Inibidores de Metaloproteinases de Matriz/uso terapêutico , Cicatrização/efeitos dos fármacos , Animais , Matriz Extracelular/efeitos dos fármacos , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologiaRESUMO
MT1-MMP and MMP2 have been implicated as pro-tumorigenic and pro-metastatic factors in a wide variety of cancers including melanoma. We have previously demonstrated that MT1-MMP is highly expressed in melanoma where it promotes melanoma cell invasion and metastasis in part through the activation of its target MMP2. Given the accessibility of MMPs, as they are either secreted (e.g. MMP2) or membrane-tethered (e.g. MT1-MMP), they represent ideal targets for specific inhibition via small molecules. Here we show that the novel small-molecule inhibitor ND-322 with high selectivity for MT1-MMP and MMP2, effectively inhibits MT1-MMP and MMP2 activity resulting in reduced in vitro melanoma cell growth, migration and invasion. Importantly, these inhibitory effects lead to significant reduction of melanoma tumor growth and metastasis. We further show that while cell migration and invasion could be similarly hampered by specific inhibition of either MT1-MMP or MMP2 via shRNAs, the growth inhibitory activity of ND-322 could only be mirrored by specific inhibition of MT1-MMP. These data support ND-322 as a novel effective inhibitor capable of counteracting both MT1-MMP and MMP2, two key proteases involved in melanoma growth and metastasis. ND-322 may therefore represent a new inhibitor in the repertoire of treatments against melanoma.
Assuntos
Arginina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Melanoma/tratamento farmacológico , Metástase Neoplásica/tratamento farmacológico , Sulfetos/farmacologia , Sulfonas/farmacologia , Animais , Arginina/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Masculino , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
The oxadiazole antibacterials, a class of newly discovered compounds that are active against Gram-positive bacteria, target bacterial cell-wall biosynthesis by inhibition of a family of essential enzymes, the penicillin-binding proteins. Ligand-based 3D-QSAR analyses by comparative molecular field analysis (CoMFA), comparative molecular shape indices analysis (CoMSIA) and Field-Based 3D-QSAR evaluated a series of 102 members of this class. This series included inactive compounds as well as compounds that were moderately to strongly antibacterial against Staphylococcus aureus. Multiple models were constructed using different types of energy minimization and charge calculations. CoMFA derived contour maps successfully defined favored and disfavored regions of the molecules in terms of steric and electrostatic properties for substitution.
Assuntos
Antibacterianos/química , Oxidiazóis/química , Relação Quantitativa Estrutura-Atividade , Antibacterianos/síntese química , Antibacterianos/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Desenho de Fármacos , Bactérias Gram-Positivas/metabolismo , Testes de Sensibilidade Microbiana , Conformação Molecular , Oxidiazóis/síntese química , Oxidiazóis/farmacologiaRESUMO
Four possible isomers of essramycin, a natural product from a marine Streptomyces species isolated from the Egyptian Mediterranean coast, were synthesized. The structures for the isomers were assigned unequivocally by (1)H NMR, (13)C NMR, high-resolution mass spectrometry, and X-ray crystal structure determinations. Notwithstanding the earlier report of broad-spectrum antibacterial activity for the natural product, none of the four isomers exhibited any such activity.
Assuntos
Antibacterianos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Pirimidinonas/química , Pirimidinonas/farmacologia , Streptomyces/química , Triazóis/química , Triazóis/farmacologia , Antibacterianos/química , Egito , Biologia Marinha , Mar Mediterrâneo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , EstereoisomerismoRESUMO
The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the ß-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to ß-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the ß-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to ß-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with ß-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain--a remarkable 60 Å distant from the DD-transpeptidase active site--discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA ß-lactam antibiotic. The ability of an anti-MRSA ß-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second ß-lactam molecule, opens an unprecedented realm for ß-lactam antibiotic structure-based design.
Assuntos
Resistência a Meticilina/fisiologia , Staphylococcus aureus Resistente à Meticilina/enzimologia , Proteínas de Ligação às Penicilinas/química , Acilação/fisiologia , Regulação Alostérica/fisiologia , Domínio Catalítico , Cefalosporinas/química , Cefalosporinas/metabolismo , Cristalografia por Raios X , Staphylococcus aureus Resistente à Meticilina/genética , Ácidos Murâmicos/química , Ácidos Murâmicos/metabolismo , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo , Especificidade por Substrato/fisiologia , CeftarolinaRESUMO
The transpeptidases involved in the synthesis of the bacterial cell wall (also known as penicillin-binding proteins, PBPs) have evolved to bind the acyl-D-Ala-D-Ala segment of the stem peptide of the nascent peptidoglycan for the physiologically important cross-linking of the cell wall. The Tipper-Strominger hypothesis stipulates that ß-lactam antibiotics mimic the acyl-D-Ala-D-Ala moiety of the stem and, thus, are recognized by the PBPs with bactericidal consequences. We document that this mimicry exists also at the allosteric site of PBP2a of methicillin-resistant Staphylococcus aureus (MRSA). Interactions of different classes of ß-lactam antibiotics, as mimics of the acyl-D-Ala-D-Ala moiety at the allosteric site, lead to a conformational change, across a distance of 60 Å to the active site. We directly visualize this change using an environmentally sensitive fluorescent probe affixed to the protein loops that frame the active site. This conformational mobility, documented in real time, allows antibiotic access to the active site of PBP2a. Furthermore, we document that this allosteric trigger enables synergy between two different ß-lactam antibiotics, wherein occupancy at the allosteric site by one facilitates occupancy by a second at the transpeptidase catalytic site, thus lowering the minimal-inhibitory concentration. This synergy has important implications for the mitigation of facile emergence of resistance to these antibiotics by MRSA.