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[This corrects the article DOI: 10.1371/journal.pone.0253364.].
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During RNA replication, coronaviruses require proofreading to maintain the integrity of their large genomes. Nsp14 associates with viral polymerase complex to excise the mismatched nucleotides. Aside from the exonuclease activity, nsp14 methyltransferase domain mediates cap methylation, facilitating translation initiation and protecting viral RNA from recognition by the innate immune sensors. The nsp14 exonuclease activity is modulated by a protein co-factor nsp10. While the nsp10/nsp14 complex structure is available, the mechanistic basis for nsp10-mediated modulation remains unclear in the absence of the nsp14 structure. Here, we provide a crystal structure of nsp14 in an apo-form. Comparative analysis of the apo- and nsp10-bound structures explain the modulatory role of the co-factor protein and reveal the allosteric nsp14 control mechanism essential for drug discovery. Further, the flexibility of the N-terminal lid of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp14 structure presented in this study rationalizes the recently proposed idea of nsp14/nsp10/nsp16 ternary complex.
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Exorribonucleasas , Proteínas no Estructurales Virales , Proteínas Reguladoras y Accesorias Virales , Exonucleasas , Exorribonucleasas/química , Metiltransferasas/química , Pliegue de Proteína , ARN Viral/metabolismo , SARS-CoV-2 , Proteínas no Estructurales Virales/química , Proteínas Reguladoras y Accesorias Virales/químicaRESUMEN
Motivation: The complement pathway plays a critical role in innate immune defense against infections. Dysregulation between activation and regulation of the complement pathway is widely known to contribute to several diseases. Nevertheless, very few drugs that target complement proteins have made it to the final regulatory approval because of factors such as high concentrations and dosing requirements for complement proteins and serious side effects from complement inhibition. Methods: A quantitative systems pharmacology (QSP) model of the complement pathway has been developed to evaluate potential drug targets to inhibit complement activation in autoimmune diseases. The model describes complement activation via the alternative and terminal pathways as well as the dynamics of several regulatory proteins. The QSP model has been used to evaluate the effect of inhibiting complement targets on reducing pathway activation caused by deficiency in factor H and CD59. The model also informed the feasibility of developing small-molecule or large-molecule antibody drugs by predicting the drug dosing and affinity requirements for potential complement targets. Results: Inhibition of several complement proteins was predicted to lead to a significant reduction in complement activation and cell lysis. The complement proteins that are present in very high concentrations or have high turnover rates (C3, factor B, factor D, and C6) were predicted to be challenging to engage with feasible doses of large-molecule antibody compounds (≤20 mg/kg). Alternatively, complement fragments that have a short half-life (C3b, C3bB, and C3bBb) were predicted to be challenging or infeasible to engage with small-molecule compounds because of high drug affinity requirements (>1 nM) for the inhibition of downstream processes. The drug affinity requirements for disease severity reduction were predicted to differ more than one to two orders of magnitude than affinities needed for the conventional 90% target engagement (TE) for several proteins. Thus, the QSP model analyses indicate the importance for accounting for TE requirements for achieving reduction in disease severity endpoints during the lead optimization stage.
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Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.
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Antivirales/farmacología , Inhibidores de Proteasas/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Anticuerpos de Cadena Única/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Células A549 , Complejo Antígeno-Anticuerpo , Humanos , Concentración 50 Inhibidora , ARN Polimerasa Dependiente del ARN/inmunología , ARN Polimerasa Dependiente del ARN/metabolismo , Anticuerpos de Cadena Única/inmunología , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismoRESUMEN
In search of novel drugs against tuberculosis, we previously discovered and profiled a novel hydantoin-based family that demonstrated highly promising in vitro potency against Mycobacterium. tuberculosis. The compounds were found to be noncovalent inhibitors of DprE1, a subunit of decaprenylphosphoryl-ß-d-ribose-2'-epimerase. This protein, localized in the periplasmic space of the mycobacterial cell wall, was shown to be an essential and vulnerable antimycobacterial drug target. Here, we report the further SAR exploration of this chemical family through more than 80 new analogues. Among these, the most active representatives combined submicromolar cellular potency and nanomolar target affinity with balanced physicochemical properties and low human cytotoxicity. Moreover, we demonstrate in vivo activity in an acute Mtb infection model and provide further proof of DprE1 being the target of the hydantoins. Overall, the hydantoin family of DprE1 inhibitors represents a promising noncovalent lead series for the discovery of novel antituberculosis agents.
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Oxidorreductasas de Alcohol/antagonistas & inhibidores , Antituberculosos/química , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Hidantoínas/química , Hidantoínas/farmacología , Oxidorreductasas de Alcohol/metabolismo , Animales , Antituberculosos/metabolismo , Proteínas Bacterianas/metabolismo , Femenino , Células Hep G2 , Humanos , Hidantoínas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Tuberculosis/tratamiento farmacológico , Tuberculosis/metabolismoRESUMEN
In the course of optimizing a novel indazole sulfonamide series that inhibits ß-ketoacyl-ACP synthase (KasA) of Mycobacterium tuberculosis, a mutagenic aniline metabolite was identified. Further lead optimization efforts were therefore dedicated to eliminating this critical liability by removing the embedded aniline moiety or modifying its steric or electronic environment. While the narrow SAR space against the target ultimately rendered this goal unsuccessful, key structural knowledge around the binding site of this underexplored target for TB was generated to inform future discovery efforts.
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3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/antagonistas & inhibidores , Compuestos de Anilina/farmacología , Mycobacterium tuberculosis , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Sitios de Unión , Daño del ADN , Mycobacterium tuberculosis/enzimologíaRESUMEN
The complement system (CS) is an integral part of innate immunity and can be activated via three different pathways. The alternative pathway (AP) has a central role in the function of the CS. The AP of complement system is implicated in several human disease pathologies. In the absence of triggers, the AP exists in a time-invariant resting state (physiological steady state). It is capable of rapid, potent and transient activation response upon challenge with a trigger. Previous models of AP have focused on the activation response. In order to understand the molecular machinery necessary for AP activation and regulation of a physiological steady state, we built parsimonious AP models using experimentally supported kinetic parameters. The models further allowed us to test quantitative roles played by negative and positive regulators of the pathway in order to test hypotheses regarding their mechanisms of action, thus providing more insight into the complex regulation of AP.
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Vía Alternativa del Complemento , Modelos Inmunológicos , Complemento C3b/inmunología , Factor B del Complemento/inmunología , Factor H de Complemento/inmunología , Simulación por Computador , Humanos , Inmunidad Innata , Cinética , Conceptos Matemáticos , Properdina/inmunologíaRESUMEN
Tuberculosis is the leading cause of death worldwide from infectious diseases. With the development of drug-resistant strains of Mycobacterium tuberculosis, there is an acute need for new medicines with novel modes of action. Herein, we report the discovery and profiling of a novel hydantoin-based family of antimycobacterial inhibitors of the decaprenylphospho-ß-d-ribofuranose 2-oxidase (DprE1). In this study, we have prepared a library of more than a 100 compounds and evaluated them for their biological and physicochemical properties. The series is characterized by high enzymatic and whole-cell activity, low cytotoxicity, and a good overall physicochemical profile. In addition, we show that the series acts via reversible inhibition of the DprE1 enzyme. Overall, the novel compound family forms an attractive base for progression to further stages of optimization and may provide a promising drug candidate in the future.
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Oxidorreductasas de Alcohol/antagonistas & inhibidores , Antituberculosos/química , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Hidantoínas/química , Actinobacteria/efectos de los fármacos , Oxidorreductasas de Alcohol/metabolismo , Proteínas Bacterianas/metabolismo , Estabilidad de Medicamentos , Inhibidores Enzimáticos/química , Células Hep G2 , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Macrófagos/microbiología , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Reproducibilidad de los Resultados , Relación Estructura-Actividad , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Two new tricyclic ß-aminoacrylate derivatives (2e and 3e) have been found to be inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) with Ki 0.037 and 0.15µM respectively. 1H and 13C NMR spectroscopic data show that these compounds undergo ready cis-trans isomerisation at room temperature in polar solvents. In silico docking studies indicate that for both molecules there is neither conformation nor double bond configuration which bind preferentially to PfDHODH. This flexibility is favourable for inhibitors of this channel that require extensive positioning to reach their binding site.
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Acrilatos/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Plasmodium falciparum/enzimología , Acrilatos/síntesis química , Acrilatos/química , Dihidroorotato Deshidrogenasa , Relación Dosis-Respuesta a Droga , Simulación del Acoplamiento Molecular , Estructura Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Relación Estructura-ActividadRESUMEN
Wollamide B is a cationic antimycobacterial cyclohexapeptide that exhibits activity against Mycobacterium bovis (M. bovis) (IC50 of 3.1 µM). Aiming to define its structural activity relationship (SAR), optimizing potency and pharmacokinetic properties, libraries of analogues were synthesized following a standard Fmoc-based solid phase peptide synthesis approach. The antimycobacterial activities of wollamide B and all the synthesized analogues were tested against Mycobacterium tuberculosis (Mtb) H37Rv. Parallely, in vitro drug metabolism and pharmacokinetic (ADME) profiling was done for the synthesized compounds to evaluate their drug likeness. Among the 25 synthesized wollamides five of them showed potent activities with MICs ≤ 3.1 µM and found to be nontoxic against human HepG2 cells up to 100 µM. The results of the in vitro ADME profiling revealed the remarkable plasma stability and very good aqueous solubility of the class in general while the metabolic stability was found to be moderate to low. Of particular note, compounds 7c (MIC = 1.1 µM) and 13c (0.6 µM) that exhibited good balance of antimycobacterial activity vs. optimal pharmacokinetic properties could be used as a new lead for further development.
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Antituberculosos/síntesis química , Mycobacterium tuberculosis/efectos de los fármacos , Péptidos Cíclicos/síntesis química , Animales , Antituberculosos/sangre , Antituberculosos/farmacocinética , Antituberculosos/farmacología , Supervivencia Celular/efectos de los fármacos , Diseño de Fármacos , Semivida , Células Hep G2 , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/crecimiento & desarrollo , Péptidos Cíclicos/sangre , Péptidos Cíclicos/farmacocinética , Péptidos Cíclicos/farmacología , Especificidad de la Especie , Relación Estructura-ActividadRESUMEN
During the construction of bioactive molecules, regioselective alkylation of heterocyclic, N/O ambident nucleophiles is a frequently encountered synthetic transformation. In this framework, specific attention is required to unambiguously determine the structures of obtained reaction products. As part of our project on quinoloxyacetamide based antimycobacterial agents, a series of N- or O- alkylated quinolin-4-ol, 1,5-naphthyridin-4-ol and quinazolin-4-ol derivatives were prepared during the course of which we observed unexpected selectivity issues. After finding that no consistent procedure existed in the literature for assigning regioisomers of this type, we applied three readily accessible NMR experiment types (13C NMR, HSQC/HMBC and NOE) to resolve any uncertainties regarding the obtained regioisomeric structures. Furthermore, the antimycobacterial activity of all final compounds was evaluated with the best compound 23 showing potent antitubercular activity (MIC = 1.25 µM) without cytotoxic effects.
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Antibacterianos/química , Espectroscopía de Resonancia Magnética/métodos , Mycobacteriaceae/efectos de los fármacos , Naftiridinas/farmacología , Quinazolinas/farmacología , Alquilación , Antibacterianos/farmacología , Antituberculosos/química , Supervivencia Celular/efectos de los fármacos , Estructura Molecular , Naftiridinas/química , Quinazolinas/químicaRESUMEN
While modern cephalosporins developed for broad spectrum antibacterial activities have never been pursued for tuberculosis (TB) therapy, we identified first generation cephalosporins having clinically relevant inhibitory concentrations, both alone and in synergistic drug combinations. Common chemical patterns required for activity against Mycobacterium tuberculosis were identified using structure-activity relationships (SAR) studies. Numerous cephalosporins were synergistic with rifampicin, the cornerstone drug for TB therapy, and ethambutol, a first-line anti-TB drug. Synergy was observed even under intracellular growth conditions where beta-lactams typically have limited activities. Cephalosporins and rifampicin were 4- to 64-fold more active in combination than either drug alone; however, limited synergy was observed with rifapentine or rifabutin. Clavulanate was a key synergistic partner in triple combinations. Cephalosporins (and other beta-lactams) together with clavulanate rescued the activity of rifampicin against a rifampicin resistant strain. Synergy was not due exclusively to increased rifampicin accumulation within the mycobacterial cells. Cephalosporins were also synergistic with new anti-TB drugs such as bedaquiline and delamanid. Studies will be needed to validate their in vivo activities. However, the fact that cephalosporins are orally bioavailable with good safety profiles, together with their anti-mycobacterial activities reported here, suggest that they could be repurposed within new combinatorial TB therapies.
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In this study, a new series of more than 60 quinoline derivatives has been synthesized and evaluated against Mycobacterium tuberculosis (H37Rv). Apart from the SAR exploration around the initial hits, the optimization process focused on the improvement of the physicochemical properties, cytotoxicity, and metabolic stability of the series. The best compounds obtained exhibited MIC values in the low micromolar range, excellent intracellular antimycobacterial activity, and an improved physicochemical profile without cytotoxic effects. Further investigation revealed that the amide bond was the source for the poor blood stability observed, while some of the compounds exhibited hERG affinity. Compound 83 which contains a benzoxazole ring instead of the amide group was found to be a good alternative, with good blood stability and no hERG affinity, providing new opportunities for the series. Overall, the obtained results suggest that further optimization of solubility and microsomal stability of the series could provide a strong lead for a new anti-TB drug development program.
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Antituberculosos/farmacología , Benzoxazoles/farmacología , Diseño de Fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Quinolinas/farmacología , Antituberculosos/síntesis química , Antituberculosos/química , Benzoxazoles/síntesis química , Benzoxazoles/química , Relación Dosis-Respuesta a Droga , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Quinolinas/síntesis química , Quinolinas/química , Relación Estructura-ActividadRESUMEN
The translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data from Mycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models of in vivo Mycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans.
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Antituberculosos/uso terapéutico , Evaluación Preclínica de Medicamentos/métodos , Investigación Biomédica Traslacional/métodos , Tuberculosis/tratamiento farmacológico , Animales , Larva/efectos de los fármacosRESUMEN
Toxoplasma gondii is a major food pathogen and neglected parasitic infection that causes eye disease, birth defects, and fetal abortion and plays a role as an opportunistic infection in AIDS. In this study, we investigated pantothenic acid (vitamin B5) biosynthesis in T. gondii. Genes encoding the full repertoire of enzymes for pantothenate synthesis and subsequent metabolism to coenzyme A were identified and are expressed in T. gondii. A panel of inhibitors developed to target Mycobacterium tuberculosis pantothenate synthetase were tested and found to exhibit a range of values for inhibition of T. gondii growth. Two inhibitors exhibited lower effective concentrations than the currently used toxoplasmosis drug pyrimethamine. The inhibition was specific for the pantothenate pathway, as the effect of the pantothenate synthetase inhibitors was abrogated by supplementation with pantothenate. Hence, T. gondii encodes and expresses the enzymes for pantothenate synthesis, and this pathway is essential for parasite growth. These promising findings increase our understanding of growth and metabolism in this important parasite and highlight pantothenate synthetase as a new drug target.
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Ácido Pantoténico/biosíntesis , Péptido Sintasas/antagonistas & inhibidores , Toxoplasma/enzimología , Toxoplasmosis/tratamiento farmacológico , Secuencia de Aminoácidos , Línea Celular , Clonación Molecular , Coenzima A/biosíntesis , Humanos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Infecciones Oportunistas/tratamiento farmacológico , Ácido Pantoténico/metabolismo , Ácido Pantoténico/farmacología , Alineación de Secuencia , Toxoplasma/efectos de los fármacos , Toxoplasma/genética , Toxoplasmosis/parasitologíaRESUMEN
In light of the low success rate of target-based genomics and HTS (High Throughput Screening) approaches in anti-infective drug discovery, in silico structure-based drug design (SBDD) is becoming increasingly prominent at the forefront of drug discovery. In silico SBDD can be used to identify novel enzyme inhibitors rapidly, where the strength of this approach lies with its ability to model and predict the outcome of protein-ligand binding. Over the past 10 years, our group have applied this approach to a diverse number of anti-infective drug targets ranging from bacterial D-ala-D-ala ligase to Plasmodium falciparum DHODH. Our search for new inhibitors has produced lead compounds with both enzyme and whole-cell activity with established on-target mode of action. This has been achieved with greater speed and efficiency compared with the more traditional HTS initiatives and at significantly reduced cost and manpower.
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Antiinfecciosos/química , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Péptido Sintasas/antagonistas & inhibidores , Proteínas Protozoarias/antagonistas & inhibidores , Antiinfecciosos/farmacología , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Dihidroorotato Deshidrogenasa , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento/economía , Ensayos Analíticos de Alto Rendimiento/estadística & datos numéricos , Humanos , Ligandos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Simulación del Acoplamiento Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Interfaz Usuario-ComputadorRESUMEN
The de novo pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase is an emerging drug target for the treatment of malaria. In this context a key property of Plasmodium falciparum DHODH (PfDHODH) is that it can be selectively inhibited over its human homologue (HsDHODH). However, HsDHODH is also a validated drug target for autoimmune diseases such as arthritis. Here a series of novel inhibitors is described that includes compounds that switch specificity between the two enzymes as a result of small alterations in chemical structure. Structure-activity relationship (SAR), crystallography, docking, and mutagenesis studies are used to examine the binding modes of the compounds within the two enzymes and to reveal structural changes induced by inhibitor binding. Within this series, compounds with therapeutically relevant HsDHODH activity are described and their binding modes characterized using X-ray crystallography, which reveals a novel conformational shift within the inhibitor binding site.