RESUMEN
The application of dyes to understanding the aetiology of infection inspired antimicrobial chemotherapy and the first wave of antibacterial drugs. The second wave of antibacterial drug discovery was driven by rapid discovery of natural products, now making up 69% of current antibacterial drugs. But now with the most prevalent natural products already discovered, â¼107 new soil-dwelling bacterial species must be screened to discover one new class of natural product. Therefore, instead of a third wave of antibacterial drug discovery, there is now a discovery bottleneck. Unlike natural products which are curated by billions of years of microbial antagonism, the vast synthetic chemical space still requires artificial curation through the therapeutics science of antibacterial drugs - a systematic understanding of how small molecules interact with bacterial physiology, effect desired phenotypes, and benefit the host. Bacterial molecular genetics can elucidate pathogen biology relevant to therapeutics development, but it can also be applied directly to understanding mechanisms and liabilities of new chemical agents with new mechanisms of action. Therefore, the next phase of antibacterial drug discovery could be enabled by integrating chemical expertise with systematic dissection of bacterial infection biology. Facing the ambitious endeavour to find new molecules from nature or new-to-nature which cure bacterial infections, the capabilities furnished by modern chemical biology and molecular genetics can be applied to prospecting for chemical modulators of new targets which circumvent prevalent resistance mechanisms.
Asunto(s)
Antibacterianos , Bacterias , Descubrimiento de Drogas , Antibacterianos/farmacología , Antibacterianos/química , Descubrimiento de Drogas/métodos , Bacterias/genética , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Humanos , Productos Biológicos/farmacología , Productos Biológicos/química , Productos Biológicos/metabolismo , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiologíaRESUMEN
The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD <20â µM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.
Asunto(s)
Mycobacterium tuberculosis , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Nucleotidiltransferasas/metabolismo , Antituberculosos/farmacologíaRESUMEN
The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a K D <20â µM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.
RESUMEN
With increasing prevalence of antimicrobial resistance, a fundamental goal of antibiotic discovery is to uncover new small molecules that prevent growth of pathogenic bacteria through diverse mechanisms of action. This goal is particularly pertinent for tuberculosis, caused by Mycobacterium tuberculosis. In this chapter, we describe the application of a chemical-genetic method, PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), for sensitively detecting small molecule bioactivity using a pooled panel of hypomorphs (strains depleted in a particular essential gene) of M. tuberculosis. We describe statistical and heuristic approaches to assign small molecule mechanism of action from the resulting chemical-genetic interaction profiles.
Asunto(s)
Antituberculosos/clasificación , Antituberculosos/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Descubrimiento de Drogas , Genes Esenciales , Mycobacterium tuberculosis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple , Humanos , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismo , Bibliotecas de Moléculas Pequeñas/clasificación , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Especificidad por SustratoRESUMEN
The efficacies of all antibiotics against tuberculosis are eventually eroded by resistance. New strategies to discover drugs or drug combinations with higher barriers to resistance are needed. Previously, we reported the application of a large-scale chemical-genetic interaction screening strategy called PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) for the discovery of new Mycobacterium tuberculosis inhibitors, which resulted in the identification of the small molecule BRD-8000, an inhibitor of a novel target, EfpA [ Johnson et al. ( 2019 ) Nature 517 , 72 ]. Leveraging the chemical genetic interaction profile of BRD-8000, we identified BRD-9327, another structurally distinct small molecule EfpA inhibitor. We show that the two compounds are synergistic and display collateral sensitivity because of their distinct modes of action and resistance mechanisms. High-level resistance to one increases the sensitivity to and reduces the emergence of resistance to the other. Thus, the combination of BRD-9327 and BRD-8000 represents a proof-of-concept for the novel strategy of leveraging chemical genetics in the design of antimicrobial combination chemotherapy in which mutual collateral sensitivity is exploited.
Asunto(s)
Antituberculosos/química , Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Antituberculosos/aislamiento & purificación , Proteínas Bacterianas/antagonistas & inhibidores , Sinergismo Farmacológico , Quimioterapia Combinada , Proteínas de Transporte de Membrana , Mutación , Prueba de Estudio ConceptualRESUMEN
For the past several decades, chemical biologists have been leveraging chemical principles for understanding biology, tackling disease, and biomanufacturing, while systems biologists have holistically applied computation and genome-scale experimental tools to the same problems. About a decade ago, the benefit of combining the philosophies of chemical biology with systems biology into systems chemical biology was advocated, with the potential to systematically understand the way small molecules affect biological systems. Recently, there has been an explosion in new technologies that permit massive expansion in the scale of biological experimentation, increase access to more diverse chemical space, and enable powerful computational interpretation of large datasets. Fueled by these rapidly increasing capabilities, systems chemical biology is now at an inflection point, poised to enter a new era of more holistic and integrated scientific discovery. Systems chemical biology is primed to reveal an integrated understanding of fundamental biology and to discover new chemical probes to comprehensively dissect and systematically understand that biology, thereby providing a path to novel strategies for discovering therapeutics, designing drug combinations, avoiding toxicity, and harnessing beneficial polypharmacology. In this Review, we examine the emergence of new capabilities driving us to this inflection point in systems chemical biology, and highlight holistic approaches and opportunities that are arising from integrating chemical biology with a systems-level understanding of the intersection of biology and chemistry.
Asunto(s)
Biología de Sistemas , Combinación de Medicamentos , Sistemas de Liberación de Medicamentos , Humanos , PolifarmacologíaRESUMEN
New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.
Asunto(s)
Antituberculosos/clasificación , Antituberculosos/aislamiento & purificación , Descubrimiento de Drogas/métodos , Eliminación de Gen , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Antituberculosos/farmacología , Girasa de ADN/metabolismo , Farmacorresistencia Microbiana , Ácido Fólico/biosíntesis , Terapia Molecular Dirigida , Mycobacterium tuberculosis/citología , Mycobacterium tuberculosis/enzimología , Ácidos Micólicos/metabolismo , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/clasificación , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Especificidad por Sustrato , Inhibidores de Topoisomerasa II/aislamiento & purificación , Inhibidores de Topoisomerasa II/farmacología , Triptófano/biosíntesis , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon-hydrogen bonds in diverse endogenous and exogenous organic compounds by atmospheric oxygen. This C-H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of diverse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron-electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The structure is different from the two known Class I CYP-Fdx structures: CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues in the [Fe2S2] cluster-binding loop and the α3 helix and the C-terminus residue interact with CYP199A2 residues in the proximal loop and the C helix. These residue contacts are consistent with biochemical data on CYP199A2-ferredoxin binding and electron transfer. Electron-tunneling calculations indicate an efficient electron-transfer pathway from the [Fe2S2] cluster to the heme. This new structural model of a CYP-Fdx complex provides the basis for tailoring CYP enzymes for which the cognate ferredoxin is not known, to accept electrons from HaPux and display monooxygenase activity.
RESUMEN
Cytochrome P450 (CYP) enzymes catalyze the insertion of oxygen into carbon-hydrogen bonds and have great potential for enzymatic synthesis. Application development of class I CYPs is hampered by their dependence on two redox partners (a ferredoxin and ferredoxin reductase), slowing catalysis compared to self-sufficient CYPs such as CYP102A1 (P450BM3). Previous attempts to address this have fused all three components in several permutations and geometries, with much reduced activity compared to the native system. We report here the new approach of fusing putidaredoxin reductase (PdR) to the carboxy-terminus of CYP101A1 (P450cam) via a linker peptide and reconstituting camphor hydroxylase activity with free putidaredoxin (Pdx). Initial purification of a P450cam-PdR fusion yielded 2.0% heme incorporation. Co-expression of E. coli ferrochelatase, lengthening the linker from 5 to 20 residues, and altering culture conditions for enzyme production furnished 85% heme content. Fusion co-expression with Pdx gave a functional system with comparable in vivo camphor oxidation activity as the native system. In vitro, the fused system's steady state NADH oxidation rate was two-fold faster than that of the native system. In contrast to the native system, NADH oxidation rates for the fusion enzyme showed non-hyperbolic dependence on Pdx concentration, suggesting a role for the PdR domain; these data were consistent with a kinetic model based on two-site binding of Pdx by P450cam-PdR and inactive dimer formation of the fusion. P450cam-PdR is the first example of a class I P450 fusion that exhibits significantly more favorable behavior than that of the native system.
RESUMEN
A ferredoxin associated with biological Fe-S cluster assembly has been remodelled to transfer electrons to a P450 enzyme and support substrate oxidation at 80% of the physiological ferredoxin activity, opening up the possibility of tailoring ferredoxins to reconstitute the activity of P450 enzymes for which the electron transfer partner proteins are not known.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Ferredoxinas/metabolismo , Transporte de Electrón , Ferredoxinas/química , Ferredoxinas/genética , Hierro/química , Cinética , Mutación , Oxidación-Reducción , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Azufre/químicaRESUMEN
The ubiquitin-proteasome system targets selected proteins for degradation by the 26S proteasome. Rpn12 is an essential component of the 19S regulatory particle and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. In the present paper we report the crystal structure of Rpn12, a proteasomal PCI-domain-containing protein. The structure helps to define a core structural motif for the PCI domain and identifies potential sites through which Rpn12 might form protein-protein interactions. We demonstrate that mutating residues at one of these sites impairs Rpn12 binding to Rpn10 in vitro and reduces Rpn10 incorporation into proteasomes in vivo.
Asunto(s)
Proteínas Portadoras/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Animales , Proteínas de Arabidopsis/química , Complejo del Señalosoma COP9 , Proteínas Portadoras/química , Dicroismo Circular , Cristalografía por Rayos X , Proteínas de Drosophila/química , Proteínas Asociadas a Microtúbulos/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas de Unión al ARN , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Relación Estructura-Actividad , Ubiquitina/metabolismo , Factores de Transcripción Winged-Helix/químicaRESUMEN
The crystal structures of the 4-methoxybenzoate bound forms of cytochrome P450 enzymes CYP199A2 and CYP199A4 from the Rhodopseudomonas palustris strains CGA009 and HaA2 have been solved. The structures of these two enzymes, which share 86% sequence identity, are very similar though some differences are found on the proximal surface. In these structures the enzymes have a closed conformation, in contrast to the substrate-free form of CYP199A2 where an obvious substrate access channel is observed. The switch from an open to a closed conformation arises from pronounced residue side-chain movements and alterations of ion pair and hydrogen bonding interactions at the entrance of the access channel. A chloride ion bound just inside the protein surface caps the entrance to the active site and protects the substrate and the heme from the external solvent. In both structures the substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of these enzymes for oxidative demethylation of the substrate. Mutagenesis studies on CYP199A4 highlight the involvement of hydrophobic (Phe185) and hydrophilic (Arg92, Ser95 and Arg243) amino acid residues in the binding of para-substituted benzoates by these enzymes.
Asunto(s)
Proteínas Bacterianas/química , Sistema Enzimático del Citocromo P-450/química , Hidroxibenzoatos/química , Sitios de Unión , Cloruros/química , Cristalización , Éteres de Hidroxibenzoatos , Conformación Proteica , Rhodopseudomonas/enzimología , Especificidad por SustratoRESUMEN
CYP199A4 (RPB3613) from Rhodopseudomonas palustris HaA2 is a heme monooxygenase that catalyzes the hydroxylation of para-substituted benzoic acids. Monooxygenase activity of CYP199A4 can be reconstituted in a Class I electron transfer chain with an associated [2Fe-2S] ferredoxin, HaPux, (RPB3614) and the flavin-dependent reductase, HaPuR, (RPB3656) that is not associated with a CYP gene. CYP199A4 and the ferredoxin HaPux are produced in greater quantities using recombinant Escherichia coli expression systems when compared to the equivalent proteins in the closely related CYP199A2-Pux-PuR Class I system from R. palustris CGA009. HaPuR and HaPux can also replace PuR and Pux in supporting the CYP199A2 enzyme turnover with high activity. Whole-cell in vivo substrate oxidation systems for CYP199A4 and CYP199A2 with HaPux and HaPuR as the electron transfer proteins have been constructed. These E. coli systems were capable of selectively demethylating veratric acid at the para position to produce vanillic acid at rates of up to 15.3 microM (g-cdw)(-1) min(-1) and yields of up to 1.2 g L(-1).
Asunto(s)
Proteínas Bacterianas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Rhodopseudomonas/metabolismo , Ácido Vanílico/análogos & derivados , Ácido Vanílico/metabolismo , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ferredoxinas/metabolismo , Cinética , Metilación , Oxigenasas de Función Mixta/genética , Rhodopseudomonas/genéticaRESUMEN
CYP199A2 from Rhodopseudomonas palustris CGA009 is a heme monooxygenase that catalyzes the oxidation of para-substituted benzoic acids. CYP199A2 activity is reconstituted by a class I electron transfer chain consisting of the associated [2Fe-2S] ferredoxin palustrisredoxin (Pux) and a flavoprotein palustrisredoxin reductase (PuR). Another [2Fe-2S] ferredoxin, palustrisredoxin B (PuxB; RPA3956) has been identified in the genome. PuxB shares sequence identity and motifs with vertebrate-type ferredoxins involved in Fe-S cluster assembly but also 50% identity with Pux and it mediates electron transfer from PuR to CYP199A2, albeit with lower steady-state turnover activity: 99 nmol (nmol P450)(-1)min(-1) for 4-methoxybenzoic acid oxidation compared with 1,438 nmol (nmol P450)(-1 )min(-1) for Pux. This difference mainly arises from weak CYP199A2-PuxB binding (K (m) 34.3 vs. 0.45 microM for Pux) rather than slow electron transfer (k (cat) 19.1 vs. 37.9 s(-1) for Pux). Comparison of the 2.0-A-resolution crystal structure of the PuxB A105R mutant with other vertebrate-type, P450-associated ferredoxins revealed similar protein folds but also significant differences in some loop regions. Therefore, PuxB offers a platform for studying ferredoxin-P450 recognition in class I P450 systems. Substitution of PuxB residues at key locations with those in Pux shows that Ala42, Cys43, and Ala44 in the [2Fe-2S] cluster binding loop and Met66 are important in electron transfer from PuxB to CYP199A2, whereas Phe73 and the C-terminal Ala105 were involved in both protein binding and electron transfer.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Ferredoxinas/química , Ferredoxinas/metabolismo , Rhodopseudomonas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Transporte de Electrón , Ferredoxinas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Secundaria de Proteína , Rhodopseudomonas/química , Alineación de SecuenciaRESUMEN
Cytochrome P450-199A2 from Rhodopseudomonas palustris oxidizes para-substituted benzoic acids and may play a role in lignin and aromatic acid degradation pathways in the bacterium. CYP199A2 has an associated [2Fe-2S] ferredoxin, palustrisredoxin (Pux) but not a ferredoxin reductase. A genome search identified the palustrisredoxin reductase (PuR) gene. PuR was produced in Escherichia coli and shown to be a flavin-dependent protein that supports efficient electron transfer from NADH to Pux, thus reconstituting CYP199A2 monooxygenase activity (k(cat) = 37.9 s(-1) with 4-methoxybenzoic acid). The reduction of Pux by PuR shows K(m) = 4.2 microM and k(cat) = 262 s(-1) in 50 mM Tris, pH 7.4. K(m) is increased to 154 microM in the presence of 200 mM KCl, indicating the importance of ionic interactions in PuR/Pux binding. The crystal structure of PuR has been determined at 2.2 A resolution and found to be closely related to that of other oxygenase-coupled NADH-dependent ferredoxin reductases. Residues on the surface that had been proposed to be involved in ferredoxin reductase-ferredoxin binding are conserved in PuR. However, Lys328 in PuR lies over the FAD isoalloxazine ring and, together with His11 and Gln41, render the electrostatic potential of the surface more positive and may account for the greater involvement of electrostatic interactions in ferredoxin binding by PuR. Consistent with these observations the K328G mutation weakened Pux binding and virtually eliminated the dependence of PuR/Pux binding on salt concentration, thus confirming that the FAD si side surface in the vicinity of Lys328 is the ferredoxin binding site.