RESUMEN
Development of γ-secretase inhibitors (GSIs) and modulators (GSMs) represents an attractive therapeutic opportunity for Alzheimer's disease (AD) and cancers. However, how these GSIs and GSMs target γ-secretase has remained largely unknown. Here, we report the cryoelectron microscopy (cryo-EM) structures of human γ-secretase bound individually to two GSI clinical candidates, Semagacestat and Avagacestat, a transition state analog GSI L685,458, and a classic GSM E2012, at overall resolutions of 2.6-3.1 Å. Remarkably, each of the GSIs occupies the same general location on presenilin 1 (PS1) that accommodates the ß strand from amyloid precursor protein or Notch, interfering with substrate recruitment. L685,458 directly coordinates the two catalytic aspartate residues of PS1. E2012 binds to an allosteric site of γ-secretase on the extracellular side, potentially explaining its modulating activity. Structural analysis reveals a set of shared themes and variations for inhibitor and modulator recognition that will guide development of the next-generation substrate-selective inhibitors.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Preparaciones Farmacéuticas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/ultraestructura , Azepinas/farmacología , Sitios de Unión , Microscopía por Crioelectrón , Células HEK293 , Humanos , Modelos Biológicos , Modelos Moleculares , Oxadiazoles/química , Oxadiazoles/farmacología , Presenilina-1/química , Presenilina-1/metabolismo , Unión Proteica/efectos de los fármacos , Conformación Proteica , Relación Estructura-Actividad , Especificidad por Sustrato/efectos de los fármacos , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.
Asunto(s)
Factor de Transcripción Asociado a Microftalmía/metabolismo , NADP Transhidrogenasas/metabolismo , Pigmentación de la Piel/efectos de la radiación , Rayos Ultravioleta , Animales , Línea Celular , Estudios de Cohortes , AMP Cíclico/metabolismo , Daño del ADN , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Predisposición Genética a la Enfermedad , Humanos , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Melanosomas/efectos de los fármacos , Melanosomas/metabolismo , Melanosomas/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , NADP Transhidrogenasas/antagonistas & inhibidores , Oxidación-Reducción/efectos de los fármacos , Oxidación-Reducción/efectos de la radiación , Polimorfismo de Nucleótido Simple/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Proteolisis/efectos de la radiación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Pigmentación de la Piel/efectos de los fármacos , Pigmentación de la Piel/genética , Ubiquitina/metabolismo , Pez CebraRESUMEN
Myosins are among the most fascinating enzymes in biology. As extremely allosteric chemomechanical molecular machines, myosins are involved in myriad pivotal cellular functions and are frequently sites of mutations leading to disease phenotypes. Human ß-cardiac myosin has proved to be an excellent target for small-molecule therapeutics for heart muscle diseases, and, as we describe here, other myosin family members are likely to be potentially unique targets for treating other diseases as well. The first part of this review focuses on how myosins convert the chemical energy of ATP hydrolysis into mechanical movement, followed by a description of existing therapeutic approaches to target human ß-cardiac myosin. The next section focuses on the possibility of targeting nonmuscle members of the human myosin family for several diseases. We end the review by describing the roles of myosin in parasites and the therapeutic potential of targeting them to block parasitic invasion of their hosts.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Miosinas/metabolismo , Neoplasias/tratamiento farmacológico , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Infecciones por Protozoos/tratamiento farmacológico , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Fenómenos Biomecánicos , Cryptosporidium/efectos de los fármacos , Cryptosporidium/enzimología , Inhibidores Enzimáticos/química , Expresión Génica , Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Humanos , Familia de Multigenes , Mutación , Miosinas/antagonistas & inhibidores , Miosinas/clasificación , Miosinas/genética , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Enfermedades del Sistema Nervioso/enzimología , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/patología , Plasmodium/efectos de los fármacos , Plasmodium/enzimología , Infecciones por Protozoos/enzimología , Infecciones por Protozoos/genética , Infecciones por Protozoos/patología , Toxoplasma/efectos de los fármacos , Toxoplasma/enzimologíaRESUMEN
Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.
Asunto(s)
Antibacterianos/química , Antineoplásicos/química , Infecciones por Escherichia coli/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Nucleótidos/metabolismo , Ribonucleótido Reductasas/química , Antibacterianos/uso terapéutico , Antineoplásicos/uso terapéutico , Biocatálisis , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Infecciones por Escherichia coli/enzimología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Humanos , Simulación del Acoplamiento Molecular , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Nucleótidos/química , Oxidación-Reducción , Estructura Secundaria de Proteína , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Relación Estructura-ActividadRESUMEN
Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Trastornos Congénitos de Glicosilación/metabolismo , Inhibidores Enzimáticos/farmacología , N-Acetilglucosaminiltransferasas/química , Animales , Antibióticos Antituberculosos/química , Sitios de Unión , Trastornos Congénitos de Glicosilación/genética , Inhibidores Enzimáticos/química , Femenino , Células HEK293 , Células Hep G2 , Humanos , Metabolismo de los Lípidos , Ratones , Simulación del Acoplamiento Molecular , Mutación , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Unión Proteica , Células Sf9 , Spodoptera , Tunicamicina/química , Tunicamicina/farmacología , Uridina Difosfato Ácido Glucurónico/química , Uridina Difosfato Ácido Glucurónico/metabolismoRESUMEN
All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins' physiological target is Midasin, an essential â¼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin's role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Subunidades Ribosómicas Grandes de Eucariotas/efectos de los fármacos , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Schizosaccharomyces pombe/antagonistas & inhibidores , Triazinas/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Indoles/química , Indoles/aislamiento & purificación , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/metabolismo , Relación Estructura-Actividad , Triazinas/química , Triazinas/aislamiento & purificaciónRESUMEN
While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.
Asunto(s)
Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Terapia Molecular Dirigida , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Diferenciación Celular , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento , Proteínas de Homeodominio/genética , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Células Mieloides/patología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Pirimidinas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.
Asunto(s)
Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , National Institutes of Health (U.S.) , Estados UnidosRESUMEN
WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.
Asunto(s)
Regulación Alostérica , Descubrimiento de Drogas , Inhibidores Enzimáticos , Proteómica , Helicasa del Síndrome de Werner , Animales , Femenino , Humanos , Masculino , Ratones , Regulación Alostérica/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Cisteína/efectos de los fármacos , Cisteína/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inestabilidad de Microsatélites , Modelos Moleculares , Helicasa del Síndrome de Werner/antagonistas & inhibidores , Helicasa del Síndrome de Werner/química , Helicasa del Síndrome de Werner/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Muerte Celular/efectos de los fármacos , Adenosina Trifosfato/metabolismoRESUMEN
The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.
Asunto(s)
Antineoplásicos , Descubrimiento de Drogas , Inhibidores Enzimáticos , Inestabilidad de Microsatélites , Neoplasias , Mutaciones Letales Sintéticas , Helicasa del Síndrome de Werner , Animales , Femenino , Humanos , Ratones , Administración Oral , Regulación Alostérica/efectos de los fármacos , Antineoplásicos/efectos adversos , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Daño del ADN/efectos de los fármacos , Inhibidores Enzimáticos/efectos adversos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismo , Dominios Proteicos , Reproducibilidad de los Resultados , Supresión Genética , Mutaciones Letales Sintéticas/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Helicasa del Síndrome de Werner/antagonistas & inhibidores , Helicasa del Síndrome de Werner/genética , Helicasa del Síndrome de Werner/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Eukaryotic and prokaryotic organisms possess huge numbers of uncharacterized enzymes. Selective inhibitors offer powerful probes for assigning functions to enzymes in native biological systems. Here, we discuss how the chemical proteomic platform activity-based protein profiling (ABPP) can be implemented to discover selective and in vivo-active inhibitors for enzymes. We further describe how these inhibitors have been used to delineate the biochemical and cellular functions of enzymes, leading to the discovery of metabolic and signaling pathways that make important contributions to human physiology and disease. These studies demonstrate the value of selective chemical probes as drivers of biological inquiry.
Asunto(s)
Química Farmacéutica/métodos , Diseño de Fármacos , Inhibidores Enzimáticos/química , Proteómica/métodos , Animales , Unión Competitiva , Línea Celular Tumoral , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Humanos , Lactonas/química , Fenotipo , Fotoquímica/métodos , ProteomaRESUMEN
4-Hydroxyphenylpyruvate dioxygenase (HPPD) plays a key role in tyrosine metabolism and has been identified as a promising target for herbicide and drug discovery. The structures of HPPD complexed with different types of inhibitors have been determined previously. We summarize the structures of HPPD complexed with structurally diverse molecules, including inhibitors, natural products, substrates, and catalytic intermediates; from these structures, the detailed inhibitory mechanisms of different inhibitors were analyzed and compared, and the key structural factors determining the slow-binding behavior of inhibitors were identified. Further, we propose four subpockets that accommodate different inhibitor substructures. We believe that these analyses will facilitate in-depth understanding of the enzymatic reaction mechanism and enable the design of new inhibitors with higher potency and selectivity.
Asunto(s)
4-Hidroxifenilpiruvato Dioxigenasa , Herbicidas , 4-Hidroxifenilpiruvato Dioxigenasa/química , 4-Hidroxifenilpiruvato Dioxigenasa/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Herbicidas/farmacología , Herbicidas/química , Catálisis , BiologíaRESUMEN
Isoprenoids are vital for all organisms, in which they maintain membrane stability and support core functions such as respiration1. IspH, an enzyme in the methyl erythritol phosphate pathway of isoprenoid synthesis, is essential for Gram-negative bacteria, mycobacteria and apicomplexans2,3. Its substrate, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), is not produced in metazoans, and in humans and other primates it activates cytotoxic Vγ9Vδ2 T cells at extremely low concentrations4-6. Here we describe a class of IspH inhibitors and refine their potency to nanomolar levels through structure-guided analogue design. After modification of these compounds into prodrugs for delivery into bacteria, we show that they kill clinical isolates of several multidrug-resistant bacteria-including those from the genera Acinetobacter, Pseudomonas, Klebsiella, Enterobacter, Vibrio, Shigella, Salmonella, Yersinia, Mycobacterium and Bacillus-yet are relatively non-toxic to mammalian cells. Proteomic analysis reveals that bacteria treated with these prodrugs resemble those after conditional IspH knockdown. Notably, these prodrugs also induce the expansion and activation of human Vγ9Vδ2 T cells in a humanized mouse model of bacterial infection. The prodrugs we describe here synergize the direct killing of bacteria with a simultaneous rapid immune response by cytotoxic γδ T cells, which may limit the increase of antibiotic-resistant bacterial populations.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/inmunología , Activación de Linfocitos/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Oxidorreductasas/antagonistas & inhibidores , Linfocitos T Citotóxicos/efectos de los fármacos , Animales , Farmacorresistencia Microbiana , Resistencia a Múltiples Medicamentos , Inhibidores Enzimáticos/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Femenino , Semivida , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Oxidorreductasas/deficiencia , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Profármacos/farmacocinética , Profármacos/farmacología , Especificidad por Sustrato , Porcinos/sangre , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides using radical-based chemistry. For class Ia RNRs, the radical species is stored in a separate subunit (ß2) from the subunit housing the active site (α2), requiring the formation of a short-lived α2ß2 complex and long-range radical transfer (RT). RT occurs via proton-coupled electron transfer (PCET) over a long distance (~32-Å) and involves the formation and decay of multiple amino acid radical species. Here, we use cryogenic electron microscopy and a mechanism-based inhibitor 2'-azido-2'-deoxycytidine-5'-diphosphate (N3CDP) to trap a wild-type α2ß2 complex of Escherichia coli class Ia RNR. We find that one α subunit has turned over and that the other is trapped, bound to ß in a midturnover state. Instead of N3CDP in the active site, forward RT has resulted in N2 loss, migration of the third nitrogen from the ribose C2' to C3' positions, and attachment of this nitrogen to the sulfur of cysteine-225. In this study, an inhibitor has been visualized as an adduct to an RNR. Additionally, this structure reveals the positions of PCET residues following forward RT, complementing the previous structure that depicted a preturnover PCET pathway and suggesting how PCET is gated at the α-ß interface. This N3CDP-trapped structure is also of sufficient resolution (2.6 Å) to visualize water molecules, allowing us to evaluate the proposal that water molecules are proton acceptors and donors as part of the PCET process.
Asunto(s)
Microscopía por Crioelectrón , Escherichia coli , Ribonucleótido Reductasas , Microscopía por Crioelectrón/métodos , Ribonucleótido Reductasas/química , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/metabolismo , Escherichia coli/enzimología , Escherichia coli/metabolismo , Dominio Catalítico , Citidina Difosfato/química , Citidina Difosfato/metabolismo , Modelos Moleculares , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidoresRESUMEN
Experimental analysis of enzymatic transition-state structures uses kinetic isotope effects (KIEs) to report on bonding and geometry differences between reactants and the transition state. Computational correlation of experimental values with chemical models permits three-dimensional geometric and electrostatic assignment of transition states formed at enzymatic catalytic sites. The combination of experimental and computational access to transition-state information permits (a) the design of transition-state analogs as powerful enzymatic inhibitors, (b) exploration of protein features linked to transition-state structure, (c) analysis of ensemble atomic motions involved in achieving the transition state, (d) transition-state lifetimes, and (e) separation of ground-state (Michaelis complexes) from transition-state effects. Transition-state analogs with picomolar dissociation constants have been achieved for several enzymatic targets. Transition states of closely related isozymes indicate that the protein's dynamic architecture is linked to transition-state structure. Fast dynamic motions in catalytic sites are linked to transition-state generation. Enzymatic transition states have lifetimes of femtoseconds, the lifetime of bond vibrations. Binding isotope effects (BIEs) reveal relative reactant and transition-state analog binding distortion for comparison with actual transition states.
Asunto(s)
Enzimas/química , Enzimas/metabolismo , Conformación Proteica , Animales , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Isótopos/química , Cinética , Modelos Moleculares , Estructura Molecular , Nucleósidos/química , Electricidad Estática , TermodinámicaRESUMEN
Telomere replication is essential for continued proliferation of human cells, such as stem cells and cancer cells. Telomerase lengthens the telomeric G-strand, while C-strand replication is accomplished by CST-polymerase α-primase (CST-PP). Replication of both strands is inhibited by formation of G-quadruplex (GQ) structures in the G-rich single-stranded DNA. TMPyP4 and pyridostatin (PDS), which stabilize GQ structures in both DNA and RNA, inhibit telomerase in vitro, and in human cells they cause telomere shortening that has been attributed to telomerase inhibition. Here, we show that TMPyP4 and PDS also inhibit C-strand synthesis by stabilizing DNA secondary structures and thereby preventing CST-PP from binding to telomeric DNA. We also show that these small molecules inhibit CST-PP binding to a DNA sequence containing no consecutive guanine residues, which is unlikely to form GQs. Thus, while these "telomerase inhibitors" indeed inhibit telomerase, they are also robust inhibitors of telomeric C-strand synthesis. Furthermore, given their binding to GQ RNA and their limited specificity for GQ structures, they may disrupt many other protein-nucleic acid interactions in human cells.
Asunto(s)
Inhibidores Enzimáticos , G-Cuádruplex , Telomerasa , Telómero , Telomerasa/antagonistas & inhibidores , Telomerasa/metabolismo , Telomerasa/genética , Humanos , Telómero/metabolismo , G-Cuádruplex/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Ácidos Picolínicos/farmacología , Ácidos Picolínicos/química , Replicación del ADN/efectos de los fármacos , ADN Polimerasa I/antagonistas & inhibidores , ADN Polimerasa I/metabolismo , ADN/metabolismo , Aminoquinolinas , Porfirinas , ADN PrimasaRESUMEN
The development of cancer therapeutics is often hindered by the fact that specific oncogenes cannot be directly pharmaceutically addressed. Targeting deubiquitylases that stabilize these oncogenes provides a promising alternative. USP28 and USP25 have been identified as such target deubiquitylases, and several small-molecule inhibitors indiscriminately inhibiting both enzymes have been developed. To obtain insights into their mode of inhibition, we structurally and functionally characterized USP28 in the presence of the three different inhibitors AZ1, Vismodegib and FT206. The compounds bind into a common pocket acting as a molecular sink. Our analysis provides an explanation why the two enzymes are inhibited with similar potency while other deubiquitylases are not affected. Furthermore, a key glutamate residue at position 366/373 in USP28/USP25 plays a central structural role for pocket stability and thereby for inhibition and activity. Obstructing the inhibitor-binding pocket by mutation of this glutamate may provide a tool to accelerate future drug development efforts for selective inhibitors of either USP28 or USP25 targeting distinct binding pockets.
Asunto(s)
Ubiquitina Tiolesterasa , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Humanos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Sitios de Unión , Piridinas/química , Piridinas/farmacología , Unión Proteica , Modelos MolecularesRESUMEN
A promising yet clinically unexploited antibiotic target in difficult-to-treat Gram-negative bacteria is LpxC, the key enzyme in the biosynthesis of lipopolysaccharides, which are the major constituents of the outer membrane. Despite the development of dozens of chemically diverse LpxC inhibitor molecules, it is essentially unknown how bacteria counteract LpxC inhibition. Our study provides comprehensive insights into the response against five different LpxC inhibitors. All compounds bound to purified LpxC from Escherichia coli. Treatment of E. coli with these compounds changed the cell shape and stabilized LpxC suggesting that FtsH-mediated proteolysis of the inactivated enzyme is impaired. LpxC inhibition sensitized E. coli to vancomycin and rifampin, which poorly cross the outer membrane of intact cells. Four of the five compounds led to an accumulation of lyso-phosphatidylethanolamine, a cleavage product of phosphatidylethanolamine, generated by the phospholipase PldA. The combined results suggested an imbalance in lipopolysaccharides and phospholipid biosynthesis, which was corroborated by the global proteome response to treatment with the LpxC inhibitors. Apart from LpxC itself, FabA and FabB responsible for the biosynthesis of unsaturated fatty acids were consistently induced. Upregulated compound-specific proteins are involved in various functional categories, such as stress reactions, nucleotide, or amino acid metabolism and quorum sensing. Our work shows that antibiotics targeting the same enzyme do not necessarily elicit identical cellular responses. Moreover, we find that the response of E. coli to LpxC inhibition is distinct from the previously reported response in Pseudomonas aeruginosa.
Asunto(s)
Amidohidrolasas , Inhibidores Enzimáticos , Escherichia coli , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/metabolismo , Antibacterianos/farmacología , Antibacterianos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Lipopolisacáridos/biosíntesis , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Farmacorresistencia Bacteriana/efectos de los fármacos , Membrana Celular/efectos de los fármacosRESUMEN
The process of virtual screening relies heavily on the databases, but it is disadvantageous to conduct virtual screening based on commercial databases with patent-protected compounds, high compound toxicity and side effects. Therefore, this paper utilizes generative recurrent neural networks (RNN) containing long short-term memory (LSTM) cells to learn the properties of drug compounds in the DrugBank, aiming to obtain a new and virtual screening compounds database with drug-like properties. Ultimately, a compounds database consisting of 26,316 compounds is obtained by this method. To evaluate the potential of this compounds database, a series of tests are performed, including chemical space, ADME properties, compound fragmentation, and synthesizability analysis. As a result, it is proved that the database is equipped with good drug-like properties and a relatively new backbone, its potential in virtual screening is further tested. Finally, a series of seedling compounds with completely new backbones are obtained through docking and binding free energy calculations.
Asunto(s)
Aprendizaje Profundo , Simulación del Acoplamiento Molecular , Simulación del Acoplamiento Molecular/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Humanos , Bases de Datos Farmacéuticas , Redes Neurales de la Computación , Bases de Datos de Compuestos QuímicosRESUMEN
ATP-citrate lyase (ACLY) is a central metabolic enzyme and catalyses the ATP-dependent conversion of citrate and coenzyme A (CoA) to oxaloacetate and acetyl-CoA1-5. The acetyl-CoA product is crucial for the metabolism of fatty acids6,7, the biosynthesis of cholesterol8, and the acetylation and prenylation of proteins9,10. There has been considerable interest in ACLY as a target for anti-cancer drugs, because many cancer cells depend on its activity for proliferation2,5,11. ACLY is also a target against dyslipidaemia and hepatic steatosis, with a compound currently in phase 3 clinical trials4,5. Many inhibitors of ACLY have been reported, but most of them have weak activity5. Here we report the development of a series of low nanomolar, small-molecule inhibitors of human ACLY. We have also determined the structure of the full-length human ACLY homo-tetramer in complex with one of these inhibitors (NDI-091143) by cryo-electron microscopy, which reveals an unexpected mechanism of inhibition. The compound is located in an allosteric, mostly hydrophobic cavity next to the citrate-binding site, and requires extensive conformational changes in the enzyme that indirectly disrupt citrate binding. The observed binding mode is supported by and explains the structure-activity relationships of these compounds. This allosteric site greatly enhances the 'druggability' of ACLY and represents an attractive target for the development of new ACLY inhibitors.