RESUMO
The highly conserved and essential Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as the leading target for vaccines against the disease-causing blood stage of malaria. However, the features of the human vaccine-induced antibody response that confer highly potent inhibition of malaria parasite invasion into red blood cells are not well defined. Here, we characterize 236 human IgG monoclonal antibodies, derived from 15 donors, induced by the most advanced PfRH5 vaccine. We define the antigenic landscape of this molecule and establish that epitope specificity, antibody association rate, and intra-PfRH5 antibody interactions are key determinants of functional anti-parasitic potency. In addition, we identify a germline IgG gene combination that results in an exceptionally potent class of antibody and demonstrate its prophylactic potential to protect against P. falciparum parasite challenge in vivo. This comprehensive dataset provides a framework to guide rational design of next-generation vaccines and prophylactic antibodies to protect against blood-stage malaria.
Assuntos
Anticorpos Monoclonais , Anticorpos Antiprotozoários , Antígenos de Protozoários , Imunoglobulina G , Vacinas Antimaláricas , Malária Falciparum , Plasmodium falciparum , Proteínas de Protozoários , Humanos , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/imunologia , Anticorpos Antiprotozoários/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Malária Falciparum/parasitologia , Animais , Proteínas de Protozoários/imunologia , Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Antígenos de Protozoários/imunologia , Camundongos , Epitopos/imunologia , Eritrócitos/parasitologia , Eritrócitos/imunologia , Feminino , Proteínas de Transporte/imunologiaRESUMO
Pantothenamides inhibit blood-stage Plasmodium falciparum with potencies (50% inhibitory concentration [IC50], â¼20 nM) similar to that of chloroquine. They target processes dependent on pantothenate, a precursor of the essential metabolic cofactor coenzyme A. However, their antiplasmodial activity is reduced due to degradation by serum pantetheinase. Minor modification of the pantothenamide structure led to the identification of α-methyl-N-phenethyl-pantothenamide, a pantothenamide resistant to degradation, with excellent antiplasmodial activity (IC50, 52 ± 6 nM), target specificity, and low toxicity.
Assuntos
Amidoidrolases/metabolismo , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Cloroquina/farmacologia , Proteínas Ligadas por GPI/metabolismo , Plasmodium falciparum/efeitos dos fármacosRESUMO
Chikungunya virus (CHIKV) causes severe fever, rash and debilitating joint pain that can last for months1,2or even years. Millions of people have been infected with CHIKV, mostly in low and middle-income countries, and the virus continues to spread into new areas due to the geographical expansion of its mosquito hosts. Its genome encodes a macrodomain, which functions as an ADP-ribosyl hydrolase, removing ADPr from viral and host-cell proteins interfering with the innate immune response. Mutational studies have shown that the CHIKV nsP3 macrodomain is necessary for viral replication, making it a potential target for the development of antiviral therapeutics. We, therefore, performed a high-throughput crystallographic fragment screen against the CHIKV nsP3 macrodomain, yielding 109 fragment hits covering the ADPr-binding site and two adjacent subsites that are absent in the homologous macrodomain of SARS-CoV-2 but may be present in other alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV). Finally, a subset of overlapping fragments was used to manually design three fragment merges covering the adenine and oxyanion subsites. The rich dataset of chemical matter and structural information discovered from this fragment screen is publicly available and can be used as a starting point for developing a CHIKV nsP3 macrodomain inhibitor.
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Zika virus (ZIKV) infections cause microcephaly in new-borns and Guillain-Barre syndrome in adults raising a significant global public health concern, yet no vaccines or antiviral drugs have been developed to prevent or treat ZIKV infections. The viral protease NS3 and its co-factor NS2B are essential for the cleavage of the Zika polyprotein precursor into individual structural and non-structural proteins and is therefore an attractive drug target. Generation of a robust crystal system of co-expressed NS2B-NS3 protease has enabled us to perform a crystallographic fragment screening campaign with 1076 fragments. 48 binders with diverse chemical scaffolds were identified in the active site of the protease, with another 6 fragment hits observed in a potential allosteric binding site. Our work provides potential starting points for the development of potent NS2B-NS3 protease inhibitors. Furthermore, we have structurally characterized a potential allosteric binding pocket, identifying opportunities for allosteric inhibitor development.
RESUMO
Enteroviruses are the causative agents of paediatric hand-foot-and-mouth disease, and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak. The 2A protease of these viruses is responsible for the self-cleavage of the poly protein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. These 2A proteases are highly conserved between Enterovirus species, such as Enterovirus A71 and Coxsackievirus A16 . Inhibition of the 2A protease deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. Herein, we describe a crystallographic fragment screening campaign that identified 75 fragments which bind to the 2A protease including 38 unique compounds shown to bind within the active site. These fragments reveal a path for the development of non-peptidomimetic inhibitors of the 2A protease with broad-spectrum anti-enteroviral activity.
RESUMO
The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barré syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3Hel) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3Hel. Here we applied high-throughput crystallographic fragment screening on ZIKV NS3Hel, which yielded structures that reveal 3D binding poses of 46 fragments at multiple sites of the protein, including 11 unique fragments in the RNA-cleft site. These fragment structures provide templates for direct design of hit compounds and should thus assist the development of novel direct-acting antivirals against ZIKV and related flaviviruses, thus opening a promising avenue for combating future outbreaks.
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A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunity-driven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development.
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The research and development of a new antimicrobial drug using a target-based approach raises the question of whether any resulting hits will also show activity against the homologous target in other closely related organisms. While an assessment of the similarities of the predicted interactions between the identified inhibitor and the various targets is an obvious first step in answering this question, no clear and consistent framework has been proposed for how this should be done. Here we developed Multifaceted Target Specificity Analysis (MTSA) and applied it to typeâ III pantothenate kinase (PanKIII ) - an essential enzyme required for coenzyme A biosynthesis in a wide range of pathogenic bacteria - as a case study to establish if targeting a specific organism's PanKIII would lead to a narrow- or broad-spectrum agent. We propose that MTSA is a useful tool and aid for directing new target-based antimicrobial drug development initiatives.
Assuntos
Anti-Infecciosos , Ácido Pantotênico , Ácido Pantotênico/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool) , BactériasRESUMO
The main protease from SARS-CoV-2 (Mpro) is responsible for cleavage of the viral polyprotein. Mpro self-processing is called maturation, and it is crucial for enzyme dimerization and activity. Here we use C145S Mpro to study the structure and dynamics of N-terminal cleavage in solution. Native mass spectroscopy analysis shows that mixed oligomeric states are composed of cleaved and uncleaved particles, indicating that N-terminal processing is not critical for dimerization. A 3.5 Å cryo-EM structure provides details of Mpro N-terminal cleavage outside the constrains of crystal environment. We show that different classes of inhibitors shift the balance between oligomeric states. While non-covalent inhibitor MAT-POS-e194df51-1 prevents dimerization, the covalent inhibitor nirmatrelvir induces the conversion of monomers into dimers, even with intact N-termini. Our data indicates that the Mpro dimerization is triggered by induced fit due to covalent linkage during substrate processing rather than the N-terminal processing.
Assuntos
Proteases 3C de Coronavírus , SARS-CoV-2 , Antivirais , Inibidores de Proteases/farmacologia , SARS-CoV-2/enzimologia , Proteases 3C de Coronavírus/químicaRESUMO
We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.
Assuntos
Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus , Inibidores de Protease de Coronavírus , Descoberta de Drogas , SARS-CoV-2 , Humanos , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Simulação de Acoplamento Molecular , Inibidores de Protease de Coronavírus/síntese química , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Relação Estrutura-Atividade , Cristalografia por Raios XRESUMO
New antibiotics with either a novel mode of action or novel mode of inhibition are urgently needed to overcome the threat of drug-resistant tuberculosis (TB). The present study profiles new spiropyrimidinetriones (SPTs), DNA gyrase inhibitors having activity against drug-resistant Mycobacterium tuberculosis (Mtb), the causative agent of TB. While the clinical candidate zoliflodacin has progressed to phase 3 trials for the treatment of gonorrhea, compounds herein demonstrated higher inhibitory potency against Mtb DNA gyrase (e.g., compound 42 with IC50 = 2.0) and lower Mtb minimum inhibitor concentrations (0.49 µM for 42). Notably, 42 and analogues showed selective Mtb activity relative to representative Gram-positive and Gram-negative bacteria. DNA gyrase inhibition was shown to involve stabilization of double-cleaved DNA, while on-target activity was supported by hypersensitivity against a gyrA hypomorph. Finally, a docking model for SPTs with Mtb DNA gyrase was developed, and a structural hypothesis was built for structure-activity relationship expansion.
Assuntos
Mycobacterium tuberculosis , Inibidores da Topoisomerase II , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Antituberculosos/química , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , DNA Girase/genética , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Testes de Sensibilidade Microbiana , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Inibidores da Topoisomerase II/uso terapêuticoRESUMO
The pentafluorosulfanyl (-SF5 ) functional group is of increasing interest as a bioisostere in medicinal chemistry. A library of SF5 -containing compounds, including amide, isoxazole, and oxindole derivatives, was synthesised using a range of solution-based and solventless methods, including microwave and ball-mill techniques. The library was tested against targets including human dihydroorotate dehydrogenase (HDHODH). A subsequent focused approach led to synthesis of analogues of the clinically used disease modifying anti-rheumatic drugs (DMARDs), Teriflunomide and Leflunomide, considered for potential COVID-19 use, where SF5 bioisostere deployment led to improved inhibition of HDHODH compared with the parent drugs. The results demonstrate the utility of the SF5 group in medicinal chemistry.
Assuntos
Química Farmacêutica , Di-Hidro-Orotato Desidrogenase , Amidas , Di-Hidro-Orotato Desidrogenase/antagonistas & inibidores , HumanosRESUMO
The pantothenate analogue hopantenate (HoPan) is widely used as a modulator of coenzyme A (CoA) levels in cell biology and disease modelsâespecially for pantothenate kinase associated neurodegeneration (PKAN), a genetic disease rooted in impaired CoA metabolism. This use of HoPan was based on reports that it inhibits pantothenate kinase (PanK), the first enzyme of CoA biosynthesis. Using a combination of in vitro enzyme kinetic studies, crystal structure analysis, and experiments in a typical PKAN cell biology model, we demonstrate that instead of inhibiting PanK, HoPan relies on it for metabolic activation. Once phosphorylated, HoPan inhibits the next enzyme in the CoA pathwayâphosphopantothenoylcysteine synthetase (PPCS)âthrough formation of a nonproductive substrate complex. Moreover, the obtained structure of the human PPCS in complex with the inhibitor and activating nucleotide analogue provides new insights into the catalytic mechanism of PPCS enzymesâincluding the elusive binding mode for cysteineâand reveals the functional implications of mutations in the human PPCS that have been linked to severe dilated cardiomyopathy. Taken together, this study demonstrates that the molecular mechanism of action of HoPan is more complex than previously thought, suggesting that the results of studies in which it is used as a tool compound must be interpreted with care. Moreover, our findings provide a clear framework for evaluating the various factors that contribute to the potency of CoA-directed inhibitors, one that will prove useful in the future rational development of potential therapies of both human genetic and infectious diseases.
Assuntos
Coenzima A/metabolismo , Inibidores Enzimáticos/farmacologia , Ácido Pantotênico/análogos & derivados , Peptídeo Sintases/antagonistas & inibidores , Ácido gama-Aminobutírico/análogos & derivados , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células Cultivadas , Cristalização , Drosophila melanogaster , Cinética , Conformação Molecular , Ácido Pantotênico/farmacologia , Peptídeo Sintases/metabolismo , Especificidade por Substrato , Ácido gama-Aminobutírico/farmacologiaRESUMO
Despite the tremendous success of X-ray cryo-crystallography in recent decades, the transfer of crystals from the drops in which they are grown to diffractometer sample mounts remains a manual process in almost all laboratories. Here, the Shifter, a motorized, interactive microscope stage that transforms the entire crystal-mounting workflow from a rate-limiting manual activity to a controllable, high-throughput semi-automated process, is described. By combining the visual acuity and fine motor skills of humans with targeted hardware and software automation, it was possible to transform the speed and robustness of crystal mounting. Control software, triggered by the operator, manoeuvres crystallization plates beneath a clear protective cover, allowing the complete removal of film seals and thereby eliminating the tedium of repetitive seal cutting. The software, either upon request or working from an imported list, controls motors to position crystal drops under a hole in the cover for human mounting at a microscope. The software automatically captures experimental annotations for uploading to the user's data repository, removing the need for manual documentation. The Shifter facilitates mounting rates of 100-240 crystals per hour in a more controlled process than manual mounting, which greatly extends the lifetime of the drops and thus allows a dramatic increase in the number of crystals retrievable from any given drop without loss of X-ray diffraction quality. In 2015, the first in a series of three Shifter devices was deployed as part of the XChem fragment-screening facility at Diamond Light Source, where they have since facilitated the mounting of over 120â 000 crystals. The Shifter was engineered to have a simple design, providing a device that could be readily commercialized and widely adopted owing to its low cost. The versatile hardware design allows use beyond fragment screening and protein crystallography.
Assuntos
Desenho de Equipamento , Microscopia , Proteínas/química , Software , Cristalização , Cristalografia por Raios XRESUMO
Fluorine-containing amino acids have been used with great success as mechanism-based inhibitors of pyridoxal phosphate (PLP)-dependent enzymes, and the influence of fluorine on the conformation of molecules has also been extensively studied and practically exploited. In this study, we sought to use these unique characteristics to probe the reactivity and binding of aspartate decarboxylase (ADC) enzymes, which are members of the small class of pyruvoyl-dependant decarboxylases. Since ADC activity has been shown to be essential to the virulence of Mycobacterium tuberculosis, information gained in this manner could be used for the development of inhibitors that selectively target pyruvoyl-dependent enzymes such as ADC, without affecting PLP-dependent enzymes in the host. For this purpose, we synthesized the L-erythro and L-threo isomers of 3-fluoroaspartate and tested their ability to act as substrates and/or inhibitors of the M. tuberculosis and Escherichia coli ADC enzymes. Trapping and MS-based binding analysis was additionally used to confirm that both isomers enter the enzymes' active sites. Our studies show that both isomers undergo single turnover decarboxylation and fluorine elimination reactions to give enamine products that can be trapped within the active site. Interestingly, the enamine/ADC complex that forms from the L-erythro (but not the L-threo) isomer is sufficiently stable that it can be observed even without any trapping. This finding suggests that the two 3-fluoroaspartates maintain different conformations within the ADC active site, which leads to the enamine products with configurations of different stabilities. Taken together, our results provide new insights for the development of cofactor-specific inhibitors, and confirm the utility of fluorine as a unique tool for probing reactivity and binding profiles within enzymes.
Assuntos
Ácido Aspártico/análogos & derivados , Carboxiliases/química , Carboxiliases/metabolismo , Ácido Pirúvico/química , Sequência de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Sítios de Ligação , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Ácido Pirúvico/metabolismoRESUMO
Even with the emergence of antibiotic resistance, penicillin and the wider family of ß-lactams have remained the single most important family of antibiotics. The periplasmic/extra-cytoplasmic targets of penicillin are a family of enzymes with a highly conserved catalytic activity involved in the final stage of bacterial cell wall (peptidoglycan) biosynthesis. Named after their ability to bind penicillin, rather than their catalytic activity, these key targets are called penicillin-binding proteins (PBPs). Resistance is predominantly mediated by reducing the target drug concentration via ß-lactamases; however, naturally transformable bacteria have also acquired target-mediated resistance by inter-species recombination. Here we focus on structural based interpretations of amino acid alterations associated with the emergence of resistance within clinical isolates and include new PBP3 structures along with new, and improved, PBP-ß-lactam co-structures.
Assuntos
Proteínas de Ligação às Penicilinas/química , D-Ala-D-Ala Carboxipeptidase Tipo Serina/química , Resistência beta-Lactâmica/fisiologia , beta-Lactamas/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Haemophilus influenzae/enzimologia , Modelos Moleculares , Mutação , Neisseria gonorrhoeae/enzimologia , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/química , Peptidoglicano Glicosiltransferase/genética , Peptidoglicano Glicosiltransferase/metabolismo , Conformação Proteica , Domínios Proteicos , Pseudomonas aeruginosa/enzimologia , Alinhamento de Sequência , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismo , beta-Lactamas/farmacologiaRESUMO
N-substituted pantothenamides are analogues of pantothenic acid, the vitamin precursor of CoA, and constitute a class of well-studied bacterial growth inhibitors that show potential as new antibacterial agents. Previous studies have highlighted the importance of pantothenate kinase (PanK; EC 2.7.1.33) (the first enzyme of CoA biosynthesis) in mediating pantothenamide-induced growth inhibition by one of two proposed mechanisms: first, by acting on the pantothenamides as alternate substrates (allowing their conversion into CoA antimetabolites, with subsequent effects on CoA- and acyl carrier protein-dependent processes) or, second, by being directly inhibited by them (causing a reduction in CoA biosynthesis). In the present study we used structurally modified pantothenamides to probe whether PanKs interact with these compounds in the same manner. We show that the three distinct types of eubacterial PanKs that are known to exist (PanKI , PanKII and PanKIII ) respond very differently and, consequently, are responsible for determining the pantothenamide mode of action in each case: although the promiscuous PanKI enzymes accept them as substrates, the highly selective PanKIII s are resistant to their inhibitory effects. Most unexpectedly, Staphylococcus aureus PanK (the only known example of a bacterial PanKII ) experiences uncompetitive inhibition in a manner that is described for the first time. In addition, we show that pantetheine, a CoA degradation product that closely resembles the pantothenamides, causes the same effect. This suggests that, in S. aureus, pantothenamides may act by usurping a previously unknown role of pantetheine in the regulation of CoA biosynthesis, and validates its PanK as a target for the development of new antistaphylococcal agents.