RESUMO
The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.
Assuntos
Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , SARS-CoV-2/metabolismo , Ubiquitina/metabolismo , Animais , Sítios de Ligação , Chlorocebus aethiops , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/genética , Cristalografia por Raios X , Citocinas/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Reposicionamento de Medicamentos , Polarização de Fluorescência , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Inibidores de Proteases/farmacologia , Conformação Proteica , SARS-CoV-2/química , SARS-CoV-2/genética , Ubiquitinas/genética , Células VeroRESUMO
Necroptosis is a mode of programmed, lytic cell death that is executed by the mixed lineage kinase domain-like (MLKL) pseudokinase following activation by the upstream kinases, receptor-interacting serine/threonine protein kinase (RIPK)-1 and RIPK3. Dysregulated necroptosis has been implicated in the pathophysiology of many human diseases, including inflammatory and degenerative conditions, infectious diseases and cancers, provoking interest in pharmacological targeting of the pathway. To identify small molecules impacting on the necroptotic machinery, we performed a phenotypic screen using a mouse cell line expressing an MLKL mutant that kills cells in the absence of upstream death or pathogen detector receptor activation. This screen identified the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitor, ABT-869 (Linifanib), as a small molecule inhibitor of necroptosis. We applied a suite of cellular, biochemical and biophysical analyses to pinpoint the apical necroptotic kinase, RIPK1, as the target of ABT-869 inhibition. Our study adds to the repertoire of established protein kinase inhibitors that additionally target RIPK1 and raises the prospect that serendipitous targeting of necroptosis signalling may contribute to their clinical efficacy in some settings.
Assuntos
Proteínas Quinases , Humanos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Necroptose , Fator A de Crescimento do Endotélio Vascular/metabolismo , Apoptose , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Activating the intrinsic apoptosis pathway with small molecules is now a clinically validated approach to cancer therapy. In contrast, blocking apoptosis to prevent the death of healthy cells in disease settings has not been achieved. Caspases have been favored, but they act too late in apoptosis to provide long-term protection. The critical step in committing a cell to death is activation of BAK or BAX, pro-death BCL-2 proteins mediating mitochondrial damage. Apoptosis cannot proceed in their absence. Here we show that WEHI-9625, a novel tricyclic sulfone small molecule, binds to VDAC2 and promotes its ability to inhibit apoptosis driven by mouse BAK. In contrast to caspase inhibitors, WEHI-9625 blocks apoptosis before mitochondrial damage, preserving cellular function and long-term clonogenic potential. Our findings expand on the key role of VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically tractable.
Assuntos
Apoptose/fisiologia , Bibliotecas de Moléculas Pequenas/metabolismo , Canal de Ânion 2 Dependente de Voltagem/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Animais , Camundongos , Ligação Proteica , Canal de Ânion 2 Dependente de Voltagem/metabolismoRESUMO
Malaria is a devastating parasitic disease caused by parasites from the genus Plasmodium. Therapeutic resistance has been reported against all clinically available antimalarials, threatening our ability to control the disease and therefore there is an ongoing need for the development of novel antimalarials. Towards this goal, we identified the 2-(N-phenyl carboxamide) triazolopyrimidine class from a high throughput screen of the Janssen Jumpstarter library against the asexual stages of the P. falciparum parasite. Here we describe the structure activity relationship of the identified class and the optimisation of asexual stage activity while maintaining selectivity against the human HepG2 cell line. The most potent analogues from this study were shown to exhibit equipotent activity against P. falciparum multidrug resistant strains and P. knowlesi asexual parasites. Asexual stage phenotyping studies determined the triazolopyrimidine class arrests parasites at the trophozoite stage, but it is likely these parasites are still metabolically active until the second asexual cycle, and thus have a moderate to slow onset of action. Non-NADPH dependent degradation of the central carboxamide and low aqueous solubility was observed in in vitro ADME profiling. A significant challenge remains to correct these liabilities for further advancement of the 2-(N-phenyl carboxamide) triazolopyrimidine scaffold as a potential moderate to slow acting partner in a curative or prophylactic antimalarial treatment.
Assuntos
Antimaláricos/farmacologia , Eritrócitos/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium knowlesi/efeitos dos fármacos , Purinas/farmacologia , Antimaláricos/síntese química , Antimaláricos/química , Relação Dose-Resposta a Droga , Eritrócitos/parasitologia , Humanos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Purinas/síntese química , Purinas/química , Relação Estrutura-AtividadeRESUMO
Malaria is a devastating disease caused by Plasmodium parasites. Emerging resistance against current antimalarial therapeutics has engendered the need to develop antimalarials with novel structural classes. We recently described the identification and initial optimization of the 2-anilino quinazoline antimalarial class. Here, we refine the physicochemical properties of this antimalarial class with the aim to improve aqueous solubility and metabolism and to reduce adverse promiscuity. We show the physicochemical properties of this class are intricately balanced with asexual parasite activity and human cell cytotoxicity. Structural modifications we have implemented improved LipE, aqueous solubility and in vitro metabolism while preserving fast acting P. falciparum asexual stage activity. The lead compounds demonstrated equipotent activity against P. knowlesi parasites and were not predisposed to resistance mechanisms of clinically used antimalarials. The optimized compounds exhibited modest activity against early-stage gametocytes, but no activity against pre-erythrocytic liver parasites. Confoundingly, the refined physicochemical properties installed in the compounds did not engender improved oral efficacy in a P. berghei mouse model of malaria compared to earlier studies on the 2-anilino quinazoline class. This study provides the framework for further development of this antimalarial class.
Assuntos
Compostos de Anilina/química , Compostos de Anilina/farmacologia , Antimaláricos/química , Antimaláricos/farmacologia , Malária/tratamento farmacológico , Plasmodium/efeitos dos fármacos , Quinazolinas/química , Quinazolinas/farmacologia , Aminação , Compostos de Anilina/uso terapêutico , Animais , Antimaláricos/uso terapêutico , Feminino , Humanos , Malária/parasitologia , Camundongos , Plasmodium/fisiologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/fisiologia , Quinazolinas/uso terapêuticoRESUMO
BACKGROUND AND AIMS: Proteomics holds promise for individualizing cancer treatment. We analyzed to what extent the proteomic landscape of human colorectal cancer (CRC) is maintained in established CRC cell lines and the utility of proteomics for predicting therapeutic responses. METHODS: Proteomic and transcriptomic analyses were performed on 44 CRC cell lines, compared against primary CRCs (n=95) and normal tissues (n=60), and integrated with genomic and drug sensitivity data. RESULTS: Cell lines mirrored the proteomic aberrations of primary tumors, in particular for intrinsic programs. Tumor relationships of protein expression with DNA copy number aberrations and signatures of post-transcriptional regulation were recapitulated in cell lines. The 5 proteomic subtypes previously identified in tumors were represented among cell lines. Nonetheless, systematic differences between cell line and tumor proteomes were apparent, attributable to stroma, extrinsic signaling, and growth conditions. Contribution of tumor stroma obscured signatures of DNA mismatch repair identified in cell lines with a hypermutation phenotype. Global proteomic data showed improved utility for predicting both known drug-target relationships and overall drug sensitivity as compared with genomic or transcriptomic measurements. Inhibition of targetable proteins associated with drug responses further identified corresponding synergistic or antagonistic drug combinations. Our data provide evidence for CRC proteomic subtype-specific drug responses. CONCLUSIONS: Proteomes of established CRC cell line are representative of primary tumors. Proteomic data tend to exhibit improved prediction of drug sensitivity as compared with genomic and transcriptomic profiles. Our integrative proteogenomic analysis highlights the potential of proteome profiling to inform personalized cancer medicine.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Proteínas de Neoplasias/metabolismo , Medicina de Precisão , Proteoma , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Cromatografia Líquida , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Bases de Dados de Proteínas , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Proteínas de Neoplasias/genética , Seleção de Pacientes , Polimorfismo de Nucleotídeo Único , Proteômica/métodos , Transdução de Sinais , Células Estromais/metabolismo , Espectrometria de Massas em Tandem , Transcriptoma , Microambiente TumoralRESUMO
The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum-infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target.
Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Proteases/antagonistas & inibidores , Oligopeptídeos/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Sulfonamidas/farmacologia , Retículo Endoplasmático/metabolismo , Eritrócitos/parasitologia , Humanos , Transporte Proteico/efeitos dos fármacos , Proteínas de Protozoários/metabolismoRESUMO
The BimEL tumor suppressor is a potent proapoptotic BH3-only protein. We found that, in response to survival signals, BimEL was rapidly phosphorylated on three serine residues in a conserved degron, facilitating binding and degradation via the F box protein betaTrCP. Phosphorylation of the BimEL degron was executed by Rsk1/2 and promoted by the Erk1/2-mediated phosphorylation of BimEL on Ser69. Compared to wild-type BimEL, a BimEL phosphorylation mutant unable to bind betaTrCP was stabilized and consequently potent at inducing apoptosis by the intrinsic mitochondrial pathway. Moreover, although non-small cell lung cancer (NSCLC) cells often become resistant to gefitinib (a clinically relevant tyrosine kinase inhibitor that induces apoptosis through BimEL), silencing of either betaTrCP or Rsk1/2 resulted in BimEL-mediated apoptosis of both gefitinib-sensitive and gefitinib-insensitive NSCLC cells. Our findings reveal that betaTrCP promotes cell survival in cooperation with the ERK-RSK pathway by targeting BimEL for degradation.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Animais , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular , Humanos , Camundongos , Estabilidade ProteicaRESUMO
The use of arginine isosteres is a known strategy to overcome poor membrane permeability commonly associated with peptides or peptidomimetics that possess this highly polar amino acid. Here, we apply this strategy to peptidomimetics that are potent inhibitors of the malarial protease, plasmepsin V, with the aim of enhancing their activity against Plasmodium parasites, and exploring the structure-activity relationship of the P3 arginine within the S3 pocket of plasmepsin V. Of the arginine isosteres trialled in the P3 position, we discovered that canavanine was the ideal and that this peptidomimetic potently inhibits plasmepsin V, efficiently blocks protein export and inhibits parasite growth. Structure studies of the peptidomimetics bound to plasmepsin V provided insight into the structural basis for the enzyme activity observed in vitro and provides further evidence why plasmepsin V is highly sensitive to substrate modification.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Peptidomiméticos/química , Plasmodium vivax/enzimologia , Animais , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The prosurvival BCL-2 family protein BCL-X(L) is often overexpressed in solid tumors and renders malignant tumor cells resistant to anticancer therapeutics. Enhancing apoptotic responses by inhibiting BCL-X(L) will most likely have widespread utility in cancer treatment and, instead of inhibiting multiple prosurvival BCL-2 family members, a BCL-X(L)-selective inhibitor would be expected to minimize the toxicity to normal tissues. We describe the use of a high-throughput screen to discover a new series of small molecules targeting BCL-X(L) and their structure-guided development by medicinal chemistry. The optimized compound, WEHI-539 (7), has high affinity (subnanomolar) and selectivity for BCL-X(L) and potently kills cells by selectively antagonizing its prosurvival activity. WEHI-539 will be an invaluable tool for distinguishing the roles of BCL-X(L) from those of its prosurvival relatives, both in normal cells and notably in malignant tumor cells, many of which may prove to rely upon BCL-X(L) for their sustained growth.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/química , Animais , Apoptose , Benzotiazóis/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Hidrazonas/química , Cinética , Camundongos , Modelos Químicos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Papain-like protease (PLpro) is an attractive drug target for SARS-CoV-2 because it is essential for viral replication, cleaving viral poly-proteins pp1a and pp1ab, and has de-ubiquitylation and de-ISGylation activities, affecting innate immune responses. We employ Deep Mutational Scanning to evaluate the mutational effects on PLpro enzymatic activity and protein stability in mammalian cells. We confirm features of the active site and identify mutations in neighboring residues that alter activity. We characterize residues responsible for substrate binding and demonstrate that although residues in the blocking loop are remarkably tolerant to mutation, blocking loop flexibility is important for function. We additionally find a connected network of mutations affecting activity that extends far from the active site. We leverage our library to identify drug-escape variants to a common PLpro inhibitor scaffold and predict that plasticity in both the S4 pocket and blocking loop sequence should be considered during the drug design process.
Assuntos
Mutação , SARS-CoV-2 , SARS-CoV-2/genética , Humanos , Proteases Semelhantes à Papaína de Coronavírus/genética , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/química , Domínio Catalítico , Antivirais/farmacologia , Proteases 3C de Coronavírus/genética , Proteases 3C de Coronavírus/metabolismo , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , COVID-19/virologia , Tratamento Farmacológico da COVID-19 , Modelos Moleculares , Células HEK293RESUMO
Malaria remains a global health threat and growing resistance to artemisinin-based therapies calls for therapeutic agents with novel mechanisms of action. The Plasmodium spp M1 and M17 metalloaminopeptidases have been identified as attractive new antimalarial drug targets as inhibition of these enzymes results in antiplasmodial activity. Previously identified novel hydroxamic acid 2 as a moderate inhibitor of PfA-M1 and PfA-M17 and a potent inhibitor of P. falciparum. This study has sought to improve the enzymatic inhibitory properties in addition to increasing the drug-likeness of this scaffold by introducing polar moieties into the S1' region of the active site. Structural biology studies on the co-crystallised structures of potent dual-inhibitor 9aa bound to PfA-M1 and PfA-M17 have revealed that there are few direct interactions between the inhibitor and the S1' domain of these enzymes. Structure-based compound design led to the identification of a variety of novel hydroxamic acids that show improved inhibitory activity against PfA-M1 and PfA-M17, in addition to displaying antiplasmodial activity. Notably, compounds with substitutions on the aniline ring resulted in a loss of potency (Ki > 500 nM) toward PfA-M1 and PfA-M17. ioisosteric replacement of the S1-region biaryl ring system with a bromophenyl moiety resulted in increased potency compared to parent 9aa. Elaboration of 9aa to bioisosterically replace the S1 moiety with an aryl bromide, combined with substituted anilines has resulted in potent selective PfA-M1 inhibitors which show strong activity against Pf-3D7, with meta- and para-fluoroaniline groups of 15ag and 15ah forming hydrogen-bonds with residues within the active site. These findings establish the importance of the previously under-utilised S1' domain and will aid the design of future PfA-M1 and PfA-M17 inhibitors.
Assuntos
Antimaláricos , Malária Falciparum , Plasmodium , Humanos , Plasmodium falciparum , Aminopeptidases , Antimaláricos/química , Malária Falciparum/tratamento farmacológicoRESUMO
There is an urgent need to populate the antimalarial clinical portfolio with new candidates because of resistance against frontline antimalarials. To discover new antimalarial chemotypes, we performed a high-throughput screen of the Janssen Jumpstarter library against the Plasmodium falciparum asexual blood-stage parasite and identified the 2,3-dihydroquinazolinone-3-carboxamide scaffold. We defined the SAR and found that 8-substitution on the tricyclic ring system and 3-substitution of the exocyclic arene produced analogues with potent activity against asexual parasites equivalent to clinically used antimalarials. Resistance selection and profiling against drug-resistant parasite strains revealed that this antimalarial chemotype targets PfATP4. Dihydroquinazolinone analogues were shown to disrupt parasite Na+ homeostasis and affect parasite pH, exhibited a fast-to-moderate rate of asexual kill, and blocked gametogenesis, consistent with the phenotype of clinically used PfATP4 inhibitors. Finally, we observed that optimized frontrunner analogue WJM-921 demonstrates oral efficacy in a mouse model of malaria.
Assuntos
Antimaláricos , Malária Falciparum , Malária , Animais , Camundongos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Plasmodium falciparum , Homeostase , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologiaRESUMO
Necroptosis is a form of caspase-independent programmed cell death that arises from disruption of cell membranes by the mixed lineage kinase domain-like (MLKL) pseudokinase after its activation by the upstream kinases, receptor interacting protein kinase (RIPK)-1 and RIPK3, within a complex known as the necrosome. Dysregulated necroptosis has been implicated in numerous inflammatory pathologies. As such, new small molecule necroptosis inhibitors are of great interest, particularly ones that operate downstream of MLKL activation, where the pathway is less well defined. To better understand the mechanisms involved in necroptosis downstream of MLKL activation, and potentially uncover new targets for inhibition, we screened known kinase inhibitors against an activated mouse MLKL mutant, leading us to identify the lymphocyte-specific protein tyrosine kinase (Lck) inhibitor AMG-47a as an inhibitor of necroptosis. We show that AMG-47a interacts with both RIPK1 and RIPK3, that its ability to protect from cell death is dependent on the strength of the necroptotic stimulus, and that it blocks necroptosis most effectively in human cells. Moreover, in human cell lines, we demonstrate that AMG-47a can protect against cell death caused by forced dimerisation of MLKL truncation mutants in the absence of any upstream signalling, validating that it targets a process downstream of MLKL activation. Surprisingly, however, we also found that the cell death driven by activated MLKL in this model was completely dependent on the presence of RIPK1, and to a lesser extent RIPK3, although it was not affected by known inhibitors of these kinases. Together, these results suggest an additional role for RIPK1, or the necrosome, in mediating human necroptosis after MLKL is phosphorylated by RIPK3 and provide further insight into reported differences in the progression of necroptosis between mouse and human cells.
Assuntos
Necroptose , Proteínas Quinases , Animais , Apoptose , Morte Celular , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Camundongos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de SinaisRESUMO
Plasmepsins IX (PMIX) and X (PMX) are essential aspartyl proteases for Plasmodium spp. egress, invasion, and development. WM4 and WM382 inhibit PMIX and PMX in Plasmodium falciparum and P. vivax. WM4 inhibits PMX, while WM382 is a dual inhibitor of PMIX and PMX. To understand their function, we identified protein substrates. Enzyme kinetic and structural analyses identified interactions responsible for drug specificity. PMIX and PMX have similar substrate specificity; however, there are distinct differences for peptide and protein substrates. Differences in WM4 and WM382 binding for PMIX and PMX map to variations in the S' region and engagement of the active site S3 pocket. Structures of PMX reveal interactions and mechanistic detail of drug binding important for development of clinical candidates against these targets.
Assuntos
Ácido Aspártico Endopeptidases , Plasmodium falciparum , Ácido Aspártico Endopeptidases/química , Cinética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Especificidade por SubstratoRESUMO
Background: RH5 is the leading vaccine candidate for the Plasmodium falciparum blood stage and has shown impact on parasite growth in the blood in a human clinical trial. RH5 binds to Ripr and CyRPA at the apical end of the invasive merozoite form, and this complex, designated RCR, is essential for entry into human erythrocytes. RH5 has advanced to human clinical trials, and the impact on parasite growth in the blood was encouraging but modest. This study assessed the potential of a protein-in-adjuvant blood stage malaria vaccine based on a combination of RH5, Ripr and CyRPA to provide improved neutralizing activity against P. falciparum in vitro. Methods: Mice were immunized with the individual RCR antigens to down select the best performing adjuvant formulation and rats were immunized with the individual RCR antigens to select the correct antigen dose. A second cohort of rats were immunized with single, double and triple antigen combinations to assess immunogenicity and parasite neutralizing activity in growth inhibition assays. Results: The DPX® platform was identified as the best performing formulation in potentiating P. falciparum inhibitory antibody responses to these antigens. The three antigens derived from RH5, Ripr and CyRPA proteins formulated with DPX induced highly inhibitory parasite neutralising antibodies. Notably, RH5 either as a single antigen or in combination with Ripr and/or CyRPA, induced inhibitory antibodies that outperformed CyRPA, Ripr. Conclusion: An RCR combination vaccine may not induce substantially improved protective immunity as compared with RH5 as a single immunogen in a clinical setting and leaves the development pathway open for other antigens to be combined with RH5 as a next generation malaria vaccine.
Assuntos
Vacinas Antimaláricas , Malária Falciparum , Humanos , Camundongos , Ratos , Animais , Antígenos de Protozoários , Proteínas de Protozoários/metabolismo , Malária Falciparum/parasitologia , Plasmodium falciparum , Anticorpos Antiprotozoários , Vacinas CombinadasRESUMO
Drug resistance to first-line antimalarials-including artemisinin-is increasing, resulting in a critical need for the discovery of new agents with novel mechanisms of action. In collaboration with the Walter and Eliza Hall Institute and with funding from the Wellcome Trust, a phenotypic screen of Merck's aspartyl protease inhibitor library identified a series of plasmepsin X (PMX) hits that were more potent than chloroquine. Inspired by a PMX homology model, efforts to optimize the potency resulted in the discovery of leads that, in addition to potently inhibiting PMX, also inhibit another essential aspartic protease, plasmepsin IX (PMIX). Further potency and pharmacokinetic profile optimization efforts culminated in the discovery of WM382, a very potent dual PMIX/X inhibitor with robust in vivo efficacy at multiple stages of the malaria parasite life cycle and an excellent resistance profile.
RESUMO
The COVID-19 pandemic continues unabated, emphasizing the need for additional antiviral treatment options to prevent hospitalization and death of patients infected with SARS-CoV-2. The papain-like protease (PLpro) domain is part of the SARS-CoV-2 non-structural protein (nsp)-3, and represents an essential protease and validated drug target for preventing viral replication. PLpro moonlights as a deubiquitinating (DUB) and deISGylating enzyme, enabling adaptation of a DUB high throughput (HTS) screen to identify PLpro inhibitors. Drug repurposing has been a major focus through the COVID-19 pandemic as it may provide a fast and efficient route for identifying clinic-ready, safe-in-human antivirals. We here report our effort to identify PLpro inhibitors by screening the ReFRAME library of 11,804 compounds, showing that none inhibit PLpro with any reasonable activity or specificity to justify further progression towards the clinic. We also report our latest efforts to improve piperidine-scaffold inhibitors, 5c and 3k, originally developed for SARS-CoV PLpro. We report molecular details of binding and selectivity, as well as in vitro absorption, distribution, metabolism and excretion (ADME) studies of this scaffold. A co-crystal structure of SARS-CoV-2 PLpro bound to inhibitor 3k guides medicinal chemistry efforts to improve binding and ADME characteristics. We arrive at compounds with improved and favorable solubility and stability characteristics that are tested for inhibiting viral replication. Whilst still requiring significant improvement, our optimized small molecule inhibitors of PLpro display decent antiviral activity in an in vitro SARS-CoV-2 infection model, justifying further optimization.
RESUMO
Limited therapeutic options are available for the treatment of human schistosomiasis caused by the parasitic Schistosoma flatworm. The B cell lymphoma-2 (BCL-2)-regulated apoptotic cell death pathway in schistosomes was recently characterized and shown to share similarities with the intrinsic apoptosis pathway in humans. Here, we exploit structural differences in the human and schistosome BCL-2 (sBCL-2) pro-survival proteins toward a novel treatment strategy for schistosomiasis. The benzothiazole hydrazone scaffold previously employed to target human BCL-XL was repurposed as a starting point to target sBCL-2. We utilized X-ray structural data to inform optimization and then applied a scaffold-hop strategy to identify the 5-carboxamide thiazole hydrazone scaffold (43) with potent sBCL-2 activity (IC50 30 nM). Human BCL-XL potency (IC50 13 nM) was inadvertently preserved during the optimization process. The lead analogues from this study exhibit on-target activity in model fibroblast cell lines dependent on either sBCL-2 or human BCL-XL for survival. Further optimization of the thiazole hydrazone class is required to exhibit activity in schistosomes and enhance the potential of this strategy for treating schistosomiasis.
Assuntos
Hidrazonas , Schistosoma , Animais , Apoptose , Benzotiazóis , Humanos , Hidrazonas/farmacologia , Proteína bcl-X/genéticaRESUMO
The emerging resistance to combination therapies comprised of artemisinin derivatives has driven a need to identify new antimalarials with novel mechanisms of action. Central to the survival and proliferation of the malaria parasite is the invasion of red blood cells by Plasmodium merozoites, providing an attractive target for novel therapeutics. A screen of the Medicines for Malaria Venture Pathogen Box employing transgenic P. falciparum parasites expressing the nanoluciferase bioluminescent reporter identified the phenylsulfonyl piperazine class as a specific inhibitor of erythrocyte invasion. Here, we describe the optimization and further characterization of the phenylsulfonyl piperazine class. During the optimization process we defined the functionality required for P. falciparum asexual stage activity and determined the alpha-carbonyl S-methyl isomer was important for antimalarial potency. The optimized compounds also possessed comparable activity against multidrug resistant strains of P. falciparum and displayed weak activity against sexual stage gametocytes. We determined that the optimized compounds blocked erythrocyte invasion consistent with the asexual activity observed and therefore the phenylsulfonyl piperazine analogues described could serve as useful tools for studying Plasmodium erythrocyte invasion.