RESUMO
Visceral leishmaniasis causes considerable mortality and morbidity in many parts of the world. There is an urgent need for the development of new, effective treatments for this disease. Here we describe the development of an anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound from this series (7, DDD853651/GSK3186899) is efficacious in a mouse model of visceral leishmaniasis, has suitable physicochemical, pharmacokinetic and toxicological properties for further development, and has been declared a preclinical candidate. Detailed mode-of-action studies indicate that compounds from this series act principally by inhibiting the parasite cdc-2-related kinase 12 (CRK12), thus defining a druggable target for visceral leishmaniasis.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/enzimologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Terapia de Alvo Molecular , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Quinase 9 Dependente de Ciclina/química , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Simulação de Acoplamento Molecular , Proteoma/efeitos dos fármacos , Proteômica , Pirazóis/química , Pirazóis/uso terapêutico , Pirimidinas/química , Pirimidinas/uso terapêutico , Reprodutibilidade dos Testes , Especificidade por SubstratoRESUMO
BRCA2 is a well-established cancer driver in several human malignancies. While the remarkable success of PARP inhibitors proved the clinical potential of targeting BRCA deficiencies, the emergence of resistance mechanisms underscores the importance of seeking novel Synthetic Lethal (SL) targets for future drug development efforts. In this work, we performed a BRCA2-centric SL screen with a collection of plant-derived compounds from South America. We identified the steroidal alkaloid Solanocapsine as a selective SL inducer, and we were able to substantially increase its potency by deriving multiple analogs. The use of two complementary chemoproteomic approaches led to the identification of the nucleotide salvage pathway enzyme deoxycytidine kinase (dCK) as Solanocapsine's target responsible for its BRCA2-linked SL induction. Additional confirmatory evidence was obtained by using the highly specific dCK inhibitor (DI-87), which induces SL in multiple BRCA2-deficient and KO contexts. Interestingly, dCK-induced SL is mechanistically different from the one induced by PARP inhibitors. dCK inhibition generates substantially lower levels of DNA damage, and cytotoxic phenotypes are associated exclusively with mitosis, thus suggesting that the fine-tuning of nucleotide supply in mitosis is critical for the survival of BRCA2-deficient cells. Moreover, by using a xenograft model of contralateral tumors, we show that dCK impairment suffices to trigger SL in-vivo. Taken together, our findings unveil dCK as a promising new target for BRCA2-deficient cancers, thus setting the ground for future therapeutic alternatives to PARP inhibitors.
Assuntos
Antineoplásicos , Desoxicitidina Quinase , Humanos , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Nucleotídeos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteína BRCA2/genéticaRESUMO
Epigenetic regulatory mechanisms are central to the development and survival of all eukaryotic organisms. These mechanisms critically depend on the marking of chromatin domains with distinctive histone tail modifications (PTMs) and their recognition by effector protein complexes. Here we used quantitative proteomic approaches to unveil interactions between PTMs and associated reader protein complexes of Plasmodium falciparum, a unicellular parasite causing malaria. Histone peptide pull-downs with the most prominent and/or parasite-specific PTMs revealed the binding preference for 14 putative and novel reader proteins. Amongst others, they highlighted the acetylation-level-dependent recruitment of the BDP1/BDP2 complex and identified an PhD-finger protein (PHD 1, PF3D7_1008100) that could mediate a cross-talk between H3K4me2/3 and H3K9ac marks. Tagging and interaction proteomics of 12 identified proteins unveiled the composition of 5 major epigenetic complexes, including the elusive TBP-associated-factor complex as well as two distinct GCN5/ADA2 complexes. Furthermore, it has highlighted a remarkable degree of interaction between these five (sub)complexes. Collectively, this study provides an extensive inventory of PTM-reader interactions and composition of epigenetic complexes. It will not only fuel further explorations of gene regulation amongst ancient eukaryotes, but also provides a stepping stone for exploration of PTM-reader interactions for antimalarial drug development.
Assuntos
Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Histonas/metabolismo , Plasmodium falciparum/genética , Processamento de Proteína Pós-Traducional/genética , Cromatina/metabolismo , Humanos , Malária Falciparum/genética , Malária Falciparum/parasitologia , MetilaçãoRESUMO
N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing four different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared with parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.
Assuntos
Defeitos Congênitos da Glicosilação , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Regulação da Expressão Gênica , Humanos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Alternative mode-of-inhibition of clinically validated targets is an effective strategy for circumventing existing clinical drug resistance. Herein, we report 1,3-diarylpyrazolyl-acylsulfonamides as potent inhibitors of HadAB/BC, a 3-hydroxyl-ACP dehydratase complex required to iteratively elongate the meromycolate chain of mycolic acids in Mycobacterium tuberculosis (Mtb). Mutations in compound 1-resistant Mtb mutants mapped to HadC (Rv0637; K157R), while chemoproteomics confirmed the compound's binding to HadA (Rv0635), HadB (Rv0636), and HadC. The compounds effectively inhibited the HadAB and HadBC enzyme activities and affected mycolic acid biosynthesis in Mtb, in a concentration-dependent manner. Unlike known 3-hydroxyl-ACP dehydratase complex inhibitors of clinical significance, isoxyl and thioacetazone, 1,3-diarylpyrazolyl-acylsulfonamides did not require activation by EthA and thus are not liable to EthA-mediated resistance. Further, the crystal structure of a key compound in a complex with Mtb HadAB revealed unique binding interactions within the active site of HadAB, providing a useful tool for further structure-based optimization of the series.
Assuntos
Mycobacterium tuberculosis , Tioacetazona , Proteínas de Bactérias/metabolismo , Ácidos Micólicos/química , Tioacetazona/metabolismo , Tioacetazona/farmacologia , Hidroliases/química , Hidroliases/metabolismo , Hidroliases/farmacologiaRESUMO
Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human "mammalian target of rapamycin" (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite Plasmodium spp. Chemoproteomics studies revealed multiple potential Plasmodium kinase targets, and potent inhibition of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4Kß) and cyclic guanosine monophosphate-dependent protein kinase (PKG) was confirmed in vitro. Conditional knockdown of PI4Kß in ABS cultures modulated parasite sensitivity to sapanisertib, and laboratory-generated P. falciparum sapanisertib resistance was mediated by mutations in PI4Kß. Parasite metabolomic perturbation profiles associated with sapanisertib and other known PI4Kß and/or PKG inhibitors revealed similarities and differences between chemotypes, potentially caused by sapanisertib targeting multiple parasite kinases. The multistage activity of sapanisertib and its in vivo antimalarial efficacy, coupled with potent inhibition of at least two promising drug targets, provides an opportunity to reposition this pyrazolopyrimidine for malaria.
Assuntos
Antimaláricos , Plasmodium , Animais , Humanos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Plasmodium falciparum , Inibidores de MTOR , 1-Fosfatidilinositol 4-Quinase , Guanosina Monofosfato , Estágios do Ciclo de Vida , Serina-Treonina Quinases TOR , Sirolimo , MamíferosRESUMO
The sensitivity of Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis, which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects.
Assuntos
Mycobacterium tuberculosis , Pró-Fármacos , Tuberculose , Animais , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Etionamida/química , Etionamida/farmacologia , Etionamida/uso terapêutico , Camundongos , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Tuberculose/tratamento farmacológicoRESUMO
The emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) ensures that drug discovery efforts remain at the forefront of TB research. There are multiple different experimental approaches that can be employed in the discovery of anti-TB agents. Notably, inhibitors of MmpL3 are numerous and structurally diverse in Mtb and have been discovered through the generation of spontaneous resistant mutants and subsequent whole genome sequencing studies. However, this approach is not always reliable and can lead to incorrect target assignment and requires orthogonal confirmatory approaches. In fact, many of these inhibitors have also been shown to act as multi-target agents, with secondary targets in Mtb, as well as in other non-MmpL3-containing pathogens. Herein, we have investigated further the cellular targets of the MmpL3-inhibitor BM212 and a number of BM212 analogues. To determine the alternative targets of BM212, which may have been masked by MmpL3 mutations, we have applied a combination of chemo-proteomic profiling using bead-immobilised BM212 derivatives and protein extracts, along with whole-cell and biochemical assays. The study identified EthR2 (Rv0078) as a protein that binds BM212 analogues. We further demonstrated binding of BM212 to EthR2 through an in vitro tryptophan fluorescence assay, which showed significant quenching of tryptophan fluorescence upon addition of BM212. Our studies have demonstrated the value of revisiting drugs with ambiguous targets, such as MmpL3, in an attempt to find alternative targets and the study of off-target effects to understand more precisely target engagement of new hits emerging from drug screening campaigns.
RESUMO
Calcium signaling regulated by the cGMP-dependent protein kinase (PKG) controls key life cycle transitions in the malaria parasite. However, how calcium is mobilized from intracellular stores in the absence of canonical calcium channels in Plasmodium is unknown. Here, we identify a multipass membrane protein, ICM1, with homology to transporters and calcium channels that is tightly associated with PKG in both asexual blood stages and transmission stages. Phosphoproteomic analyses reveal multiple ICM1 phosphorylation events dependent on PKG activity. Stage-specific depletion of Plasmodium berghei ICM1 prevents gametogenesis due to a block in intracellular calcium mobilization, while conditional loss of Plasmodium falciparum ICM1 is detrimental for the parasite resulting in severely reduced calcium mobilization, defective egress, and lack of invasion. Our findings suggest that ICM1 is a key missing link in transducing PKG-dependent signals and provide previously unknown insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission.
Assuntos
Malária , Parasitos , Animais , Cálcio/metabolismo , Canais de Cálcio , Gametogênese , Malária/parasitologia , Proteínas de Membrana/metabolismo , Plasmodium berghei/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-κB-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19.
Assuntos
COVID-19/metabolismo , Ativação do Complemento , Células Epiteliais/metabolismo , Janus Quinase 1/metabolismo , Janus Quinase 2/metabolismo , Pulmão/metabolismo , Sistema de Sinalização das MAP Quinases , SARS-CoV-2/metabolismo , COVID-19/patologia , Linhagem Celular Tumoral , Complemento C3a/metabolismo , Fator B do Complemento/metabolismo , Células Epiteliais/patologia , Humanos , Pulmão/patologiaRESUMO
N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.
Assuntos
Retículo Endoplasmático , Regulação da Expressão Gênica , Retículo Endoplasmático/metabolismo , Humanos , Células K562 , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Myotonic dystrophy type 1 (DM1) is an RNA-based disease with no current treatment. It is caused by a transcribed CTG repeat expansion within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Mutant repeat expansion transcripts remain in the nuclei of patients' cells, forming distinct microscopically detectable foci that contribute substantially to the pathophysiology of the condition. Here, we report small-molecule inhibitors that remove nuclear foci and have beneficial effects in the HSALR mouse model, reducing transgene expression, leading to improvements in myotonia, splicing, and centralized nuclei. Using chemoproteomics in combination with cell-based assays, we identify cyclin-dependent kinase 12 (CDK12) as a druggable target for this condition. CDK12 is a protein elevated in DM1 cell lines and patient muscle biopsies, and our results showed that its inhibition led to reduced expression of repeat expansion RNA. Some of the inhibitors identified in this study are currently the subject of clinical trials for other indications and provide valuable starting points for a drug development program in DM1.
Assuntos
Distrofia Miotônica , Animais , Quinases Ciclina-Dependentes , Modelos Animais de Doenças , Humanos , Camundongos , Distrofia Miotônica/tratamento farmacológico , Distrofia Miotônica/genética , RNA , Splicing de RNA/genética , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.
Assuntos
Antimaláricos/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Resistência a Medicamentos/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antimaláricos/química , Antimaláricos/metabolismo , Sítios de Ligação , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Feminino , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/parasitologia , Humanos , Imidazóis/química , Estágios do Ciclo de Vida/efeitos dos fármacos , Metabolômica , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Proteômica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Antimalarial drug resistance compels the quest for new compounds that target alternative pathways to current drugs. The Plasmodium cyclic GMP-dependent protein kinase (PKG) has essential functions in all of the major life cycle developmental stages. An imidazopyridine PKG inhibitor scaffold was previously shown to clear P. falciparum infection in a rodent model in vivo and blocked transmission to mosquitoes providing proof of concept for this target. To find new classes of PKG inhibitors to serve as alternative chemical starting points, we performed a high-throughput screen of the GSK Full Diversity Collection using recombinant P. falciparum PKG. We developed a robust enzymatic assay in a 1536-well plate format. Promising compounds were then tested for activity against P. falciparum asexual blood stage growth, selectivity and cytotoxicity. By using a scoring system we selected the 66 most promising PKG inhibitors (comprising nine clusters and seven singletons). Among these, thiazoles were the most potent scaffold with mid-nanomolar activity on P. falciparum blood stage and gamete development. Using Kinobeads profiling we identified additional P. falciparum protein kinases targeted by the thiazoles that mediate a faster speed of the kill than PKG-selective compounds. This scaffold represents a promising starting point to develop a new antimalarial.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células Hep G2 , Ensaios de Triagem em Larga Escala , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Inibidores de Proteínas Quinases/química , Proteínas de Protozoários/metabolismo , Tiazóis/químicaRESUMO
One of the attractive properties of artemisinins is their extremely fast-killing capability, quickly relieving malaria symptoms. Nevertheless, the unique benefits of these medicines are now compromised by the prolonged parasite clearance times and the increasing frequency of treatment failures, attributed to the increased tolerance of Plasmodium falciparum to artemisinin. This emerging artemisinin resistance threatens to undermine the effectiveness of antimalarial combination therapies. Herein, we describe the medicinal chemistry efforts focused on a cGMP-dependent protein kinase (PKG) inhibitor scaffold, leading to the identification of novel chemical entities with very potent, similar to artemisinins, fast-killing potency against asexual blood stages that cause disease, and activity against gametocyte activation that is required for transmission. Furthermore, we confirm that selective PKG inhibitors have a slow speed of kill, while chemoproteomic analysis suggests for the first time serine/arginine protein kinase 2 (SRPK2) targeting as a novel strategy for developing antimalarial compounds with extremely fast-killing properties.
Assuntos
Antimaláricos/farmacologia , Artemisininas/química , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/química , Antimaláricos/metabolismo , Artemisininas/metabolismo , Artemisininas/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/metabolismo , Humanos , Concentração Inibidora 50 , Mutagênese Sítio-Dirigida , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Solubilidade , Relação Estrutura-Atividade , Tiazóis/químicaRESUMO
The leishmaniases are diseases that affect millions of people across the world, in particular visceral leishmaniasis (VL) which is fatal unless treated. Current standard of care for VL suffers from multiple issues and there is a limited pipeline of new candidate drugs. As such, there is a clear unmet medical need to identify new treatments. This paper describes the optimization of a phenotypic hit against Leishmania donovani, the major causative organism of VL. The key challenges were to balance solubility and metabolic stability while maintaining potency. Herein, strategies to address these shortcomings and enhance efficacy are discussed, culminating in the discovery of preclinical development candidate GSK3186899/DDD853651 (1) for VL.
Assuntos
Leishmaniose Visceral/tratamento farmacológico , Morfolinas/uso terapêutico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Tripanossomicidas/uso terapêutico , Animais , Feminino , Células Hep G2 , Humanos , Leishmania donovani/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Estrutura Molecular , Morfolinas/síntese química , Morfolinas/toxicidade , Testes de Sensibilidade Parasitária , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/toxicidade , Pirazóis/síntese química , Pirazóis/toxicidade , Pirimidinas/síntese química , Pirimidinas/toxicidade , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/toxicidadeRESUMO
The folate pathway has been extensively studied in a number of organisms, with its essentiality exploited by a number of drugs. However, there has been little success in developing drugs that target folate metabolism in the kinetoplastids. Despite compounds being identified which show significant inhibition of the parasite enzymes, this activity does not translate well into cellular and animal models of disease. Understanding to which enzymes antifolates bind under physiological conditions and how this corresponds to the phenotypic response could provide insight on how to target the folate pathway in these organisms. To facilitate this, we have adopted a chemical proteomics approach to study binding of compounds to enzymes of folate metabolism. Clinical and literature antifolate compounds were immobilized onto resins to allow for "pull down" of the proteins in the "folateome". Using competition studies, proteins, which bind the beads specifically and nonspecifically, were identified in parasite lysate ( Trypanosoma brucei and Leishmania major) for each antifolate compound. Proteins were identified through tryptic digest, tandem mass tag (TMT) labeling of peptides followed by LC-MS/MS. This approach was further exploited by creating a combined folate resin (folate beads). The resin could pull down up to 9 proteins from the folateome. This information could be exploited in gaining a better understanding of folate metabolism in kinetoplastids and other organisms.
Assuntos
Antagonistas do Ácido Fólico/metabolismo , Ácido Fólico/metabolismo , Leishmania major/metabolismo , Proteômica/métodos , Trypanosoma brucei brucei/metabolismo , Extratos Celulares , Cromatografia Líquida , Células HeLa , Humanos , Proteínas Imobilizadas , Ligantes , Ligação Proteica , Pterinas/metabolismo , Espectrometria de Massas em TandemRESUMO
As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.
Assuntos
Antineoplásicos/uso terapêutico , Bases de Dados Factuais , Neoplasias Hematológicas , Leucemia Linfocítica Crônica de Células B , Modelos Biológicos , Transdução de Sinais , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 12/metabolismo , Feminino , Neoplasias Hematológicas/classificação , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Leucemia Linfocítica Crônica de Células B/classificação , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Trissomia/genéticaRESUMO
Since the appearance of resistance to the current front-line antimalarial treatments, ACTs (artemisinin combination therapies), the discovery of novel chemical entities to treat the disease is recognized as a major global health priority. From the GSK antimalarial set, we identified an aminoxadiazole with an antiparasitic profile comparable with artemisinin (1), with no cross-resistance in a resistant strains panel and a potential new mode of action. A medicinal chemistry program allowed delivery of compounds such as 19 with high solubility in aqueous media, an acceptable toxicological profile, and oral efficacy. Further evaluation of the lead compounds showed that in vivo genotoxic degradants might be generated. The compounds generated during this medicinal chemistry program and others from the GSK collection were used to build a pharmacophore model which could be used in the virtual screening of compound collections and potentially identify new chemotypes that could deliver the same antiparasitic profile.