RESUMO
Identification of allosteric inhibitors of PTPs has attracted great interest as a new strategy to overcome the challenge of discover potent and selective molecules for therapeutic intervention. YopH is a virulence factor of the genus Yersinia, validated as an antimicrobial target. The finding of a second substrate binding site in YopH has revealed a putative allosteric site that could be further exploited. Novel chalcone compounds that inhibit PTPs activity were designed and synthesized. Compound 3j was the most potent inhibitor, interestingly, with different mechanisms of inhibition for the panel of enzymes evaluated. Further, our results showed that compound 3j is an irreversible non-competitive inhibitor of YopH that binds to a site different than the catalytic site, but close to the well-known second binding site of YopH.
Assuntos
Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Chalcona/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Fatores de Virulência/antagonistas & inibidores , Sítio Alostérico/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/metabolismo , Chalcona/síntese química , Chalcona/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Estrutura Molecular , Proteínas Tirosina Fosfatases/metabolismo , Relação Estrutura-Atividade , Fatores de Virulência/metabolismoRESUMO
Leishmaniasis is a neglected disease caused by the protozoa Leishmania ssp. Environmental differences found by the parasites in the vector and the host are translated into cellular stress, leading to the production of heat shock proteins (Hsp). These are molecular chaperones involved in the folding of nascent proteins as well as in the regulation of gene expression, signalling events and proteostasis. Since Leishmania spp. use Hsp90 to trigger important transitions between their different stages of the life cycle, this protein family becomes a profitable target in anti-parasite drug discovery. In this work, we implemented a multidisciplinary strategy coupling molecular modelling with in vitro assays to identify small molecules able to inhibit Hsp90 from L. braziliensis (LbHsp90). Overall, we identified some compounds able to kill the promastigote form of the L. braziliensis, and to inhibit LbHsp90 ATPase activity.
Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Leishmania braziliensis/efeitos dos fármacos , Chaperonas Moleculares/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Antiprotozoários/síntese química , Antiprotozoários/química , Relação Dose-Resposta a Droga , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Proteínas de Choque Térmico HSP90/metabolismo , Leishmania braziliensis/química , Modelos Moleculares , Chaperonas Moleculares/síntese química , Chaperonas Moleculares/química , Estrutura Molecular , Testes de Sensibilidade Parasitária , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
In Brazil, the mucocutaneous form of leishmaniasis, caused by the parasite Leishmania braziliensis, is a widespread and very challenging disease responsible for disfiguration and, in the most severe cases, death. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone playing a pivotal role in the folding process of client proteins, and therefore its activity is fundamental for cell survival and proliferation. Since the chaperone activity requires ATP hydrolysis, molecules able to occupy the ATP binding pocket in the protein N-terminal domain (NTD) act as Hsp90 inhibitors. The development of selective molecules targeting the ATPase site of protozoan Hsp90 is tricky for the high homology with the human Hsp90 NTD (hNTD). Notably, only the human Lys112 is replaced by Arg97 in the L. braziliensis enzyme. Recently, this difference has been probed to design selective inhibitors targeting parasite Hsp90s. Here, a reliable protocol for expression and purification of LbHsp90-NTD (LbNTD) was developed but its structural characterization was unsuccessful. The role of Arg97 in LbNTD was hence probed by means of the "leishmanized" K112R variant of hNTDα. To deeply investigate the role of this residue, also the hNTDα K112A variant was generated. Structural studies performed on hNTDα and its variants using various ADP and ATP analogues and cAMP revealed that this residue is not crucial for nucleotide binding. This finding strongly suggests that Arg97 in LbNTD and more generally the conserved arginine residue in parasite Hsp90s are not exploitable for the development of selective inhibitors.
Assuntos
Proteínas de Choque Térmico HSP90/química , Leishmania braziliensis/química , Mutagênese Sítio-Dirigida , Proteínas de Protozoários/química , Trifosfato de Adenosina/química , Sítios de Ligação , Brasil , Clonagem Molecular , Proteínas de Choque Térmico HSP90/genética , Humanos , Hidrólise , Leishmania braziliensis/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Ligação Proteica , Conformação Proteica , Proteínas de Protozoários/genéticaRESUMO
New drug development for neoplasm treatment is nowadays based on molecular targets that participate in the disease pathogenesis and tumor phenotype. Herein, we describe a new specific pharmacological hematopoietic cell kinase (HCK) inhibitor (iHCK-37) that was able to reduce PI3K/AKT and MAPK/ERK pathways activation after erythropoietin induction in cells with high HCK expression: iHCK-37 treatment increased leukemic cells death and, very importantly, did not affect normal hematopoietic stem cells. We also present evidence that HCK, one of Src kinase family (SFK) member, regulates early-stage erythroid cell differentiation by acting as an upstream target of a frequently deregulated pathway in hematologic neoplasms, PI3K/AKT and MAPK/ERK. Notably, HCK levels were highly increased in stem cells from patients with some diseases, as Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML), that are associated with ineffective erythropoiesis These discoveries support the exploration of the new pharmacological iHCK-37 in future preclinical and clinical studies.
Assuntos
Inibidores Enzimáticos/farmacologia , Eritropoetina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-hck/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-hck/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto , Idoso , Morte Celular/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Feminino , Fator de Transcrição GATA1/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/metabolismo , Adulto JovemRESUMO
Glucose-6-phosphate dehydrogenase (G6PDH) plays a housekeeping role in cell metabolism by generating reducing power (NADPH) and fueling the production of nucleotide precursors (ribose-5-phosphate). Based on its indispensability for pathogenic parasites from the genus Trypanosoma, G6PDH is considered a drug target candidate. Several steroid-like scaffolds were previously reported to target the activity of G6PDH. Epiandrosterone (EA) is an uncompetitive inhibitor of trypanosomal G6PDH for which its binding site to the enzyme remains unknown. Molecular simulation studies with the structure of Trypanosoma cruzi G6PDH revealed that EA binds in a pocket close to the G6P binding-site and protrudes into the active site blocking the interaction between substrates and hence catalysis. Site directed mutagenesis revealed the important steroid-stabilizing effect of residues (L80, K83 and K84) located on helix α-1 of T. cruzi G6PDH. The higher affinity and potency of 16α-Br EA by T. cruzi G6PDH is explained by the formation of a halogen bond with the hydrogen from the terminal amide of the NADP+-nicotinamide. At variance with the human enzyme, the inclusion of a 21-hydroxypregnane-20-one moiety to a 3ß-substituted steroid is detrimental for T. cruzi G6PDH inhibition. The species-specificity of certain steroid derivatives towards the parasite G6PDH and the corresponding biochemically validated binding models disclosed in this work may prove valuable for the development of selective inhibitors against the pathogen's enzyme.
Assuntos
Androsterona/farmacocinética , Doença de Chagas/tratamento farmacológico , Glucosefosfato Desidrogenase/antagonistas & inibidores , Androsterona/metabolismo , Sítios de Ligação , Doença de Chagas/parasitologia , Glucosefosfato Desidrogenase/metabolismo , Humanos , Simulação de Acoplamento Molecular , Ribosemonofosfatos/metabolismo , Esteroides/farmacologia , Tripanossomicidas/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/patogenicidadeRESUMO
Protein tyrosine phosphatase B (PtpB) is one of the virulence factors secreted into the host cell by Mycobacterium tuberculosis. PtpB attenuates host immune defenses by interfering with signal transduction pathways in macrophages and, therefore, it is considered a promising target for the development of novel anti-tuberculosis drugs. Here we report the discovery of natural compound inhibitors of PtpB among an in house library of more than 800 natural substances by means of a multidisciplinary approach, mixing in silico screening with enzymatic and kinetics studies and MS assays. Six natural compounds proved to inhibit PtpB at low micromolar concentrations (< 30 µM) with Kuwanol E being the most potent with K i = 1.6 ± 0.1 µM. To the best of our knowledge, Kuwanol E is the most potent natural compound PtpB inhibitor reported so far, as well as it is the first non-peptidic PtpB inhibitor discovered from natural sources. Compounds herein identified may inspire the design of novel specific PtpB inhibitors.