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1.
Chembiochem ; 17(20): 1925-1930, 2016 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-27504718

RESUMO

Sulfonyl fluoride (SF)-based activity probes have become important tools in chemical biology. Herein, exploiting the relative chemical stability of SF to carry out a number of unprecedented SF-sparing functional group manipulations, we report the chemoselective synthesis of a toolbox of highly functionalized aryl SF monomers that we used to quickly prepare SF chemical biology probes. In addition to SF, the monomers bear an embedded click handle (a terminal alkyne that can perform copper(I)-mediated azide-alkyne cycloaddition). The monomers can be used either as fragments to prepare clickable SF analogues of drugs (biologically active compounds) bearing an aryl ring or, alternatively, attached to drugs as minimalist clickable aryl SF substituents.


Assuntos
Sondas Moleculares/síntese química , Ácidos Sulfínicos/síntese química , Química Click , Modelos Moleculares , Sondas Moleculares/química , Estrutura Molecular , Ácidos Sulfínicos/química
2.
Biochem J ; 460(2): 211-22, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24593284

RESUMO

ITK (interleukin-2-inducible T-cell kinase) is a critical component of signal transduction in T-cells and has a well-validated role in their proliferation, cytokine release and chemotaxis. ITK is an attractive target for the treatment of T-cell-mediated inflammatory diseases. In the present study we describe the discovery of kinase inhibitors that preferentially bind to an allosteric pocket of ITK. The novel ITK allosteric site was characterized by NMR, surface plasmon resonance, isothermal titration calorimetry, enzymology and X-ray crystallography. Initial screening hits bound to both the allosteric pocket and the ATP site. Successful lead optimization was achieved by improving the contribution of the allosteric component to the overall inhibition. NMR competition experiments demonstrated that the dual-site binders showed higher affinity for the allosteric site compared with the ATP site. Moreover, an optimized inhibitor displayed non-competitive inhibition with respect to ATP as shown by steady-state enzyme kinetics. The activity of the isolated kinase domain and auto-activation of the full-length enzyme were inhibited with similar potency. However, inhibition of the activated full-length enzyme was weaker, presumably because the allosteric site is altered when ITK becomes activated. An optimized lead showed exquisite kinome selectivity and is efficacious in human whole blood and proximal cell-based assays.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/farmacologia , Regulação Alostérica , Sítio Alostérico , Cristalização , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície
3.
J Med Chem ; 64(1): 326-342, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33356244

RESUMO

Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (ß6 Glu → Val) on the ß-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.


Assuntos
Anemia Falciforme/tratamento farmacológico , Hemoglobina A/efeitos dos fármacos , Hemoglobina Falciforme/efeitos dos fármacos , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Animais , Eritrócitos/metabolismo , Camundongos , Oxigênio/metabolismo , Quinolinas/química
4.
J Pharmacol Exp Ther ; 333(3): 707-16, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20228155

RESUMO

Rho kinase, is the most widely studied downstream effector of the small Rho GTPase RhoA. Two Rho kinase isoforms have been described and are frequently referred to in the literature as ROCK1 and ROCK2. The RhoA-Rho kinase pathway has been implicated in the recruitment of cellular infiltrates to disease loci in a number of preclinical animal models of inflammatory disease. In this study, we used biochemical enzyme assays and a cellular target biomarker assay to define PF-4950834 [N-methyl-3-{[(4-pyridin-4-ylbenzoyl)amino]methyl}benzamide] as an ATP-competitive, selective Rho kinase inhibitor. We further used PF-4950834 to study the role of Rho kinase activation in lymphocyte and neutrophil migration in addition to the endothelial cell-mediated expression of adhesion molecules and chemokines, which are essential for leukocyte recruitment. The inhibitor blocked stromal cell-derived factor-1alpha-mediated chemotaxis of T lymphocytes in vitro and the synthesis of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in activated human endothelial cells in vitro. The secretion of chemokines interleukin-8 and monocyte chemoattractant protein-1 was also inhibited in activated endothelial cells. In addition, when dosed orally, the compound potently inhibited neutrophil migration in a carrageenan-induced acute inflammation model. In summary, we have used a pharmacologic approach to link Rho kinase activation to multiple phenotypes that can contribute to leukocyte infiltration. Inhibition of this pathway therefore could be strongly anti-inflammatory and provide therapeutic benefit in chronic inflammatory diseases.


Assuntos
Anti-Inflamatórios não Esteroides , Benzamidas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Benzamidas/farmacocinética , Disponibilidade Biológica , Western Blotting , Moléculas de Adesão Celular/biossíntese , Movimento Celular/efeitos dos fármacos , Quimiocinas/biossíntese , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Citometria de Fluxo , Humanos , Inflamação/induzido quimicamente , Inflamação/prevenção & controle , Interleucina-8/biossíntese , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Masculino , Cadeias Leves de Miosina/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacocinética , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Receptores CCR2/biossíntese
7.
J Med Chem ; 62(17): 7669-7683, 2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31415173

RESUMO

The first chemical probe to primarily occupy the co-factor binding site of a Su(var)3-9, enhancer of a zeste, trithorax (SET) domain containing protein lysine methyltransferase (PKMT) is reported. Protein methyltransferases require S-adenosylmethionine (SAM) as a co-factor (methyl donor) for enzymatic activity. However, SAM itself represents a poor medicinal chemistry starting point for a selective, cell-active inhibitor given its extreme physicochemical properties and its role in multiple cellular processes. A previously untested medicinal chemistry strategy of deliberate file enrichment around molecules bearing the hallmarks of SAM, but with improved lead-like properties from the outset, yielded viable hits against SET and MYND domain-containing protein 2 (SMYD2) that were shown to bind in the co-factor site. These leads were optimized to identify a highly biochemically potent, PKMT-selective, and cell-active chemical probe. While substrate-based inhibitors of PKMTs are known, this represents a novel, co-factor-derived strategy for the inhibition of SMYD2 which may also prove applicable to lysine methyltransferase family members previously thought of as intractable.


Assuntos
Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , S-Adenosilmetionina/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Histona-Lisina N-Metiltransferase/isolamento & purificação , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , S-Adenosilmetionina/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
8.
ACS Med Chem Lett ; 10(1): 80-85, 2019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-30655951

RESUMO

Potent covalent inhibitors of Bruton's tyrosine kinase (BTK) based on an aminopyrazole carboxamide scaffold have been identified. Compared to acrylamide-based covalent reactive groups leading to irreversible protein adducts, cyanamide-based reversible-covalent inhibitors provided the highest combined BTK potency and EGFR selectivity. The cyanamide covalent mechanism with BTK was confirmed through enzyme kinetic, NMR, MS, and X-ray crystallographic studies. The lead cyanamide-based inhibitors demonstrated excellent kinome selectivity and rat pharmacokinetic properties.

9.
J Med Chem ; 61(3): 1130-1152, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29298069

RESUMO

Janus kinases (JAKs) are intracellular tyrosine kinases that mediate the signaling of numerous cytokines and growth factors involved in the regulation of immunity, inflammation, and hematopoiesis. As JAK1 pairs with JAK2, JAK3, and TYK2, a JAK1-selective inhibitor would be expected to inhibit many cytokines involved in inflammation and immune function while avoiding inhibition of the JAK2 homodimer regulating erythropoietin and thrombopoietin signaling. Our efforts began with tofacitinib, an oral JAK inhibitor approved for the treatment of rheumatoid arthritis. Through modification of the 3-aminopiperidine linker in tofacitinib, we discovered highly selective JAK1 inhibitors with nanomolar potency in a human whole blood assay. Improvements in JAK1 potency and selectivity were achieved via structural modifications suggested by X-ray crystallographic analysis. After demonstrating efficacy in a rat adjuvant-induced arthritis (rAIA) model, PF-04965842 (25) was nominated as a clinical candidate for the treatment of JAK1-mediated autoimmune diseases.


Assuntos
Doenças Autoimunes/tratamento farmacológico , Ciclobutanos/farmacologia , Janus Quinase 1/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Sulfonamidas/farmacologia , Animais , Artrite Experimental/tratamento farmacológico , Ciclobutanos/química , Ciclobutanos/farmacocinética , Ciclobutanos/uso terapêutico , Cães , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Janus Quinase 1/química , Janus Quinase 2/antagonistas & inibidores , Modelos Moleculares , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/uso terapêutico , Pirróis/química , Pirróis/farmacocinética , Pirróis/uso terapêutico , Ratos , Especificidade por Substrato , Sulfonamidas/química , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapêutico , Distribuição Tecidual
10.
J Med Chem ; 60(7): 3094-3108, 2017 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-28257199

RESUMO

The C-5 substituted 2,4-diaminoquinazoline RG3039 (compound 1), a member of a chemical series that was identified and optimized using an SMN2 promoter screen, prolongs survival and improves motor function in a mouse model of spinal muscular atrophy (SMA). It is a potent inhibitor of the mRNA decapping scavenger enzyme (DcpS), but the mechanism whereby DcpS inhibition leads to therapeutic benefit is unclear. Compound 1 is a dibasic lipophilic molecule that is predicted to accumulate in lysosomes. To understand if the in vivo efficacy is due to DcpS inhibition or other effects resulting from the physicochemical properties of the chemotype, we undertook structure based molecular design to identify DcpS inhibitors with improved physicochemical properties. Herein we describe the design, synthesis, and in vitro pharmacological characterization of these DcpS inhibitors along with the in vivo mouse CNS PK profile of PF-DcpSi (compound 24), one of the analogs found to be efficacious in SMA mouse model.


Assuntos
Desenho de Fármacos , Endorribonucleases/antagonistas & inibidores , Atrofia Muscular Espinal/tratamento farmacológico , Quinazolinas/química , Quinazolinas/uso terapêutico , RNA Mensageiro/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Endorribonucleases/genética , Endorribonucleases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Quinazolinas/farmacocinética , Quinazolinas/farmacologia , RNA Mensageiro/genética , Proteína 2 de Sobrevivência do Neurônio Motor
11.
Mol Biosyst ; 11(10): 2709-12, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25959423

RESUMO

Despite its diverse applications, such as identification of the protein binding partners of small molecules and investigation of intracellular drug-target engagement, photoaffinity labelling (PAL) is intrinsically challenging, primarily due to the difficulty in discovering functionally active photoaffinity probes. Here we describe the creation of a chemoproteomic library to discover a novel photoaffinity probe for DcpS, an mRNA decapping enzyme that is a putative target for Spinal Muscular Atrophy. This library approach expedites the discovery of photoaffinity probes and expands the chemical biology toolbox to include RNA cap-binding proteins.


Assuntos
Endorribonucleases/metabolismo , Sondas Moleculares/química , Marcadores de Fotoafinidade/química , Sítios de Ligação , Endorribonucleases/química , Biblioteca Gênica , Humanos , Modelos Moleculares , Sondas Moleculares/metabolismo , Quinazolinas/química
12.
ACS Chem Biol ; 10(4): 1094-8, 2015 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-25571984

RESUMO

This work describes the first rational targeting of tyrosine residues in a protein binding site by small-molecule covalent probes. Specific tyrosine residues in the active site of the mRNA-decapping scavenger enzyme DcpS were modified using reactive sulfonyl fluoride covalent inhibitors. Structure-based molecular design was used to create an alkyne-tagged probe bearing the sulfonyl fluoride warhead, thus enabling the efficient capture of the protein from a complex proteome. Use of the probe in competition experiments with a diaminoquinazoline DcpS inhibitor permitted the quantification of intracellular target occupancy. As a result, diaminoquinazoline upregulators of survival motor neuron protein that are used for the treatment of spinal muscular atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes designed to react with specific tyrosine residues of a protein and augments the chemical biology toolkit by these probes uses in target validation and molecular pharmacology.


Assuntos
Endorribonucleases/metabolismo , Inibidores Enzimáticos/farmacologia , Sondas Moleculares/química , Ácidos Sulfínicos/química , Tirosina/metabolismo , Domínio Catalítico , Células Cultivadas , Técnicas de Química Sintética , Cristalografia por Raios X , Endorribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Sondas Moleculares/síntese química , Terapia de Alvo Molecular/métodos , Relação Estrutura-Atividade , Tirosina/química
13.
Bioorg Med Chem ; 15(10): 3390-412, 2007 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-17387018

RESUMO

The integrin alpha(v)beta(3) is expressed in a number of cell types and is thought to play a major role in several pathological conditions. Various small molecules that inhibit the integrin have been shown to suppress tumor growth and retinal angiogenesis. The tripeptide Arg-Gly-Asp (RGD), a common binding motif in several ligands that bind to alpha(v)beta(3), has been depeptidized and optimized in our efforts toward discovering a small molecule inhibitor. We recently disclosed the synthesis and biological activity of several small molecules that did not contain any peptide bond and mimic the tripeptide RGD. The phenethyl group in one of the lead compounds was successfully replaced with a cyclopropyl moiety. The new lead compound was optimized for potency, selectivity, and for its ADME properties. We describe herein the discovery, synthesis, and optimization of cyclopropyl containing analogs that are potent and selective inhibitors of alpha(v)beta(3).


Assuntos
Acetatos/síntese química , Acetatos/farmacologia , Integrina alfaVbeta3/antagonistas & inibidores , Naftiridinas/síntese química , Naftiridinas/farmacologia , Animais , Área Sob a Curva , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Desenho de Fármacos , Meia-Vida , Humanos , Indicadores e Reagentes , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Espectrofotometria Ultravioleta , Relação Estrutura-Atividade , Transfecção
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