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1.
J Med Chem ; 64(15): 11302-11329, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34292726

RESUMO

Indoleamine 2,3-dioxygenase 1 (IDO1), a heme-containing enzyme that mediates the rate-limiting step in the metabolism of l-tryptophan to kynurenine, has been widely explored as a potential immunotherapeutic target in oncology. We developed a class of inhibitors with a conformationally constrained bicyclo[3.1.0]hexane core. These potently inhibited IDO1 in a cellular context by binding to the apoenzyme, as elucidated by biochemical characterization and X-ray crystallography. A SKOV3 tumor model was instrumental in differentiating compounds, leading to the identification of IACS-9779 (62) and IACS-70465 (71). IACS-70465 has excellent cellular potency, a robust pharmacodynamic response, and in a human whole blood assay was more potent than linrodostat (BMS-986205). IACS-9779 with a predicted human efficacious once daily dose below 1 mg/kg to sustain >90% inhibition of IDO1 displayed an acceptable safety margin in rodent toxicology and dog cardiovascular studies to support advancement into preclinical safety evaluation for human development.


Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade
2.
J Appl Crystallogr ; 54(Pt 3): 895-902, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34188616

RESUMO

Soaking of macromolecular crystals allows the formation of complexes via diffusion of molecules into a preformed crystal for structural analysis. Soaking offers various advantages over co-crystallization, e.g. small samples and high-throughput experimentation. However, this method has disadvantages, such as inducing mechanical stress on crystals and reduced success rate caused by low affinity/solubility of the ligand. To bypass these issues, the Picodropper was previously developed in the authors' laboratory. This technique aimed to deliver small volumes of compound solution in response to crystal dehydration supported by the Free Mounting System humidity control or by IR-laser-induced protein crystal transformation. Herein, a new related soaking development, the Aerosol-Generator, is introduced. This device delivers compounds onto the solution-free surface of protein crystals using an ultrasonic technique. The produced aerosol stream enables an easier and more accurate control of solution volumes, reduced crystal handling, and crystal-size-independent soaking. The Aerosol-Generator has been used to produce complexes of DPP8 crystals, where otherwise regular soaking did not achieve complex formation. These results demonstrate the potential of this device in challenging ligand-binding scenarios and contribute to further understanding of DPP8 inhibitor binding.

3.
Bioorg Med Chem ; 42: 116223, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34091303

RESUMO

Libraries of DNA-Encoded small molecules created using combinatorial chemistry and synthetic oligonucleotides are being applied to drug discovery projects across the pharmaceutical industry. The majority of reported projects describe the discovery of reversible, i.e. non-covalent, target modulators. We synthesized multiple DNA-encoded chemical libraries terminated in electrophiles and then used them to discover covalent irreversible inhibitors and report the successful discovery of acrylamide- and epoxide-terminated Bruton's Tyrosine Kinase (BTK) inhibitors. We also demonstrate their selectivity, potency and covalent cysteine engagement using a range of techniques including X-ray crystallography, thermal transition shift assay, reporter displacement assay and intact protein complex mass spectrometry. The epoxide BTK inhibitors described here are the first ever reported to utilize this electrophile for this target.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , DNA/química , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Tirosina Quinase da Agamaglobulinemia/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
4.
J Am Chem Soc ; 140(18): 5914-5924, 2018 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-29676907

RESUMO

ß-Glucocerebrosidase (GCase) mutations cause Gaucher's disease and are a high risk factor in Parkinson's disease. The implementation of a small molecule modulator is a strategy to restore proper folding and lysosome delivery of degradation-prone mutant GCase. Here, we present a potent quinazoline modulator, JZ-4109, which stabilizes wild-type and N370S mutant GCase and increases GCase abundance in patient-derived fibroblast cells. We then developed a covalent modification strategy using a lysine targeted inactivator (JZ-5029) for in vitro mechanistic studies. By using native top-down mass spectrometry, we located two potentially covalently modified lysines. We obtained the first crystal structure, at 2.2 Å resolution, of a GCase with a noniminosugar modulator covalently bound, and were able to identify the exact lysine residue modified (Lys346) and reveal an allosteric binding site. GCase dimerization was induced by our modulator binding, which was observed by native mass spectrometry, its crystal structure, and size exclusion chromatography with a multiangle light scattering detector. Finally, the dimer form was confirmed by negative staining transmission electron microscopy studies. Our newly discovered allosteric site and observed GCase dimerization provide a new mechanistic insight into GCase and its noniminosugar modulators and facilitate the rational design of novel GCase modulators for Gaucher's disease and Parkinson's disease.


Assuntos
Sítio Alostérico , Glucosilceramidase/química , Glucosilceramidase/metabolismo , Multimerização Proteica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Cristalografia por Raios X , Fibroblastos/metabolismo , Glucosilceramidase/genética , Células HEK293 , Humanos , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Mutação
5.
Proc Natl Acad Sci U S A ; 115(7): E1437-E1445, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29382749

RESUMO

Dipeptidyl peptidases 8 and 9 are intracellular N-terminal dipeptidyl peptidases (preferentially postproline) associated with pathophysiological roles in immune response and cancer biology. While the DPP family member DPP4 is extensively characterized in molecular terms as a validated therapeutic target of type II diabetes, experimental 3D structures and ligand-/substrate-binding modes of DPP8 and DPP9 have not been reported. In this study we describe crystal and molecular structures of human DPP8 (2.5 Å) and DPP9 (3.0 Å) unliganded and complexed with a noncanonical substrate and a small molecule inhibitor, respectively. Similar to DPP4, DPP8 and DPP9 molecules consist of one ß-propeller and α/ß hydrolase domain, forming a functional homodimer. However, they differ extensively in the ligand binding site structure. In intriguing contrast to DPP4, where liganded and unliganded forms are closely similar, ligand binding to DPP8/9 induces an extensive rearrangement at the active site through a disorder-order transition of a 26-residue loop segment, which partially folds into an α-helix (R-helix), including R160/133, a key residue for substrate binding. As vestiges of this helix are also seen in one of the copies of the unliganded form, conformational selection may contributes to ligand binding. Molecular dynamics simulations support increased flexibility of the R-helix in the unliganded state. Consistently, enzyme kinetics assays reveal a cooperative allosteric mechanism. DPP8 and DPP9 are closely similar and display few opportunities for targeted ligand design. However, extensive differences from DPP4 provide multiple cues for specific inhibitor design and development of the DPP family members as therapeutic targets or antitargets.


Assuntos
Dipeptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Homeostase/fisiologia , Conformação Proteica , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dipeptidases/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Humanos , Estrutura Molecular , Domínios Proteicos
6.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 5): 1224-32, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24816092

RESUMO

A method and the design of instrumentation, and its preliminary practical realisation, including test experiments, with the object of inducing phase changes of biomolecular crystals by controlled dehydration through heating with infrared (IR) light are described. The aim is to generate and select crystalline phases through transformation in the solid state which have improved order (higher resolution in X-ray diffraction experiments) and reduced mosaic spread (more uniformly aligned mosaic blocks) for diffraction data collection and analysis. The crystal is heated by pulsed and/or constant IR laser irradiation. Loss of crystal water following heating and its reabsorption through equilibration with the environment is measured optically by a video system. Heating proved superior to traditional controlled dehydration by humidity change for the test cases CODH (carbon monoxide dehydrogenase) and CLK2 (a protein kinase). Heating with IR light is experimentally simple and offers an exploration of a much broader parameter space than the traditional method, as it allows the option of varying the rate of phase changes through modification of the IR pulse strength, width and repeat frequency. It impacts the crystal instantaneously, isotropically and homogeneously, and is therefore expected to cause less mechanical stress.


Assuntos
Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/métodos , Aldeído Oxirredutases/química , Desenho de Equipamento , Raios Infravermelhos , Lasers , Complexos Multienzimáticos/química , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , Software
7.
J Med Chem ; 55(20): 8827-37, 2012 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-22984809

RESUMO

Rational structure-based design has yielded highly potent inhibitors of cathepsin K (Cat K) with excellent physical properties, selectivity profiles, and pharmacokinetics. Compounds with a 3,4-(CH3O)2Ph motif, such as 31, were found to have excellent metabolic stability and absorption profiles. Through metabolite identification studies, a reactive metabolite risk was identified with this motif. Subsequent structure-based design of isoteres culminated in the discovery of an optimized and balanced inhibitor (indazole, 38).


Assuntos
Catepsina K/antagonistas & inibidores , Cicloexanos/síntese química , Indazóis/síntese química , Animais , Proteínas Sanguíneas/metabolismo , Células Cultivadas , Cicloexanos/farmacocinética , Cicloexanos/farmacologia , Desenho de Fármacos , Hepatócitos/metabolismo , Humanos , Indazóis/farmacocinética , Indazóis/farmacologia , Masculino , Modelos Moleculares , Ligação Proteica , Ratos , Ratos Wistar , Estereoisomerismo , Relação Estrutura-Atividade
8.
Bioorg Med Chem Lett ; 22(17): 5563-8, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22858142

RESUMO

The discovery of nitrile compound 4, a potent inhibitor of Cathepsin K (Cat K) with good bioavailability in dog is described. The compound was used to demonstrate target engagement and inhibition of Cat K in an in vivo dog PD model. The margin to hERG ion channel inhibition was deemed too low for a clinical candidate and an optimisation program to find isosteres or substitutions on benzothiazole group led to the discovery of 20, 24 and 27; all three free from hERG inhibition.


Assuntos
Benzotiazóis/química , Benzotiazóis/farmacologia , Catepsina K/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Nitrilas/química , Nitrilas/farmacologia , Animais , Benzotiazóis/metabolismo , Benzotiazóis/farmacocinética , Catepsina K/metabolismo , Cães , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Nitrilas/metabolismo , Nitrilas/farmacocinética , Ratos , Relação Estrutura-Atividade
9.
J Med Chem ; 55(14): 6363-74, 2012 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-22742641

RESUMO

Directed screening of nitrile compounds revealed 3 as a highly potent cathepsin K inhibitor but with cathepsin S activity and very poor stability to microsomes. Synthesis of compounds with reduced molecular complexity, such as 7, revealed key SAR and demonstrated that baseline physical properties and in vitro stability were in fact excellent for this series. The tricycle carboline P3 unit was discovered by hypothesis-based design using existing structural information. Optimization using small substituents, knowledge from matched molecular pairs, and control of lipophilicity yielded compounds very close to the desired profile, of which 34 (AZD4996) was selected on the basis of pharmacokinetic profile.


Assuntos
Carbolinas/farmacologia , Catepsina K/antagonistas & inibidores , Indóis/farmacologia , Osteoartrite/tratamento farmacológico , Inibidores de Proteases/farmacologia , Animais , Carbolinas/metabolismo , Carbolinas/farmacocinética , Carbolinas/uso terapêutico , Catepsina K/química , Cães , Humanos , Indóis/metabolismo , Indóis/farmacocinética , Indóis/uso terapêutico , Concentração Inibidora 50 , Masculino , Modelos Moleculares , Osteoartrite/enzimologia , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/uso terapêutico , Conformação Proteica , Ratos , Especificidade por Substrato
10.
Artigo em Inglês | MEDLINE | ID: mdl-22505407

RESUMO

Factor XI (FXI) is a key enzyme in the coagulation pathway and an attractive target for the development of anticoagulant drugs. A small number of high-resolution crystal structures of FXIa in complex with small synthetic inhibitors have been published to date. All of these ligands have a basic P1 group and bind exclusively in the nonprime side of the active site of FXIa. Here, two structures of FXIa in complex with nonbasic inhibitors that occupy both the prime and nonprime sides of the active site are presented. These new structures could be valuable in the design and optimization of new FXIa synthethic inhibitors.


Assuntos
Inibidores Enzimáticos/química , Fator XIa/química , Domínios e Motivos de Interação entre Proteínas , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Fator XIa/antagonistas & inibidores , Fator XIa/metabolismo , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Homologia Estrutural de Proteína
11.
Methods Enzymol ; 493: 61-89, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21371587

RESUMO

In the past two decades, fragment-based approaches have evolved as a predominant strategy in lead discovery. The availability of structural information on the interaction geometries of binding fragments is key to successful structure-guided fragment-to-lead evolution. In this chapter, we illustrate methodological advances for protein-fragment crystal structure generation in order to offer general lessons on the importance of fragment properties and the most appropriate crystallographic setup to evaluate them. We analyze elaborate protocols, methods, and clues applied to challenging complex formation projects. The results should assist medicinal chemists to select the most promising targets and strategies for fragment-based crystallography as well as provide a tutorial to structural biologists who attempt to determine protein-fragment structures.


Assuntos
Proteínas/química , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Desenho de Fármacos , Descoberta de Drogas/métodos , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Ligação Proteica
14.
J Biol Chem ; 282(17): 13003-10, 2007 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-17303565

RESUMO

Uridine diphosphate-glucose pyrophosphorylase (UGPase) represents a ubiquitous enzyme, which catalyzes the formation of UDP-glucose, a key metabolite of the carbohydrate pathways of all organisms. In the protozoan parasite Leishmania major, which causes a broad spectrum of diseases and is transmitted to humans by sand fly vectors, UGPase represents a virulence factor because of its requirement for the synthesis of cell surface glycoconjugates. Here we present the crystal structures of the L. major UGPase in its uncomplexed apo form (open conformation) and in complex with UDP-glucose (closed conformation). The UGPase consists of three distinct domains. The N-terminal domain exhibits species-specific differences in length, which might permit distinct regulation mechanisms. The central catalytic domain resembles a Rossmann-fold and contains key residues that are conserved in many nucleotidyltransferases. The C-terminal domain forms a left-handed parallel beta-helix (LbetaH), which represents a rarely observed structural element. The presented structures together with mutagenesis analyses provide a basis for a detailed analysis of the catalytic mechanism and for the design of species-specific UGPase inhibitors.


Assuntos
Leishmania major/enzimologia , Dobramento de Proteína , Proteínas de Protozoários/química , UTP-Glucose-1-Fosfato Uridililtransferase/química , Fatores de Virulência/química , Animais , Apoenzimas/química , Apoenzimas/metabolismo , Catálise , Cristalografia por Raios X , Glicoconjugados/biossíntese , Glicoconjugados/química , Leishmania major/patogenicidade , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Fatores de Virulência/metabolismo
15.
Glycobiology ; 14(10): 43R-51R, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15201214

RESUMO

Activation of sugars into nucleotide sugars is critical for their entry into biosynthetic pathways. In eukaryotic cells, the activation of the acidic nine-carbon sugar sialic acid to CMP-sialic acid takes place in the cell nucleus, whereas all other nucleotide sugars are made in the cytoplasm. Molecular cloning of vertebrate CMP-sialic acid synthetases confirmed the nuclear localization and introduced new molecular tools for directly exploring the functional mechanisms of the enzymes, as well as the physiological relevance of their nuclear transport. Although major advances have been made in understanding structure-function relationships and defining elements involved in the nuclear transport, the riddle surrounding the physiological relevance of nuclear localization awaits resolution.


Assuntos
N-Acilneuraminato Citidililtransferase , Sequência de Aminoácidos , Animais , Clonagem Molecular , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , N-Acilneuraminato Citidililtransferase/química , N-Acilneuraminato Citidililtransferase/genética , N-Acilneuraminato Citidililtransferase/fisiologia , Filogenia , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
16.
J Mol Biol ; 334(4): 625-37, 2003 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-14636592

RESUMO

Sialic acids are activated by CMP-5-N-acetylneuraminic acid synthetase prior to their transfer onto oligo- or polysaccharides. Here, we present the crystal structure of the N-terminal catalytically active domain of the murine 5-N-acetylneuraminic acid synthetase in complex with the reaction product. In contrast to the previously solved structure of 5-N-acetylneuraminic acid synthetase from Neisseria meningitidis and the related CMP-KDO-synthetase of Escherichia coli, the murine enzyme is a tetramer, which was observed with the active sites closed. In this conformation a loop is shifted by 6A towards the active site and thus an essential arginine residue can participate in catalysis. Furthermore, a network of intermolecular salt-bridges and hydrogen bonds in the dimer as well as hydrophobic interfaces between two dimers indicate a cooperative behaviour of the enzyme. In addition, a complex regulation of the enzyme activity is proposed that includes phosphorylation and dephosphorylation.


Assuntos
N-Acilneuraminato Citidililtransferase/química , Estrutura Quaternária de Proteína , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ácido N-Acetilneuramínico do Monofosfato de Citidina/química , Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , N-Acilneuraminato Citidililtransferase/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência
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