RESUMO
Macrocyclic retinoic acid receptor-related orphan receptor C2 (RORC2) inverse agonists have been designed with favorable properties for topical administration. Inspired by the unanticipated bound conformation of an acyclic sulfonamide-based RORC2 ligand from cocrystal structure analysis, macrocyclic linker connections between the halves of the molecule were explored. Further optimization of analogues was accomplished to maximize potency and refine physiochemical properties (MW, lipophilicity) best suited for topical application. Compound 14 demonstrated potent inhibition of interleukin-17A (IL-17A) production by human Th17 cells and in vitro permeation through healthy human skin achieving high total compound concentration in both skin epidermis and dermis layers.
RESUMO
A diaryl ketone series was identified as vanin-1 inhibitors from a high-throughput screening campaign. While this novel scaffold provided valuable probe 2 that was used to build target confidence, concerns over the ketone moiety led to the replacement of this group. The successful replacement of this moiety was achieved with pyrimidine carboxamides derived from cyclic secondary amines that were extensively characterized using biophysical and crystallographic methods as competitive inhibitors of vanin-1. Through optimization of potency and physicochemical and ADME properties, and guided by co-crystal structures with vanin-1, 3 was identified with a suitable profile for advancement into preclinical development.
Assuntos
Amidoidrolases/antagonistas & inibidores , Piridinas/síntese química , Piridinas/farmacologia , Animais , Colite/induzido quimicamente , Colite/tratamento farmacológico , Cristalografia por Raios X , Sulfato de Dextrana , Cães , Descoberta de Drogas , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Cetonas/química , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Piridinas/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
Tyrosine kinase 2 (TYK2) is a member of the JAK kinase family that regulates signal transduction downstream of receptors for the IL-23/IL-12 pathways and type I interferon family, where it pairs with JAK2 or JAK1, respectively. On the basis of human genetic and emerging clinical data, a selective TYK2 inhibitor provides an opportunity to treat autoimmune diseases delivering a potentially differentiated clinical profile compared to currently approved JAK inhibitors. The discovery of an ATP-competitive pyrazolopyrazinyl series of TYK2 inhibitors was accomplished through computational and structurally enabled design starting from a known kinase hinge binding motif. With understanding of PK/PD relationships, a target profile balancing TYK2 potency and selectivity over off-target JAK2 was established. Lead optimization involved modulating potency, selectivity, and ADME properties which led to the identification of the clinical candidate PF-06826647 (22).
Assuntos
Doenças Autoimunes/enzimologia , Descoberta de Drogas/métodos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , TYK2 Quinase/antagonistas & inibidores , Animais , Doenças Autoimunes/tratamento farmacológico , Humanos , Camundongos , Camundongos Transgênicos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Inibidores de Proteínas Quinases/uso terapêutico , Estrutura Secundária de Proteína , TYK2 Quinase/química , TYK2 Quinase/metabolismoRESUMO
Translation of modulation of drug target activity to therapeutic effect is a critical aspect for all drug discovery programs. In this work we describe the profiling of a non-receptor tyrosine-protein kinase (TYK2) inhibitor which shows a functionally relevant potency shift between human and preclinical species (e.g. murine, dog, macaque) in both biochemical and cellular assays. Comparison of the structure and sequence homology of TYK2 between human and preclinical species within the ATP binding site highlights a single amino acid (I960 â V) responsible for the potency shift. Through TYK2 kinase domain mutants and a TYK2 980I knock-in mouse model, we demonstrate that this single amino acid change drives a functionally relevant potency difference that exists between human and all evaluated preclinical species, for a series of TYK2 inhibitors which target the ATP binding site.
Assuntos
Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , TYK2 Quinase/antagonistas & inibidores , TYK2 Quinase/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/efeitos dos fármacos , Cães , Humanos , Janus Quinase 1 , Macaca , Camundongos , Mutação , Domínios Proteicos/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , TYK2 Quinase/genética , TYK2 Quinase/metabolismoRESUMO
Herein, we disclose a new series of TYK2/ JAK1 inhibitors based upon a 3.1.0 azabicyclic substituted pyrimidine scaffold. We illustrate the use of structure-based drug design for the initial design and subsequent optimization of this series of compounds. One advanced example 19 met program objectives for potency, selectivity and ADME, and demonstrated oral activity in the adjuvant-induced arthritis rat model.
Assuntos
Artrite Experimental/tratamento farmacológico , Desenho de Fármacos , Janus Quinase 1/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , TYK2 Quinase/antagonistas & inibidores , Animais , Artrite Experimental/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Janus Quinase 1/metabolismo , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Ratos , Ratos Endogâmicos Lew , Relação Estrutura-Atividade , TYK2 Quinase/metabolismoRESUMO
Cytokine signaling is an important characteristic of autoimmune diseases. Many pro-inflammatory cytokines signal through the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway. JAK1 is important for the γ-common chain cytokines, interleukin (IL)-6, and type-I interferon (IFN) family, while TYK2 in addition to type-I IFN signaling also plays a role in IL-23 and IL-12 signaling. Intervention with monoclonal antibodies (mAbs) or JAK1 inhibitors has demonstrated efficacy in Phase III psoriasis, psoriatic arthritis, inflammatory bowel disease, and rheumatoid arthritis studies, leading to multiple drug approvals. We hypothesized that a dual JAK1/TYK2 inhibitor will provide additional efficacy, while managing risk by optimizing selectivity against JAK2 driven hematopoietic changes. Our program began with a conformationally constrained piperazinyl-pyrimidine Type 1 ATP site inhibitor, subsequent work led to the discovery of PF-06700841 (compound 23), which is in Phase II clinical development (NCT02969018, NCT02958865, NCT03395184, and NCT02974868).
Assuntos
Antituberculosos/farmacologia , Artrite Experimental/prevenção & controle , Janus Quinase 1/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , TYK2 Quinase/antagonistas & inibidores , Tuberculose/complicações , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/microbiologia , Feminino , Estrutura Molecular , Ratos , Ratos Endogâmicos Lew , Tuberculose/microbiologiaRESUMO
We disclose the discovery and X-ray cocrystal data of potent, selective quinazoline inhibitors of PDE1. Inhibitor ( S)-3 readily attains free plasma concentrations above PDE1 IC50 values and has restricted brain access. The racemic compound 3 inhibits >75% of PDE hydrolytic activity in soluble samples of human myocardium, consistent with heightened PDE1 activity in this tissue. These compounds represent promising new tools to probe the value of PDE1 inhibition in the treatment of cardiovascular disease.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/antagonistas & inibidores , Descoberta de Drogas , Miocárdio/enzimologia , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/farmacologia , Quinazolinas/química , AMP Cíclico/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação ProteicaRESUMO
PF-06651600, a newly discovered potent JAK3-selective inhibitor, is highly efficacious at inhibiting γc cytokine signaling, which is dependent on both JAK1 and JAK3. PF-06651600 allowed the comparison of JAK3-selective inhibition to pan-JAK or JAK1-selective inhibition, in relevant immune cells to a level that could not be achieved previously without such potency and selectivity. In vitro, PF-06651600 inhibits Th1 and Th17 cell differentiation and function, and in vivo it reduces disease pathology in rat adjuvant-induced arthritis as well as in mouse experimental autoimmune encephalomyelitis models. Importantly, by sparing JAK1 function, PF-06651600 selectively targets γc cytokine pathways while preserving JAK1-dependent anti-inflammatory signaling such as the IL-10 suppressive functions following LPS treatment in macrophages and the suppression of TNFα and IL-1ß production in IL-27-primed macrophages. Thus, JAK3-selective inhibition differentiates from pan-JAK or JAK1 inhibition in various immune cellular responses, which could potentially translate to advantageous clinical outcomes in inflammatory and autoimmune diseases.
Assuntos
Artrite Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Janus Quinase 3/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Animais , Artrite Experimental/imunologia , Modelos Animais de Doenças , Descoberta de Drogas , Encefalomielite Autoimune Experimental/imunologia , Humanos , Interleucina-10/imunologia , Interleucina-1beta/imunologia , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/metabolismo , Janus Quinase 3/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Modelos Moleculares , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Pirróis/farmacocinética , Pirróis/farmacologia , Ratos , Células Th1/citologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th17/citologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
A novel series of spirocyclic-diamine based, isoform non-selective inhibitors of acetyl-CoA carboxylase (ACC) is described. These spirodiamine derivatives were discovered by design of a library to mimic the structural rigidity and hydrogen-bonding pattern observed in the co-crystal structure of spirochromanone inhibitor I. The lead compound 3.5.1 inhibited de novo lipogenesis in rat hepatocytes, with an IC50 of 0.30 µM.
Assuntos
Acetilcoenzima A/metabolismo , Acetil-CoA Carboxilase/antagonistas & inibidores , Descoberta de Drogas , Hepatócitos/efeitos dos fármacos , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hepatócitos/enzimologia , Humanos , Concentração Inibidora 50 , Modelos Biológicos , Estrutura Molecular , Ratos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Acetyl-CoA carboxylase (ACC) inhibitors offer significant potential for the treatment of type 2 diabetes mellitus (T2DM), hepatic steatosis, and cancer. However, the identification of tool compounds suitable to test the hypothesis in human trials has been challenging. An advanced series of spirocyclic ketone-containing ACC inhibitors recently reported by Pfizer were metabolized in vivo by ketone reduction, which complicated human pharmacology projections. We disclose that this metabolic reduction can be greatly attenuated through introduction of steric hindrance adjacent to the ketone carbonyl. Incorporation of weakly basic functionality improved solubility and led to the identification of 9 as a clinical candidate for the treatment of T2DM. Phase I clinical studies demonstrated dose-proportional increases in exposure, single-dose inhibition of de novo lipogenesis (DNL), and changes in indirect calorimetry consistent with increased whole-body fatty acid oxidation. This demonstration of target engagement validates the use of compound 9 to evaluate the role of DNL in human disease.
Assuntos
Acetil-CoA Carboxilase/antagonistas & inibidores , Hepatócitos/efeitos dos fármacos , Cetonas/metabolismo , Lipogênese/efeitos dos fármacos , Microssomos/efeitos dos fármacos , Acetil-CoA Carboxilase/metabolismo , Adulto , Animais , Área Sob a Curva , Células Cultivadas , Estudos Cross-Over , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Cães , Método Duplo-Cego , Hepatócitos/citologia , Humanos , Masculino , Malonil Coenzima A/metabolismo , Microssomos/metabolismo , Pessoa de Meia-Idade , Modelos Moleculares , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Relação Estrutura-Atividade , Adulto JovemRESUMO
The synthesis and biological evaluation of novel Tie-2 kinase inhibitors are presented. Based on the pyrrolopyrimidine chemotype, several new series are described, including the benzimidazole series by linking a benzimidazole to the C5-position of the 4-amino-pyrrolopyrimidine core and the ketophenyl series synthesized by incorporating a ketophenyl group to the C5-position. Medicinal chemistry efforts led to potent Tie-2 inhibitors. Compound 15, a ketophenyl pyrrolopyrimidine urea analog with improved physicochemical properties, demonstrated favorable in vitro attributes as well as dose responsive and robust oral tumor growth inhibition in animal models.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Descoberta de Drogas , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Receptor TIE-2/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Masculino , Estrutura Molecular , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/síntese química , Pirimidinas/química , Pirróis/síntese química , Pirróis/química , Ratos , Ratos Sprague-Dawley , Receptor TIE-2/metabolismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The aspartyl protease ß-secretase, or BACE, has been demonstrated to be a key factor in the proteolytic formation of Aß-peptide, a major component of plaques in the brains of Alzheimer's disease (AD) patients, and inhibition of this enzyme has emerged as a major strategy for pharmacologic intervention in AD. An X-ray-based fragment screen of Pfizer's proprietary fragment collection has resulted in the identification of a novel BACE binder featuring spiropyrrolidine framework. Although exhibiting only weak inhibitory activity against the BACE enzyme, the small compound was verified by biophysical and NMR-based methods as a bona fide BACE inhibitor. Subsequent optimization of the lead compound, relying heavily on structure-based drug design and computational prediction of physiochemical properties, resulted in a nearly 1000-fold improvement in potency while maintaining ligand efficiency and properties predictive of good permeability and low P-gp liability.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Pirrolidinas/química , Compostos de Espiro/química , Secretases da Proteína Precursora do Amiloide/química , Ácido Aspártico Endopeptidases/química , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Indóis/síntese química , Indóis/química , Indóis/farmacologia , Modelos Moleculares , Estrutura Molecular , Pirrolidinas/síntese química , Pirrolidinas/farmacologia , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (1), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by 1 in a labile peptide-heme-peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS(4)) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that 1 was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of 1. The final orientation of 1 perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of 1 by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain.
Assuntos
Heme/química , Peroxidase/química , Peroxidase/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cromatografia Líquida , Cristalografia por Raios X , Humanos , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Neutrófilos/enzimologia , Oxirredução , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos , Espectrometria de Massas em TandemRESUMO
Inhibition of acetyl-CoA carboxylases (ACCs), a crucial enzyme for fatty acid metabolism, has been shown to promote fatty acid oxidation and reduce body fat in animal models. Therefore, ACCs are attractive targets for structure-based inhibitor design, particularly the carboxyltransferase (CT) domain, which is the primary site for inhibitor interaction. We have cloned, expressed, and purified the CT domain of human ACC2 using baculovirus-mediated insect cell expression system. However, attempts to crystallize the human ACC2 CT domain have not been successful in our hands. Hence, we have been using the available crystal structure of yeast CT domain to design human ACC inhibitors. Unfortunately, as the selectivity of the lead series has increased against the full-length human enzyme, the potency against the yeast enzyme has decreased significantly. This loss of potency against the yeast enzyme correlated with a complete lack of binding of the human-specific compounds to crystals of the yeast CT domain. Here, we address this problem by converting nine key active site residues of the yeast CT domain to the corresponding human residues. The resulting humanized yeast ACC-CT (yCT-H9) protein exhibits biochemical and biophysical properties closer to the human CT domain and binding to human specific compounds. We report high resolution crystal structures of yCT-H9 complexed with inhibitors that show a preference for the human CT domain. These structures offer insights that explain the species selectivity of ACC inhibitors and may guide future drug design programs.
Assuntos
Acetil-CoA Carboxilase/antagonistas & inibidores , Acetil-CoA Carboxilase/química , Domínio Catalítico , Inibidores Enzimáticos/química , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/química , Acetil-CoA Carboxilase/genética , Animais , Linhagem Celular , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Especificidade da Espécie , Spodoptera , Homologia Estrutural de Proteína , Relação Estrutura-AtividadeRESUMO
Lasofoxifene is a new and potent selective estrogen receptor modulator (SERM). The structural basis of its interaction with the estrogen receptor has been investigated by crystallographic analysis of its complex with the ligand-binding domain of estrogen receptor alpha at a resolution of 2.0 A. As with other SERMs, lasofoxifene diverts the receptor from its agonist-bound conformation by displacing the C-terminal AF-2 helix into the site at which the LXXLL motif of coactivator proteins would otherwise be able to bind. Lasofoxifene achieves this effect by occupying the space normally filled by residue Leu 540, as well as by modulating the conformation of residues of helix 11 (His 524, Leu 525). A well-defined salt bridge between lasofoxifene and Asp 351 suggests that charge neutralization in this region of the receptor may explain the some of the antiestrogenic effects of lasofoxifene. The results suggest general features of ERalpha/SERM recognition, and add a new dimension to efforts to rationalize differences between the biological activity profiles exhibited by these important pharmacological agents.
Assuntos
Receptor alfa de Estrogênio/química , Pirrolidinas/química , Tetra-Hidronaftalenos/química , Cristalografia por Raios X , Receptor alfa de Estrogênio/metabolismo , Ligação de Hidrogênio , Estrutura Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pirrolidinas/metabolismo , Moduladores Seletivos de Receptor Estrogênico/química , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Tetra-Hidronaftalenos/metabolismoRESUMO
The Hedgehog pathway drives proliferation and differentiation by activating the Gli/Ci family of zinc finger transcription factors. Gli/Ci proteins form Hedgehog signaling complexes with other signaling components, including the kinesin-like protein Costal-2, the serine-threonine kinase Fused, and Suppressor of Fused [Su(fu)]. In these complexes Gli/Ci proteins are regulated by cytoplasmic sequestration, phosphorylation, and proteolysis. Here we characterize structural and functional determinants of Su(fu) required for Gli regulation and show that Su(fu) contains at least two distinct domains: a highly conserved carboxy-terminal region required for binding to the amino-terminal ends of the Gli proteins and a unique amino-terminal domain that binds the carboxy-terminal tail of Gli1. While each domain is capable of binding to different Gli1 regions independently, interactions between Su(fu) and Gli1 at both sites are required for cytoplasmic tethering and repression of Gli1. Furthermore, we have solved the crystal structure of the amino-terminal domain of human Su(fu)(27-268) at 2.65 A resolution. This domain forms a concave pocket with a prominent acidic patch. Mutation at Asp(159) in the acidic patch disrupts Gli1 tethering and repression while not strongly disrupting binding, indicating that the amino-terminal domain of Su(fu) likely impacts Gli binding through a mechanism distinct from that for tethering and repression. These studies provide a structural basis for understanding the function of Su(fu).
Assuntos
Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Análise Mutacional de DNA , Regulação da Expressão Gênica/fisiologia , Genes Reporter , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteína GLI1 em Dedos de ZincoRESUMO
We have determined the 3.2 A X-ray crystal structure of the extracellular domain of the human epidermal growth factor receptor 2 (ErbB2 or HER2) in a complex with the antigen binding fragment of pertuzumab, an anti-ErbB2 monoclonal antibody also known as 2C4 or Omnitarg. Pertuzumab binds to ErbB2 near the center of domain II, sterically blocking a binding pocket necessary for receptor dimerization and signaling. The ErbB2-pertuzumab structure, combined with earlier mutagenesis data, defines the pertuzumab residues essential for ErbB2 interaction. To analyze the ErbB2 side of the interface, we have mutated a number of residues contacting pertuzumab and examined the effects of these mutations on pertuzumab binding and ErbB2-ErbB3 heterodimerization. We have also shown that conserved residues previously shown to be necessary for EGF receptor homodimerization may be dispensible for ErbB2-ErbB3 heterodimerization.
Assuntos
Anticorpos Monoclonais/química , Receptor ErbB-2/química , Transdução de Sinais , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Sítios de Ligação , Sítios de Ligação de Anticorpos , Células CHO , Cricetinae , Cristalografia por Raios X , Dimerização , Humanos , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismoRESUMO
Cyclophilins constitute a ubiquitous protein family whose functions include protein folding, transport and signaling. They possess both sequence-specific binding and proline cis-trans isomerase activities, as exemplified by the interaction between cyclophilin A (CypA) and the HIV-1 CA protein. Here, we report crystal structures of CypA in complex with HIV-1 CA protein variants that bind preferentially with the substrate proline residue in either the cis or the trans conformation. Cis- and trans-Pro substrates are accommodated within the enzyme active site by rearrangement of their N-terminal residues and with minimal distortions in the path of the main chain. CypA Arg55 guanidinium group probably facilitates catalysis by anchoring the substrate proline oxygen and stabilizing sp3 hybridization of the proline nitrogen in the transition state.
Assuntos
Ciclofilina A/química , Ciclofilina A/metabolismo , Sequência de Aminoácidos , Arginina/química , Arginina/metabolismo , Sítios de Ligação , Capsídeo/química , Capsídeo/metabolismo , Catálise , Cristalografia por Raios X , HIV-1/química , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Prolina/química , Prolina/metabolismo , Conformação Proteica , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Shotgun scanning combinatorial mutagenesis was used to study the antigen-binding site of Fab2C4, a humanized monoclonal antibody fragment that binds to the extracellular domain of the human oncogene product ErbB2. Essentially all the residues in the Fab2C4 complementarity determining regions (CDRs) were alanine-scanned using phage-displayed libraries that preferentially allowed side-chains to vary as the wild-type or alanine. A separate homolog-scan was performed using libraries that allowed side-chains to vary only as the wild-type or a similar amino acid residue. Following binding selections to isolate functional clones, DNA sequencing was used to determine the wild-type/mutant ratios at each varied position, and these ratios were used to assess the contributions of each side-chain to antigen binding. The alanine-scan revealed that most of the side-chains that contribute to antigen binding are located in the heavy chain, and the Fab2C4 three-dimensional structure revealed that these residues fall into two groups. The first group consists of solvent-exposed residues which likely make energetically favorable contacts with the antigen and thus comprise the functional-binding epitope. The second group consists of buried residues with side-chains that pack against other CDR residues and apparently act as scaffolding to maintain the functional epitope in a binding-competent conformation. The homolog-scan involved subtle mutations, and as a result, only a subset of the side-chains that were intolerant to alanine substitutions were also intolerant to homologous substitutions. In particular, the 610 A2 functional epitope surface revealed by alanine-scanning shrunk to only 369 A2 when mapped with homologous substitutions, suggesting that this smaller subset of side-chains may be involved in more precise contacts with the antigen. The results validate shotgun scanning as a rapid and accurate method for determining the functional contributions of individual side-chains involved in protein-protein interactions.