RESUMO
Dysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development.
Assuntos
Fator B do Complemento/antagonistas & inibidores , Via Alternativa do Complemento/efeitos dos fármacos , Descoberta de Drogas/métodos , Fatores Imunológicos/farmacologia , Animais , Modelos Animais de Doenças , Glomerulonefrite Membranosa/fisiopatologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos Sprague-DawleyRESUMO
Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.
Assuntos
Fator D do Complemento/antagonistas & inibidores , Via Alternativa do Complemento/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Fator D do Complemento/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Recently, we reported on the discovery of (3S,4S)-disubstituted pyrrolidines (e.g., 2) as inhibitors of the human aspartyl protease renin. In our effort to further expand the scope of this novel class of direct renin inhibitors, a new sub-series was designed in which the prime site substituents are linked to the pyrrolidine core by a (3S)-amino functional group. In particular, analogs bearing the corresponding sulfonamide spacer (50, 51 and 54a) demonstrated a pronounced increase in in vitro potency compared to compound 2.
Assuntos
Inibidores de Proteases/química , Pirrolidinas/química , Renina/antagonistas & inibidores , Ácido Aspártico Proteases/antagonistas & inibidores , Ácido Aspártico Proteases/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Meia-Vida , Humanos , Isomerismo , Simulação de Dinâmica Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Pirrolidinas/síntese química , Pirrolidinas/metabolismo , Renina/metabolismo , Relação Estrutura-AtividadeRESUMO
Inhibition of the aspartyl protease renin is considered as an efficient approach for treating hypertension. Lately, we described the discovery of a novel class of direct renin inhibitors which comprised a pyrrolidine scaffold (e.g., 2). Based on the X-ray structure of the lead compound 2 bound to renin we predicted that optimization of binding interactions to the prime site could offer an opportunity to further expand the scope of this chemotype. Pyrrolidine-based inhibitors were synthesized in which the prime site moieties are linked to the pyrrolidine core through an oxygen atom, resulting in an ether or a carbamate linker subseries. Especially the carbamate derivatives showed a pronounced increase in in vitro potency compared to 2. Here we report the structure-activity relationship of both subclasses and demonstrate blood pressure lowering effects for an advanced prototype in a hypertensive double-transgenic rat model after oral dosing.
Assuntos
Ácido Aspártico Proteases/antagonistas & inibidores , Inibidores de Proteases/química , Pirrolidinas/química , Renina/antagonistas & inibidores , Animais , Ácido Aspártico Proteases/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Modelos Animais de Doenças , Humanos , Ligação de Hidrogênio , Hipertensão/tratamento farmacológico , Isomerismo , Simulação de Dinâmica Molecular , Oxigênio/química , Inibidores de Proteases/metabolismo , Inibidores de Proteases/uso terapêutico , Ligação Proteica , Estrutura Terciária de Proteína , Pirrolidinas/síntese química , Pirrolidinas/metabolismo , Ratos , Ratos Sprague-Dawley , Renina/metabolismo , Relação Estrutura-AtividadeRESUMO
The alternative pathway (AP) of the complement system is a key contributor to the pathogenesis of several human diseases including age-related macular degeneration, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and various glomerular diseases. The serine protease factor B (FB) is a key node in the AP and is integral to the formation of C3 and C5 convertase. Despite the prominent role of FB in the AP, selective orally bioavailable inhibitors, beyond our own efforts, have not been reported previously. Herein we describe in more detail our efforts to identify FB inhibitors by high-throughput screening (HTS) and leveraging insights from several X-ray cocrystal structures during optimization efforts. This work culminated in the discovery of LNP023 (41), which is currently being evaluated clinically in several diverse AP mediated indications.
Assuntos
Ácido Benzoico/química , Fator B do Complemento/antagonistas & inibidores , Indóis/química , Síndrome Hemolítico-Urêmica Atípica/metabolismo , Síndrome Hemolítico-Urêmica Atípica/patologia , Ácido Benzoico/metabolismo , Ácido Benzoico/farmacocinética , Sítios de Ligação , Domínio Catalítico , Fator B do Complemento/metabolismo , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Indóis/metabolismo , Indóis/farmacocinética , Concentração Inibidora 50 , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Simulação de Dinâmica Molecular , Relação Estrutura-AtividadeRESUMO
Novel nonpeptide small molecule renin inhibitors bearing an N-isopropyl P(1) motif were designed based on initial lead structures 1 and aliskiren (2). (P(3)-P(1))-Benzamide derivatives such as 9a and 34, as well as the corresponding P(1) basic tertiary amine derivatives 10 and 35 were found to display low nanomolar inhibition against human renin in vitro.
Assuntos
Amidas/química , Anti-Hipertensivos/química , Benzamidas/química , Etilenos/química , Fumaratos/química , Inibidores de Proteases/química , Renina/antagonistas & inibidores , Administração Oral , Animais , Anti-Hipertensivos/síntese química , Anti-Hipertensivos/farmacologia , Benzamidas/síntese química , Benzamidas/farmacologia , Callithrix , Humanos , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Renina/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Complement Factor D, a serine protease of the S1 family and key component of the alternative pathway amplification loop, represents a promising target for the treatment of several prevalent and rare diseases linked to the innate immune system. Previously reported FD inhibitors have been shown to bind to the FD active site in its self-inhibited conformation characterized by the presence of a salt bridge at the bottom of the S1 pocket between Asp189 and Arg218. We report herein a new set of small-molecule FD ligands that harbor a basic S1 binding moiety directly binding to the carboxylate of Asp189, thereby displacing the Asp189-Arg218 ionic interaction and significantly changing the conformation of the self-inhibitory loop.
RESUMO
BACE2 is a membrane-bound aspartic protease of the A1 family with a high level of sequence homology to BACE1. While BACE1 is involved in the generation of amyloid plaques in Alzheimer's disease by cleaving Abeta-peptides from the amyloid precursor protein, the physiological function of BACE2 is not well understood. BACE2 appears to be associated with the early onset of dementia in patients with Down's syndrome, and it has been shown to be highly expressed in breast cancers. Further, it may participate in the function of normal and abnormal processes of human muscle biology. Similar to other aspartic proteases, BACE2 is expressed as an inactive zymogen requiring the cleavage of its pro-sequence during the maturation process. We have produced mature BACE2 by expression of pro-BACE2 in Escherichia coli as inclusion bodies, followed by refolding and autocatalytic activation at pH 3.4. Using a C and N-terminally truncated BACE2 variant, we were able to crystallize and determine the crystal structure of mature BACE2 in complex with a hydroxyethylamine transition-state mimetic inhibitor at 3.1 angstroms resolution. The structure of BACE2 follows the general fold of A1 aspartic proteases. However, similar to BACE1, its C-terminal domain is significantly larger than that of the other family members. Furthermore, the structure of BACE2 reveals differences in the S3, S2, S1' and S2' active site substrate pockets as compared to BACE1, and allows, therefore, for a deeper understanding of the structural features that may facilitate the design of selective BACE1 or BACE2 inhibitors.
Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Inibidores Enzimáticos/química , Etanolaminas/química , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases/genética , Sítios de Ligação , Cristalização , Desenho de Fármacos , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Corpos de Inclusão/enzimologia , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
The action of renin is the rate-limiting step of the renin-angiotensin system (RAS), a key regulator of blood pressure. Effective renin inhibitors directly block the RAS entirely at source and, thus, may provide a vital weapon for hypertension therapy. Our efforts toward identifying novel small-molecule peptidomimetic renin inhibitors have resulted in the design of transition-state isosteres such as 1 bearing an all-carbon 8-phenyl-octanecarboxamide framework. Optimization of the extended P3 portion of 1 and extensive P2' modifications provided analogues with improved in vitro potencies in the presence of plasma. X-ray resolution of rh-renin/38a in the course of SAR work surprisingly unveiled the exploitation of a previously unexplored pocket (S3sp) important for strong binding affinities. Several inhibitors demonstrated oral efficacy in sodium-depleted marmosets. The most potent, 38a, induced dose-dependently a pronounced reduction in mean arterial blood pressure, paralleled by complete blockade of active plasma renin, up to 8 h post-dose. Oral bioavailability of 38a was 16% in marmosets.
Assuntos
Amidas/síntese química , Anisóis/síntese química , Anti-Hipertensivos/síntese química , Caprilatos/síntese química , Peptídeos/química , Renina/antagonistas & inibidores , Administração Oral , Amidas/química , Amidas/farmacologia , Animais , Anisóis/química , Anisóis/farmacologia , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Disponibilidade Biológica , Pressão Sanguínea/efeitos dos fármacos , Callithrix , Caprilatos/química , Caprilatos/farmacologia , Cristalografia por Raios X , Frequência Cardíaca/efeitos dos fármacos , Humanos , Cinética , Modelos Moleculares , Mimetismo Molecular , Estrutura Molecular , Ligação Proteica , Renina/sangue , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Due to its function in the rate limiting initial step of the renin-angiotensin system, renin is a particularly promising target for drugs designed to control hypertension, a growing risk to health worldwide. Despite vast efforts over more than two decades, no orally efficacious renin inhibitor had reached the market. As a result of a structure-based topological design approach, we have identified a novel class of small-molecule inhibitors with good oral blood-pressure lowering effects in primates. Further lead optimization aimed for improvement of in vivo potency and duration of action, mainly by P2' modifications at the hydroxyethylene transition-state isostere. These efforts resulted in the discovery of aliskiren (46, CGP060536B, SPP100), a highly potent, selective inhibitor of renin, demonstrating excellent efficacy in sodium-depleted marmosets after oral administration, with sustained duration of action in reducing dose-dependently mean arterial blood pressure. Aliskiren has recently received regulatory approval by the U.S. Food and Drug Administration for the treatment of hypertension.
Assuntos
Amidas/síntese química , Anti-Hipertensivos/síntese química , Caprilatos/síntese química , Fumaratos/síntese química , Renina/antagonistas & inibidores , Administração Oral , Amidas/química , Amidas/farmacologia , Animais , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Callithrix , Caprilatos/química , Caprilatos/farmacologia , Cristalografia por Raios X , Fumaratos/química , Fumaratos/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Humanos , Cinética , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Renina/sangue , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Chronic dysregulation of alternative complement pathway activation has been associated with diverse clinical disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinurea. Factor D is a trypsin-like serine protease with a narrow specificity for arginine in the P1 position, which catalyzes the first enzymatic reaction of the amplification loop of the alternative pathway. In this article, we describe two hit finding approaches leading to the discovery of new chemical matter for this pivotal protease of the complement system: in silico active site mapping for hot spot identification to guide rational structure-based design and NMR screening of focused and diverse fragment libraries. The wealth of information gathered by these complementary approaches enabled the identification of ligands binding to different subpockets of the latent Factor D conformation and was instrumental for understanding the binding requirements for the generation of the first known potent noncovalent reversible Factor D inhibitors.
Assuntos
Inibidores de Proteases/farmacologia , Domínio Catalítico , Fator D do Complemento/química , Desenho de Fármacos , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Inibidores de Proteases/químicaRESUMO
The highly specific S1 serine protease factor D (FD) plays a central role in the amplification of the complement alternative pathway (AP) of the innate immune system. Genetic associations in humans have implicated AP activation in age-related macular degeneration (AMD), and AP dysfunction predisposes individuals to disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). The combination of structure-based hit identification and subsequent optimization of the center (S)-proline-based lead 7 has led to the discovery of noncovalent reversible and selective human factor D (FD) inhibitors with drug-like properties. The orally bioavailable compound 2 exerted excellent potency in 50% human whole blood in vitro and blocked AP activity ex vivo after oral administration to monkeys as demonstrated by inhibition of membrane attack complex (MAC) formation. Inhibitor 2 demonstrated sustained oral and ocular efficacy in a model of lipopolysaccharide (LPS)-induced systemic AP activation in mice expressing human FD.
Assuntos
Fator D do Complemento/antagonistas & inibidores , Via Alternativa do Complemento/efeitos dos fármacos , Prolina/análogos & derivados , Prolina/farmacologia , Administração Oral , Animais , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Síndrome Hemolítico-Urêmica Atípica/imunologia , Fator D do Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/antagonistas & inibidores , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Feminino , Haplorrinos , Humanos , Macaca fascicularis , Degeneração Macular/tratamento farmacológico , Degeneração Macular/imunologia , Masculino , Camundongos , Prolina/administração & dosagem , Prolina/farmacocinéticaRESUMO
A cis-configured 3,5-disubstituted piperidine direct renin inhibitor, (syn,rac)-1, was discovered as a high-throughput screening hit from a target-family tailored library. Optimization of both the prime and the nonprime site residues flanking the central piperidine transition-state surrogate resulted in analogues with improved potency and pharmacokinetic (PK) properties, culminating in the identification of the 4-hydroxy-3,5-substituted piperidine 31. This compound showed high in vitro potency toward human renin with excellent off-target selectivity, 60% oral bioavailability in rat, and dose-dependent blood pressure lowering effects in the double-transgenic rat model.
RESUMO
The small-molecule trans-3,4-disubstituted pyrrolidine 6 was identified from in silico three-dimensional (3D) pharmacophore searches based on known X-ray structures of renin-inhibitor complexes and demonstrated to be a weakly active inhibitor of the human enzyme. The unexpected binding mode of the more potent enantiomer (3S,4S)-6a in an extended conformation spanning the nonprime and S1' pockets of the recombinant human (rh)-renin active site was elucidated by X-ray crystallography. Initial structure-activity relationship work focused on modifications of the hydrophobic diphenylamine portion positioned in S1 and extending toward the S2 pocket. Replacement with an optimized P3-P1 pharmacophore interacting to the nonsubstrate S3(sp) cavity eventually resulted in significantly improved in vitro potency and selectivity. The prototype analogue (3S,4S)-12a of this new class of direct renin inhibitors exerted blood pressure lowering effects in a hypertensive double-transgenic rat model after oral administration.
Assuntos
Descoberta de Drogas , Modelos Moleculares , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Pirrolidinas/química , Pirrolidinas/farmacologia , Renina/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Biologia Computacional , Humanos , Inibidores de Proteases/administração & dosagem , Inibidores de Proteases/farmacocinética , Conformação Proteica , Pirrolidinas/administração & dosagem , Pirrolidinas/farmacocinética , Ratos , Renina/química , Relação Estrutura-AtividadeRESUMO
A small library of fragments comprising putative recognition motifs for the catalytic dyad of aspartic proteases was generated by in silico similarity searches within the corporate compound deck based on rh-renin active site docking and scoring filters. Subsequent screening by NMR identified the low-affinity hits 3 and 4 as competitive active site binders, which could be shown by X-ray crystallography to bind to the hydrophobic S3-S1 pocket of rh-renin. As part of a parallel multiple hit-finding approach, the 3,5-disubstituted piperidine (rac)-5 was discovered by HTS using a enzymatic assay. X-ray crystallography demonstrated the eutomer (3S,5R)-5 to be a peptidomimetic inhibitor binding to a nonsubstrate topography of the rh-renin prime site. The design of the potent and selective (3S,5R)-12 bearing a P3(sp)-tethered tricyclic P3-P1 pharmacophore derived from 3 is described. (3S,5R)-12 showed oral bioavailability in rats and demonstrated blood pressure lowering activity in the double-transgenic rat model.
Assuntos
Desenho de Fármacos , Piperidinas/química , Piperidinas/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Renina/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Concentração Inibidora 50 , Modelos Moleculares , Piperidinas/administração & dosagem , Piperidinas/farmacocinética , Inibidores de Proteases/administração & dosagem , Inibidores de Proteases/farmacocinética , Conformação Proteica , Ratos , Renina/químicaAssuntos
Anti-Hipertensivos/química , Hipertensão/tratamento farmacológico , Peptidomiméticos/química , Renina/antagonistas & inibidores , Animais , Anti-Hipertensivos/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Modelos Moleculares , Estrutura Molecular , Peptidomiméticos/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/metabolismo , Renina/metabolismo , Relação Estrutura-Atividade , Receptor de Pró-ReninaRESUMO
Hypertension is a major risk factor for cardiovascular diseases such as stroke, myocardial infarction, and heart failure, the leading causes of death in the Western world. Inhibitors of the renin-angiotensin system (RAS) have proven to be successful treatments for hypertension. As renin specifically catalyses the rate-limiting step of the RAS, it represents the optimal target for RAS inhibition. Several peptide-like renin inhibitors have been synthesized previously, but poor pharmacokinetic properties meant that these compounds were not clinically useful. We employed a combination of molecular modelling and crystallographic structure analysis to design renin inhibitors lacking the extended peptide-like backbone of earlier inhibitors, for improved pharmacokinetic properties. This led to the discovery of aliskiren, a highly potent and selective inhibitor of human renin in vitro, and in vivo; once-daily oral doses of aliskiren inhibit renin and lower blood pressure in sodium-depleted marmosets and hypertensive human patients. Aliskiren represents the first in a novel class of renin inhibitors with the potential for treatment of hypertension and related cardiovascular diseases.