RESUMO
Rapid emergence of tumor resistance via RAS pathway reactivation has been reported from clinical studies of covalent KRASG12C inhibitors. Thus, inhibitors with broad potential for combination treatment and distinct binding modes to overcome resistance mutations may prove beneficial. JDQ443 is an investigational covalent KRASG12C inhibitor derived from structure-based drug design followed by extensive optimization of two dissimilar prototypes. JDQ443 is a stable atropisomer containing a unique 5-methylpyrazole core and a spiro-azetidine linker designed to position the electrophilic acrylamide for optimal engagement with KRASG12C C12. A substituted indazole at pyrazole position 3 results in novel interactions with the binding pocket that do not involve residue H95. JDQ443 showed PK/PD activity in vivo and dose-dependent antitumor activity in mouse xenograft models. JDQ443 is now in clinical development, with encouraging early phase data reported from an ongoing Phase Ib/II clinical trial (NCT04699188).
Assuntos
Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Animais , Humanos , Camundongos , Modelos Animais de Doenças , Desenho de Fármacos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Pirazóis/farmacologia , Pirazóis/uso terapêuticoRESUMO
Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors. SIGNIFICANCE: JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155. This article is highlighted in the In This Issue feature, p. 1397.
Assuntos
Inibidores Enzimáticos , Indazóis , Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Indazóis/química , Indazóis/farmacologia , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
The serine protease factor XI (FXI) is a prominent drug target as it holds promise to deliver efficacious anticoagulation without an enhanced risk of major bleeds. Several efforts have been described targeting the active form of the enzyme, FXIa. Herein, we disclose our efforts to identify potent, selective, and orally bioavailable inhibitors of FXIa. Compound 1, identified from a diverse library of internal serine protease inhibitors, was originally designed as a complement factor D inhibitor and exhibited submicromolar FXIa activity and an encouraging absorption, distribution, metabolism, and excretion (ADME) profile while being devoid of a peptidomimetic architecture. Optimization of interactions in the S1, S1ß, and S1' pockets of FXIa through a combination of structure-based drug design and traditional medicinal chemistry led to the discovery of compound 23 with subnanomolar potency on FXIa, enhanced selectivity over other coagulation proteases, and a preclinical pharmacokinetics (PK) profile consistent with bid dosing in patients.
Assuntos
Fator XIa/antagonistas & inibidores , Fator XIa/genética , Inibidores do Fator Xa/administração & dosagem , Inibidores do Fator Xa/química , Administração Oral , Sequência de Aminoácidos , Animais , Disponibilidade Biológica , Cães , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Complement factor D (FD), a highly specific S1 serine protease, plays a central role in the amplification of the alternative complement pathway (AP) of the innate immune system. Dysregulation of AP activity predisposes individuals to diverse disorders such as age-related macular degeneration, atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis type II, and paroxysmal nocturnal hemoglobinuria. Previously, we have reported the screening efforts and identification of reversible benzylamine-based FD inhibitors (1 and 2) binding to the open active conformation of FD. In continuation of our drug discovery program, we designed compounds applying structure-based approaches to improve interactions with FD and gain selectivity against S1 serine proteases. We report herein the design, synthesis, and medicinal chemistry optimization of the benzylamine series culminating in the discovery of 12, an orally bioavailable and selective FD inhibitor. 12 demonstrated systemic suppression of AP activation in a lipopolysaccharide-induced AP activation model as well as local ocular suppression in intravitreal injection-induced AP activation model in mice expressing human FD.
Assuntos
Benzilaminas/farmacologia , Via Alternativa do Complemento/efeitos dos fármacos , Inibidores de Serina Proteinase/farmacologia , Animais , Benzilaminas/síntese química , Benzilaminas/metabolismo , Sítios de Ligação , Fator D do Complemento/antagonistas & inibidores , Fator D do Complemento/química , Fator D do Complemento/metabolismo , Cães , Desenho de Fármacos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Simulação de Acoplamento Molecular , Conformação Proteica , Ratos , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/metabolismoRESUMO
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
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
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
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
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
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
Fluorescence lifetime (FLT)-based assays have developed to become highly attractive tools in drug discovery. All recently published examples of FLT-based assays essentially describe their use for monitoring enzyme-mediated peptide modifications, such as proteolytic cleavage or phosphorylation/dephosphorylation. Here we report the development of competitive binding assays as novel, inhibitor-centric assays, principally employing the FLT of the acridone dye Puretime 14 (PT14) as the readout parameter. Exemplified with two case studies on human serine proteases, the details of the rationale for both the design and synthesis of probes (i.e., active site-directed low-molecular-weight inhibitors conjugated to PT14) are provided. Data obtained from testing inhibitors with the novel assay format match those obtained with alternative formats such as FLT-based protease activity and time-resolved fluorescence resonance energy transfer-based competitive binding assays.
Assuntos
Descoberta de Drogas/métodos , Inibidores de Proteases/química , Espectrometria de Fluorescência/métodos , Acridonas/química , Ligação Competitiva , Soluções Tampão , Domínio Catalítico , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Cinética , Pulmão/enzimologia , Conformação Molecular , Peso Molecular , Peptídeos/química , Ligação Proteica , Proteínas Recombinantes/química , Serina Proteases/química , Triptases/químicaRESUMO
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
The dynamic modification of proteins with ubiquitin is a key regulation paradigm in eukaryotic cells that controls stability, localization, and function of the vast majority of intracellular proteins. Here we describe a robust fluorescence intensity assay for monitoring the enzymatic activity of deubiquitinating proteases, which reverse ubiquitin modifications and comprise over 100 members in humans. The assay was developed for the catalytic domain of human ubiquitin-specific protease 2 (USP2) and human ubiquitin carboxyterminal hydrolase L3 (UCH-L3), and makes use of the novel substrate ubiquitin-rhodamine110-glycine. The latter combines the advantages of a high dynamic range and beneficial optical properties. Its enzymatic behavior is characterized by the kinetic constants K(m)=1.5 microM, k(cat) = 0.53s(-1) and k(cat)/K(m) = 3.5 x 10(5)M(-1) s(-1) for USP2 and K(m) = 34 nM, k(cat)=4.72s(-1), and k(cat)/K(m) = 1.4 x 10(8)M(-1) s(-1) for UCH-L3. This new assay is suitable for inhibitor screening and characterizations, and has been established for the 384-well plate format using protease concentrations of 120 pM for USP2 and 1 pM for UCH-L3 and substrate concentrations of 100 nM for both enzymes. Due to the low protease concentrations and high sensitivity, this assay would allow the determination of inhibitory constants in the subnanomolar range.
Assuntos
Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Corantes Fluorescentes/química , Glicina/análogos & derivados , Glicina/química , Ubiquitina/química , Bioensaio/instrumentação , Bioensaio/métodos , Cisteína Endopeptidases/química , Endopeptidases/química , Corantes Fluorescentes/metabolismo , Glicina/metabolismo , Humanos , Inteínas , Cinética , Modelos Biológicos , Rodaminas/química , Rodaminas/metabolismo , Espectrometria de Fluorescência , Especificidade por Substrato , Ubiquitina/metabolismo , Ubiquitina TiolesteraseRESUMO
The synthesis and SAR studies of a series of structurally novel small molecule inhibitors of PDE7 are discussed. The best compounds from the series displayed low nanomolar inhibitory activity and are selective versus PDE4.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Isoenzimas/antagonistas & inibidores , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/farmacologia , Tiadiazóis/síntese química , Tiadiazóis/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Linhagem Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7 , Humanos , Isoenzimas/metabolismo , Inibidores de Fosfodiesterase/química , Relação Estrutura-Atividade , Tiadiazóis/químicaRESUMO
The synthesis and optimization of pharmacokinetic parameters of structurally novel small PDE7 inhibitors is discussed.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Isoenzimas/antagonistas & inibidores , Inibidores de Fosfodiesterase/farmacocinética , Tiadiazóis/farmacocinética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Administração Oral , Animais , Disponibilidade Biológica , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7 , Isoenzimas/metabolismo , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/química , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Tiadiazóis/síntese química , Tiadiazóis/químicaRESUMO
The synthesis and SAR studies of spiroquinazolinones as novel PDE7 inhibitors are discussed. The best compounds from the series displayed nanomolar inhibitory affinity and were selective versus other PDE isoenzymes.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Isoenzimas/antagonistas & inibidores , Inibidores de Fosfodiesterase/síntese química , Quinazolinas/síntese química , Compostos de Espiro/síntese química , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Linhagem Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7 , Avaliação Pré-Clínica de Medicamentos , Humanos , Isoenzimas/metabolismo , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/farmacologia , Quinazolinas/química , Quinazolinas/farmacologia , Solubilidade , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
The optimization of 5,8-disubstituted spirocyclohexane-quinazolinones into potent, selective, soluble PDE7 inhibitors with acceptable in vivo pharmacokinetic parameters is presented.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Isoenzimas/antagonistas & inibidores , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/farmacocinética , Quinazolinas/síntese química , Compostos de Espiro/síntese química , 3',5'-AMP Cíclico Fosfodiesterases/classificação , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Animais , Disponibilidade Biológica , Linhagem Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7 , Meia-Vida , Humanos , Técnicas In Vitro , Isoenzimas/classificação , Isoenzimas/metabolismo , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Quinazolinas/química , Quinazolinas/farmacologia , Ratos , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
Various N-pent-4-enylglycine methyl esters have been submitted to carbocyclization of their zinc enolates onto the unactivated double bond. The cyclization to substituted pipecolic esters is highly stereoselective. In most cases, substitution of the pent-4-enyl moiety on various sites leads to a single isomer, hence a way to di-, tri-, tetra-, or pentasubstituted piperidines.
RESUMO
The amino-zinc-enolate cyclization allowed a new and straightforward route to polysubstituted pyrrolidines from simple starting materials. From this study, we have been able to determine, for the first time, the stereochemical influence of the substituents on the ring in the carbocyclization reaction. The diastereoselectivity thus obtained was explained by a chairlike amino-zinc-enolate transition state.