RESUMO
DNA polymerase theta (Polθ) is an attractive synthetic lethal target for drug discovery, predicted to be efficacious against breast and ovarian cancers harboring BRCA-mutant alleles. Here, we describe our hit-to-lead efforts in search of a selective inhibitor of human Polθ (encoded by POLQ). A high-throughput screening campaign of 350,000 compounds identified an 11 micromolar hit, giving rise to the N2-substituted fused pyrazolo series, which was validated by biophysical methods. Structure-based drug design efforts along with optimization of cellular potency and ADME ultimately led to the identification of RP-6685: a potent, selective, and orally bioavailable Polθ inhibitor that showed in vivo efficacy in an HCT116 BRCA2-/- mouse tumor xenograft model.
Assuntos
DNA Polimerase Dirigida por DNA , Neoplasias Ovarianas , Animais , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Desenho de Fármacos , Descoberta de Drogas , Feminino , Humanos , CamundongosRESUMO
PKMYT1 is a regulator of CDK1 phosphorylation and is a compelling therapeutic target for the treatment of certain types of DNA damage response cancers due to its established synthetic lethal relationship with CCNE1 amplification. To date, no selective inhibitors have been reported for this kinase that would allow for investigation of the pharmacological role of PKMYT1. To address this need compound 1 was identified as a weak PKMYT1 inhibitor. Introduction of a dimethylphenol increased potency on PKMYT1. These dimethylphenol analogs were found to exist as atropisomers that could be separated and profiled as single enantiomers. Structure-based drug design enabled optimization of cell-based potency. Parallel optimization of ADME properties led to the identification of potent and selective inhibitors of PKMYT1. RP-6306 inhibits CCNE1-amplified tumor cell growth in several preclinical xenograft models. The first-in-class clinical candidate RP-6306 is currently being evaluated in Phase 1 clinical trials for treatment of various solid tumors.
Assuntos
Neoplasias , Proteínas Tirosina Quinases , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Proteínas de Membrana , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina QuinasesRESUMO
The RAF family kinases function in the RAS-ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation and survival, enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAF(V600E) have shown great efficacy in the clinic, but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated. Here, we investigated a proteolysis-targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC, termed P4B, displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAF(V600E) cell lines. In addition, P4B displayed utility in cell lines harboring alternative BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a proof of concept for a substitute to conventional chemical inhibition to therapeutically constrain oncogenic BRAF.
Assuntos
Antineoplásicos , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas B-raf , Talidomida , Ubiquitina , Animais , Feminino , Humanos , Camundongos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Estrutura Molecular , Terapia de Alvo Molecular , Mutação , Fosforilação/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteólise , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais , Relação Estrutura-Atividade , Talidomida/análogos & derivados , Talidomida/química , Ubiquitina/químicaRESUMO
SET domain bifurcated protein 1 (SETDB1) is a human histone-lysine methyltransferase which is amplified in human cancers and was shown to be crucial in the growth of non-small and small cell lung carcinoma. In addition to its catalytic domain, SETDB1 harbors a unique tandem tudor domain which recognizes histone sequences containing both methylated and acetylated lysines, and likely contributes to its localization on chromatin. Using X-ray crystallography and NMR spectroscopy fragment screening approaches, we have identified the first small molecule fragment hits that bind to histone peptide binding groove of the Tandem Tudor Domain (TTD) of SETDB1. Herein, we describe the binding modes of these fragments and analogues and the biophysical characterization of key compounds. These confirmed small molecule fragments will inform the development of potent antagonists of SETDB1 interaction with histones.
Assuntos
Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Histona-Lisina N-Metiltransferase/isolamento & purificação , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/antagonistas & inibidores , Histonas/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Domínio Tudor/efeitos dos fármacosRESUMO
Improving our understanding of the role of chromatin regulators in the initiation, development, and suppression of cancer and other devastating diseases is critical, as they are integral players in regulating DNA integrity and gene expression. Developing small molecule inhibitors for this target class with cellular activity is a crucial step toward elucidating their specific functions. We specifically targeted the DNA damage response protein, 53BP1, which uses its tandem tudor domain to recognize histone H4 dimethylated on lysine 20 (H4K20me2), a modification related to double-strand DNA breaks. Through a cross-screening approach, we identified UNC2170 (1) as a micromolar ligand of 53BP1, which demonstrates at least 17-fold selectivity for 53BP1 as compared to other methyl-lysine (Kme) binding proteins tested. Structural studies revealed that the tert-butyl amine of UNC2170 anchors the compound in the methyl-lysine (Kme) binding pocket of 53BP1, making it competitive with endogenous Kme substrates. X-ray crystallography also demonstrated that UNC2170 binds at the interface of two tudor domains of a 53BP1 dimer. Importantly, this compound functions as a 53BP1 antagonist in cellular lysates and shows cellular activity by suppressing class switch recombination, a process which requires a functional 53BP1 tudor domain. These results demonstrate that UNC2170 is a functionally active, fragment-like ligand for 53BP1.
Assuntos
Benzamidas/metabolismo , Diaminas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina/metabolismo , Animais , Linfócitos B/efeitos dos fármacos , Benzamidas/química , Benzamidas/farmacologia , Sítios de Ligação , Cromatina/metabolismo , Cristalografia por Raios X , Diaminas/química , Diaminas/farmacologia , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos Endogâmicos C57BL , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-Atividade , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Carborane-based compounds are promising lead structures for development of inhibitors of carbonic anhydrases (CAs). Here, we report structural and computational analysis applicable to structure-based design of carborane compounds with selectivity toward the cancer-specific CAIX isoenzyme. We determined the crystal structure of CAII in complex with 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane at 1.0 Å resolution and used this structure to model the 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane interactions with CAIX. A virtual glycine scan revealed the contributions of individual residues to the energy of binding of 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane to CAII and CAIX, respectively.
Assuntos
Compostos de Boro/química , Compostos de Boro/farmacologia , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/química , Modelos Moleculares , Teoria Quântica , Inibidores da Anidrase Carbônica/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Glicina/química , Humanos , Especificidade por Substrato/efeitos dos fármacosRESUMO
CA inhibitors: Human carbonic anhydrases (CAs) are diagnostic and therapeutic targets. Various carborane cages are shown to act as active-site-directed inhibitors, and substitution with a sulfamide group and other substituents leads to compounds with high selectivity towards the cancer-specific isozymeâ IX. Crystal structures of the carboranes in the active site provide information that can be applied to the structure-based design of specific inhibitors.
Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Desenho de Fármacos , Descoberta de Drogas , Humanos , Relação Estrutura-AtividadeRESUMO
Isoquinolinesulfonamides inhibit human carbonic anhydrases (hCAs) and display selectivity toward therapeutically relevant isozymes. The crystal structure of hCA II in complex with 6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-2-ylsulfonamide revealed unusual inhibitor binding. Structural analyses allowed for discerning the fine details of the inhibitor binding mode to the active site, thus providing clues for the future design of even more selective inhibitors for druggable isoforms such as the cancer associated hCA IX and neuronal hCA VII.
Assuntos
Anidrase Carbônica II/química , Anidrase Carbônica II/metabolismo , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/metabolismo , Isoquinolinas/química , Isoquinolinas/metabolismo , Anidrase Carbônica II/antagonistas & inibidores , Inibidores da Anidrase Carbônica/farmacologia , Cristalografia por Raios X , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Isoquinolinas/farmacologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Especificidade por SubstratoRESUMO
This work employs an epitope mapping of carbonic anhydrase (CA), isoform I (CA I), for detection of the main immunodominant epitopes. Our interest has arisen from an observed spontaneous tumor regression in patients who developed an aplastic anemia type syndrome after a high-dose therapy with autologous stem cell transplantation and whose sera contained high titer of anti carbonic anhydrase (anti-CA) autoantibodies. There are many indications that the presence of these autoantibodies may provide significant survival benefit for the patients. Western blot analysis confirmed strong immunoreactivity of the patients' sera with several CA isoforms and the CA I has been selected for our study as a highly abundant and widely distributed isoform. The applied analytical approach consists of specific fragmentation of CA I protein followed by immunospecific isolation of peptides reacting with polyclonal anti-CA I autoantibodies of patients in spontaneous remission. We improved the standard epitope mapping schema by incorporating the benefits of magnetic carriers and biomagnetic separation techniques. Mass spectrometry has been applied for detection and identification of epitopes and the acquired results were verified by bioinformatic tools. The candidate epitopes of CA I (NVGHS, DGLAV, SSEQL, and SLKPI) are discussed herein as potential therapeutic targets. This work highlights the usefulness of the epitope mapping technique based on magnetic microspheres for effective and rapid determination of immunodominant epitopes of the target protein.
Assuntos
Autoanticorpos/imunologia , Anidrase Carbônica I/imunologia , Epitopos Imunodominantes/análise , Transplante de Células-Tronco/métodos , Sequência de Aminoácidos , Western Blotting , Eletroforese em Gel de Poliacrilamida , Mapeamento de Epitopos/métodos , Humanos , Epitopos Imunodominantes/imunologia , Espectrometria de Massas , Dados de Sequência Molecular , Mieloma Múltiplo/sangue , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/cirurgia , Prognóstico , Transplante AutólogoRESUMO
Following previous studies we herein report the exploration of the carbonic anhydrase (CA, EC 4.2.1.1) inhibitory effects and enzyme selectivity of a small class of 1-(cyclo)alkylisoquinolines containing a sulfonamide function considered a key feature for inhibiting CA. The results of enzymatic assays against human (h) CA isoforms, hCA I and hCA II (cytosolic, ubiquitous enzymes), hCA IX (transmembrane, tumor-associated), and hCA XIV (transmembrane), suggested that the presence of C-1 small substituents on isoquinoline scaffold controls both inhibitory potency and selectivity. Some derivatives showed potent hCA IX and hCA XIV inhibitory effects at nanomolar concentrations as well as low affinity for the ubiquitous hCA II. Moreover, we report the X-ray crystal structure of one of these derivatives in complex with dominant human isoform II, thus confirming the sulfonamide--zinc interactions. Finally, the results of docking experiments suggested the hypothetic interactions in the catalytic binding site for the most active and selective hCA IX and hCA XIV inhibitor.
Assuntos
Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Ligação Competitiva , Anidrase Carbônica I/antagonistas & inibidores , Anidrase Carbônica I/química , Anidrase Carbônica I/metabolismo , Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica II/química , Anidrase Carbônica II/metabolismo , Anidrase Carbônica IX , Inibidores da Anidrase Carbônica/síntese química , Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Isoquinolinas/química , Ligantes , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Sulfonamidas/síntese químicaRESUMO
Specific antibodies interfere with the function of human tumor-associated carbonic anhydrase IX (CA IX), and show potential as tools for anticancer interventions. In this work, a correlation between structural elements and thermodynamic parameters of the association of antibody fragment Fab M75 to a peptide corresponding to its epitope in the proteoglycan-like domain of CA IX, is presented. Comparisons of the crystal structures of free Fab M75 and its complex with the epitope peptide reveal major readjustments of CDR-H1 and CDR-H3. In contrast, the overall conformations and positions of CDR-H2 and CDR-L2 remain unaltered, and their positively charged residues may thus present a fixed frame for epitope recognition. Adoption of the altered CDR-H3 conformation in the structure of the complex is accompanied by an apparent local stabilization. Analysis of domain mobility with translation-libration-screw (TLS) method shows that librations of the entire heavy chain variable domain (V(H)) decrease and reorient in the complex, which correlates well with participation of the heavy chain in ligand binding. Isothermal titration microcalorimetry (ITC) experiments revealed a highly unfavorable entropy term, which can be attributed mainly to the decrease in the degrees of freedom of the system, the loss of conformational freedom of peptide and partially to a local stabilization of CDR-H3. Moreover, it was observed that one proton is transferred from the environment to the protein-ligand complex upon binding. Molecular dynamics simulations followed by molecular mechanics/generalized Born surface area (MM-GBSA) calculations of the ligand (epitope peptide) binding energy yielded energy values that were in agreement with the ITC measurements and indicated that the charged residues play crucial role in the epitope binding. Theoretical arguments presented in this work indicate that two adjacent arginine residues (ArgH50 and ArgH52) are responsible for the observed proton transfer.