RESUMO
Poor tolerance to standard therapies and multi-drug resistance complicate treatment of elderly patients with acute myeloid leukemia (AML). It is therefore imperative to explore novel tolerable agents and target alternative pathways. KX2-391 is an oral non-ATP-competitive inhibitor of Src kinase and tubulin polymerization. This multi-center phase Ib open-label safety and activity study involved elderly patients with relapsed or refractory AML, or who declined standard chemotherapy. Twenty-four patients averaging 74 years of age were enrolled. The majority previously received hypomethylating agents. Five doses were tested: 40 mg (n = 1), 80 mg (n = 2), 120 mg (n = 8), 140 mg (n = 12), and 160 mg (n = 1). Seven patients were treated for 12 days or less, nine for 15-29 days, five for 33-58 days, and three for 77-165 days. One patient receiving 120 mg for 165 days had reduced splenomegaly and survived 373 days. Another had no evidence of disease progression for 154 days. One patient receiving 160 mg for 12 days remained treatment-free for about 18 months. Dose-limiting toxicities occurred in eight patients at: 120 mg (transaminitis, hyperbilirubinemia), 140 mg (mucositis, allergic reaction, transaminitis, acute kidney injury), and 160 mg (mucositis). The maximum tolerated dose for KX2-391 was 120 mg once daily. KX2-391 bone marrow concentrations were approximately similar to plasma concentrations. This is the first study to evaluate the safety of KX2-391 in elderly patients with AML. Further studies are warranted, including alternative dosing phase I trials evaluating shorter courses at higher doses and phase II trials. (Clinical Trial Registration:The study was registered at ClinicalTrials.gov: NCT01397799 (July 20, 2011)).
Assuntos
Leucemia Mieloide Aguda , Mucosite , Acetamidas , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Dose Máxima Tolerável , Morfolinas/uso terapêutico , Mucosite/tratamento farmacológico , PiridinasRESUMO
PURPOSE: A major challenge to developing new therapies for patients with malignant brain tumors is that relatively few small molecule anticancer drugs penetrate the blood-brain barrier (BBB) well enough to provide therapeutically effective concentrations in brain tissue before drug exposure in non-CNS tissues results in unacceptable toxicity. METHODS: KX2-361, a member of a novel family of compounds with Src-kinase and tubulin polymerization inhibitory activity, demonstrates good oral bioavailability and readily crosses the BBB in mice. The objective of this study was to investigate the activity of KX2-361 against human and murine glioma cells and assess its therapeutic effect in a syngeneic orthotopic model of glioblastoma. RESULTS: In addition to reducing the level of Src autophosphorylation in the GL261 murine glioblastoma cell line, KX2-361 binds directly to tubulin and disrupts microtubule architecture in glioma cells maintained in culture. CONCLUSIONS: The drug is active in vivo against orthotopic GL261 gliomas in syngeneic C57BL/6 mice. Long term survival is not observed in mice lacking an adaptive immune system, indicating that KX2-361 works in concert with the host immune system to control tumor growth and promote long-term survival in the GL261 glioma model.
Assuntos
Acetamidas/administração & dosagem , Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Morfolinas/administração & dosagem , Piridinas/administração & dosagem , Moduladores de Tubulina/administração & dosagem , Quinases da Família src/antagonistas & inibidores , Animais , Apoptose , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Modelos Animais de Doenças , Glioblastoma/tratamento farmacológico , Humanos , Camundongos Endogâmicos C57BL , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagemRESUMO
Despite a demonstrated role for TNF-α in promoting muscle wasting and cachexia, the associated molecular mechanisms and signaling pathways of myoblast differentiation dysregulated by TNF-α remain poorly understood. This study presents well-controlled proteomic profiling as a means to investigate the mechanisms of TNF-α-regulated myogenic differentiation. Primary human muscle precursor cells (MPCs) cultured in growth medium (GM), differentiation medium (DM) to induce myogenic differentiation, and DM with 20 ng/mL of TNF-α (n = 5/group) were comparatively analyzed by an ion current-based quantitative platform consisting of reproducible sample preparation/on-pellet digestion, a long-column nano-LC separation, and ion current-based differential analysis. The inhibition of myogenic differentiation by TNF-α was confirmed by reduced formation of multinucleated myotubes and the recovered expression of altered myogenic proteins such as MYOD and myogenin during myogenic differentiation. Functional analysis and validation by immunoassay analysis suggested that the cooperation of NF-κB and STAT proteins is responsible for dysregulated differentiation in MPCs by TNF-α treatment. Increased MHC class I components such as HLA-A, HLA-B, HLA-C, and beta-2-microglobulin were also observed in cultures in DM treated with TNF-α. Interestingly, inhibition of the cholesterol biosynthesis pathway during myogenic differentiation induced by serum starvation was not recovered by TNF-α treatment, which combined with previous reports, implies that this process may be an early event of myogenesis. This finding could lay the foundation for the potential use of statins in modulating myogenesis through cholesterol, for example, in stem cell-based myocardial infarction treatment, where differentiation of myoblasts and stem cells into force-generating mature muscle cells is a key step to the therapeutic capacity. In conclusion, the landscapes of altered transcription regulators, metabolic processes, and signaling pathways in MPCs are revealed in the regulation of myogenic differentiation by TNF-α, which is valuable for myogenic cellular therapeutics.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Proteômica/métodos , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , Humanos , Metabolismo/efeitos dos fármacos , Mioblastos , Proteínas/análise , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Ligand-protein binding is a complex process that involves the formation of number of non-covalent interactions, e.g. H-bonds and hydrophobic interactions, between the ligand and the protein host. Upon binding, ligand functional groups can act synergistically (positive cooperativity) to improve the overall ligand binding affinity beyond what would be expected from their individual contributions. In this study, using thrombin as a protein model system, we evaluated the effect of the bioisosteric replacement of a carbonyl functionality with a sulphonyl functionality on positive cooperativity between their H-bonds with thrombin and hydrophobic binding in the adjacent S3 pocket. The positive cooperativity observed was greatly reduced when replacing the carbonyl group with a sulphonyl group. Evaluating how bioisosteric replacements affect cooperativity is important for making better informed ligand optimization SAR decisions.
Assuntos
Antitrombinas/química , Trombina/antagonistas & inibidores , Antitrombinas/metabolismo , Sítios de Ligação , Domínio Catalítico , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Relação Estrutura-Atividade , Dióxido de Enxofre/química , Termodinâmica , Trombina/metabolismoRESUMO
BACKGROUND: Src kinase is central to tumor cell proliferation, apoptosis, and metastasis. KX2-391 is a synthetic, orally bioavailable small molecule inhibitor of Src tyrosine kinase (TK) signaling and tubulin polymerization. This compound is distinct from other Src kinase inhibitors by targeting the peptide substrate rather than the ATP binding site; the binding site on hetero-dimeric tubulin is novel and distinct from the taxanes and other known tubulin inhibitors. METHODS: This multicenter Phase I trial utilized a 4 + 2 study design to determine the maximum tolerated dose (MTD), safety, and pharmacokinetics (PK) of KX2-391 in patients with refractory solid tumors. RESULTS: Forty-four (44) patients (18 M, 26 F; median age, 59) were enrolled in 9 dose cohorts. Dose-limiting toxicities, all reversible within 7 days, occurred in 7 patients and consisted of elevated AST (n = 4), ALT (n = 2), neutropenia (n = 1), thrombocytopenia (n = 1), failure to thrive (n = 1) and anorexia (n = 1). The MTD is 40 mg BID continuously. Eleven patients had stable disease for ≥ 4 months, including patients with ovarian, carcinoid, papillary thyroid, prostate, pancreas and head and neck cancer. Patients with prostate and pancreatic cancer also had significant biomarker decreases (PSA, 205 ng/mL to 39 ng/mL; CA19-9, 38,838 U/mL to 267 U/mL). The ovarian cancer patient has had stable disease > 12 months. KX2-391 was orally available, rapidly absorbed, and exposure was proportional to dose across the range investigated. CONCLUSIONS: KX2-391 has a favorable pharmacokinetic profile, is well tolerated, demonstrates preliminary evidence of biologic activity, and warrants further evaluation in Phase II trials.
Assuntos
Acetamidas/uso terapêutico , Trifosfato de Adenosina/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/uso terapêutico , Quinases da Família src/antagonistas & inibidores , Acetamidas/efeitos adversos , Acetamidas/farmacocinética , Acetamidas/farmacologia , Sítios de Ligação , Demografia , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Morfolinas , Estadiamento de Neoplasias , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Piridinas/efeitos adversos , Piridinas/farmacocinética , Piridinas/farmacologia , Especificidade por Substrato/efeitos dos fármacos , Quinases da Família src/metabolismoRESUMO
KX-01 is the first clinical Src inhibitor of the novel peptidomimetic class that targets the peptide substrate site of Src providing more specificity toward Src kinase. The present study was designed to evaluate the effects of KX-01 as a single agent and in combination with tamoxifen (TAM) on cell growth and apoptosis of ERα positive breast cancer in vitro and in vivo. Flow cytometry demonstrated that KX-01 induced cell cycle arrest in G2/M phase. Immunofluorescent staining for mitotic phase markers and TUNEL staining indicated that cells had arrested in the mitotic phase and mitotic arrested cells were undergoing apoptosis. KX-01 induced nuclear accumulation of cyclin B1, and activation of CDK1, MPM2, and Cdc25C that is required for progression past the G2/M checkpoint. Apoptosis resulted from activation of caspases 6, 7, 8, and 9. Combinational index analysis revealed that combinations of KX-01 with TAM resulted in synergistic growth inhibition of breast cancer cell lines. KX-01 combined with TAM resulted in decreased ERα phosphorylation at Src-regulated phosphorylation sites serines 118 and 167 that were associated with reduced ERα transcriptional activity. Orally administered KX-01 resulted in a dose dependent growth inhibition of MCF-7 tumor xenografts, and in combination with TAM exhibited synergistic growth inhibition. Immunohistochemical analysis revealed that combinational treatment reduced angiogenesis, and ERα signaling in tumors compared to either drug alone that may underlie the synergistic tumor growth inhibition. Combinations of KX-01 with endocrine therapy present a promising new strategy for clinical management of ERα positive breast cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Receptor alfa de Estrogênio/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Domínio Catalítico , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Citometria de Fluxo , Imunofluorescência , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Nus , Mitose/efeitos dos fármacos , Neovascularização Patológica/enzimologia , Neovascularização Patológica/prevenção & controle , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologia , Fatores de Tempo , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/metabolismoRESUMO
The expressions of c-Src and focal adhesion kinase (FAK) were studied in 65 Chinese patients with hepatocellular carcinoma (HCC) by immunohistochemistry using rabbit monoclonal antibodies. Expressions of total Src, an active form of Src, and FAK were found in 44/65 (67.7%), 36/45 (55.4%), and 33/56 (58.9%) HCC cases, respectively. There was a good correlation between the expression of total Src, active form of Src, and FAK in these HCC cases (P < 0.001). Expression of Src was not correlated to any clinical parameters, cancer cell phenotypic markers, and pathologic features apart from a positive correlation with alpha-fetoprotein (P < 0.01). The expression of FAK was correlated with earlier onset and the expression of Ki-67 but not proliferating cell nuclear antigen (PCNA) in these HCC cases. Four liver-cancer-derived cell lines (three derived from HCC and one from hepatoblastoma) were then tested with inhibitors against Src. A small molecule, KX2-391, designed to target the substrate binding pocket of Src, was found to have more broad-spectrum activity and better potency than Dasatinib, an adenosine triphosphate (ATP)-competitive inhibitor in vitro. Our data indicates that Src and FAK expression are both elevated and active in Chinese patients with HCC and that Src may play a key role in supporting HCC progression. Src antagonism with specific inhibitors may be an attractive treatment paradigm for patients with HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Adulto , Idoso , Carcinoma Hepatocelular/patologia , Proliferação de Células , Dasatinibe , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Fígado/patologia , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Antígeno Nuclear de Célula em Proliferação/metabolismo , Pirimidinas/farmacologia , Tiazóis/farmacologiaRESUMO
The discovery of potent, peptide site directed, tyrosine kinase inhibitors has remained an elusive goal. Herein we describe the discovery of two such clinical candidates that inhibit the tyrosine kinase Src. Compound 1 is a phase 3 clinical trial candidate that is likely to provide a first in class topical treatment for actinic keratosis (AK) with good efficacy and dramatically less toxicity compared to existing standard therapy. Compound 2 is a phase 1 clinical trial candidate that is likely to provide a first in class treatment of malignant glioblastoma and induces 30% long-term complete tumor remission in animal models. The discovery strategy for these compounds iteratively utilized molecular modeling, along with the synthesis and testing of increasingly elaborated proof of concept compounds, until the final clinical candidates were arrived at. This was followed with mechanism of action (MOA) studies that revealed tubulin polymerization inhibition as the second MOA.
Assuntos
Acetamidas/farmacologia , Descoberta de Drogas , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Moduladores de Tubulina/farmacologia , Quinases da Família src/antagonistas & inibidores , Acetamidas/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Linhagem Celular Tumoral , Humanos , Simulação de Acoplamento Molecular , Morfolinas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Piridinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Moduladores de Tubulina/metabolismo , Quinases da Família src/química , Quinases da Família src/metabolismoRESUMO
Understanding subtle aspects of hydrogen bonding is a challenging but crucial task to improve our ability to design ligands with high affinity for protein hosts. To gain a deeper understanding of these aspects, we investigated a series of thrombin inhibitors in which the basicity of the ligand's group that accepts an H-bond from Gly216 was modulated via bioisosterism; e.g., a C=O acceptor was made electron deficient or rich via bioisosteric replacements of the adjacent moiety. Although the ligand's binding affinity was anticipated to improve when the H-bond basicity is increased (due to stronger H-bonding with the protein), we herein present data that unexpectedly revealed an opposite trend. This trend was attributed to a dominating role played by desolvation in determining the relative binding affinity. For example, a decrease in the H-bond basicity reduces the desolvation penalty and, as experimentally observed, improves the binding affinity, given that the reduction in the desolvation penalty dominates the change in the net contribution of the ligand's interactions with the protein. The current study, therefore, reveals that desolvation can be a major underlying cause for the apparently counterintuitive structure-activity relationship (SAR) outcomes, and indicates that understanding this factor can improve our ability to predict binding affinity and to design more potent ligands.
Assuntos
Inibidores Enzimáticos/metabolismo , Solventes/farmacologia , Trombina/metabolismo , Animais , Humanos , Ligação de Hidrogênio , Ligantes , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Trombina/antagonistas & inibidoresRESUMO
Unlike breast cancer that is positive for estrogen receptor-α (ERα), there are no targeted therapies for triple-negative breast cancer (TNBC). ERα is silenced in TNBC through epigenetic changes including DNA methylation and histone acetylation. Restoring ERα expression in TNBC may sensitize patients to endocrine therapy. Expression of c-Src and ERα are inversely correlated in breast cancer suggesting that c-Src inhibition may lead to reexpression of ERα in TNBC. KX-01 is a peptide substrate-targeted Src/pretubulin inhibitor in clinical trials for solid tumors. KX-01 (1 mg/kg body weight-twice daily) inhibited growth of tamoxifen-resistant MDA-MB-231 and MDA-MB-157 TNBC xenografts in nude mice that was correlated with Src kinase inhibition. KX-01 also increased ERα mRNA and protein, as well as increased the ERα targets progesterone receptor (PR), pS2 (TFF1), cyclin D1 (CCND1), and c-myc (MYC) in MDA-MB-231 and MDA-MB-468, but not MDA-MB-157 xenografts. MDA-MB-231 and MDA-MB-468 tumors exhibited reduction in mesenchymal markers (vimentin, ß-catenin) and increase in epithelial marker (E-cadherin) suggesting mesenchymal-to-epithelial transition (MET). KX-01 sensitized MDA-MB-231 and MDA-MB-468 tumors to tamoxifen growth inhibition and tamoxifen repression of the ERα targets pS2, cyclin D1, and c-myc. Chromatin immunoprecipitation (ChIP) of the ERα promoter in KX-01-treated tumors demonstrated enrichment of active transcription marks (acetyl-H3, acetyl-H3Lys9), dissociation of HDAC1, and recruitment of RNA polymerase II. Methylation-specific PCR and bisulfite sequencing demonstrated no alteration in ERα promoter methylation by KX-01. These data demonstrate that in addition to Src kinase inhibition, peptidomimetic KX-01 restores ERα expression in TNBC through changes in histone acetylation that sensitize tumors to tamoxifen.Implications: Src kinase/pretubulin inhibitor KX-01 restores functional ERα expression in ERα- breast tumors, a novel treatment strategy to treat triple-negative breast cancer. Mol Cancer Res; 15(11); 1491-502. ©2017 AACR.
Assuntos
Acetamidas/administração & dosagem , Receptor alfa de Estrogênio/metabolismo , Piridinas/administração & dosagem , Tamoxifeno/administração & dosagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Acetamidas/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Morfolinas , Piridinas/farmacologia , Tamoxifeno/farmacologia , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: KX-01 is a novel dual inhibitor of Src and tubulin. Unlike previous Src inhibitors that failed to show clinical benefit during treatment of breast cancer, KX-01 can potentially overcome the therapeutic limitations of current Src inhibitors through inhibition of both Src and tubulin. The present study further evaluates the activity and mechanism of KX-01 in vitro and in vivo. MATERIALS AND METHODS: The antitumor effect of KX-01 in triple negative breast cancer (TNBC) cell lines was determined by MTT assay. Wound healing and immunofluorescence assays were performed to evaluate the action mechanisms of KX-01. Changes in the cell cycle and molecular changes induced by KX-01 were also evaluated. A MDA-MB-231 mouse xenograft model was used to demonstrate the in vivo effects. RESULTS: KX-01 effectively inhibited the growth of breast cancer cell lines. The expression of phospho-Src and proliferative-signaling molecules were down-regulated in KX-01-sensitive TNBC cell lines. In addition, migration inhibition was observed by wound healing assay. KX-01-induced G2/M cell cycle arrest and increased the aneuploid cell population in KX-01-sensitive cell lines. Multi-nucleated cells were significantly increased after KX-01 treatment. Furthermore, KX-01 effectively delayed tumor growth in a MDA-MB-231 mouse xenograft model. CONCLUSION: KX-01 effectively inhibited cell growth and migration of TNBC cells. Moreover, this study demonstrated that KX-01 showed antitumor effects through the inhibition of Src signaling and the induction of mitotic catastrophe. The antitumor effects of KX-01 were also demonstrated in vivo using a mouse xenograft model.
Assuntos
Acetamidas/farmacologia , Antineoplásicos/farmacologia , Mitose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Aneuploidia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Morfolinas , Fosforilação , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/metabolismoRESUMO
Studies from our lab show that noise exposure initiates cell death by multiple pathways [Nicotera, T.M., Hu, B.H., Henderson, D., 2003. The caspase pathway in noise-induced apoptosis of the chinchilla cochlea. J. Assoc. Res. Otolaryngol. 4, 466-477] therefore, protection against noise may be most effective with a multifaceted approach. The Src protein tyrosine kinase (PTK) signaling cascade may be involved in both metabolic and mechanically induced initiation of apoptosis in sensory cells of the cochlea. The current study compares three Src-PTK inhibitors, KX1-004, KX1-005 and KX1-174 as potential protective drugs for NIHL. Chinchillas were used as subjects. A 30 microl drop of one of the Src inhibitors was placed on the round window membrane of the anesthetized chinchilla; the vehicle (DMSO and buffered saline) alone was placed on the other ear. After the drug application, the middle ear was sutured and the subjects were exposed to noise. Hearing was measured before and several times after the noise exposure and treatment using evoked responses. At 20 days post-exposure, the animals were anesthetized their cochleae extracted and cochleograms were constructed. All three Src inhibitors provided protection from a 4 h, 4 kHz octave band noise at 106 dB. The most effective drug, KX1-004 was further evaluated by repeating the exposure with different doses, as well as, substituting an impulse noise exposure. For all conditions, the results suggest a role for Src-PTK activation in noise-induced hearing loss (NIHL), and that therapeutic intervention with a Src-PTK inhibitor may offer a novel approach in the treatment of NIHL.
Assuntos
Perda Auditiva Provocada por Ruído/prevenção & controle , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinases da Família src/antagonistas & inibidores , Animais , Limiar Auditivo/efeitos dos fármacos , Chinchila , Relação Dose-Resposta a Droga , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/patologia , Perda Auditiva Provocada por Ruído/enzimologia , Perda Auditiva Provocada por Ruído/patologia , Perda Auditiva Provocada por Ruído/fisiopatologia , Indóis/administração & dosagem , Indóis/química , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidoresRESUMO
One of the underappreciated non-covalent binding factors, which can significantly affect ligand-protein binding affinity, is the cooperativity between ligand functional groups. Using four different series of thrombin inhibitors, we reveal a strong positive cooperativity between an H-bond accepting carbonyl functionality and the adjacent P3 hydrophobic side chain. Adding an H-bond donating amine adjacent to the P3 hydrophobic side chain further increases this positive cooperativity thereby improving the Ki by as much as 546-fold. In contrast, adding an amidine multiple H-bond/salt bridge group in the distal S1 pocket does not affect this cooperativity. An analysis of the crystallographic B-factors of the ligand groups inside the binding site indicates that the strong cooperativity is mainly due to a significant mutual reduction in the residual mobility of the hydrophobic side chain and the H-bonding functionalities that is absent when the separation distance is large. This type of cooperativity is important to encode in binding affinity prediction software, and to consider in SAR studies.
Assuntos
Antitrombinas/farmacologia , Trombina/antagonistas & inibidores , Antitrombinas/química , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Trombina/metabolismoRESUMO
Predicting how binding affinity responds to ligand structural modifications in structure-activity relationship studies (SAR) is a major challenge in medicinal chemistry. This is particularly true when two or more of these modifications are carried out simultaneously. In this study, we present binding affinity data from several series of thermolysin inhibitors in which simultaneous structural modifications were investigated to determine whether they are cooperative or additive. Data revealed that, while additivity is at work in some cases, cooperativity is more commonly demonstrated. Cooperativity and additivity were then correlated with ligand descriptors, such as the spacing and the topological features of the modified groups, in a manner that may provide guidance as to when each model should be utilized. Cooperativity was particularly associated with contiguous groups and small unbranched hydrophobic side chain. Additivity, on the other hand, was associated with moderately distant hydrophobic group combinations and side chain branching. Such correlations can improve the predictability of SAR studies and can provide a starting point for additional investigations that may lead to further significant enhancements in the current scoring functions.
Assuntos
Inibidores de Metaloproteinases de Matriz/farmacologia , Termolisina/antagonistas & inibidores , Ligantes , Inibidores de Metaloproteinases de Matriz/química , Modelos Moleculares , Relação Estrutura-Atividade , Termodinâmica , Termolisina/metabolismoRESUMO
The thermodynamic consequences of systematic modifications in a ligand side chain that binds in a shallow hydrophobic pocket, in the presence and absence of a neighboring ligand carboxylate group, were evaluated using isothermal titration calorimetry (ITC). Data revealed that the carboxylate significantly changes the relative thermodynamic signatures of these modifications, likely via altering the H-bonding/organization status of the hydration waters both in the unbound and the bound states. This carboxylate group was found to be proenthalpic, antientropic in some cases, and antienthalpic, proentropic in others. A remarkable enthalpy-entropy compensation relationship was also observed, reflecting the fact that the hydrophobic effect is governed by the thermodynamic status of the associated aqueous environment. This study could improve our understanding of the hydrophobic effect and may enhance our ability to design potent ligands that are capable of modulating biological processes.
Assuntos
Ligação de Hidrogênio , Ligação Proteica/fisiologia , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Entropia , Indicadores e Reagentes , Ligantes , Modelos Moleculares , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
PURPOSE: KX2-391 is an oral non-ATP-competitive inhibitor of Src kinase and tubulin polymerization. In phase 1 trials, prostate-specific antigen (PSA) declines were seen in patients with advanced prostate cancer. We conducted a single-arm phase 2 study evaluating KX2-391 in men with chemotherapy-naïve bone-metastatic castration-resistant prostate cancer (CRPC). METHODS: We treated 31 patients with oral KX2-391 (40 mg twice-daily) until disease progression or unacceptable toxicity. The primary endpoint was 24-week progression-free survival (PFS); a 50 % success rate was pre-defined as clinically significant. Secondary endpoints included PSA progression-free survival (PPFS) and PSA response rates. Exploratory outcomes included pharmacokinetic studies, circulating tumor cell (CTC) enumeration, and analysis of markers of bone resorption [urinary N-telopeptide (uNTx); C-telopeptide (CTx)] and formation [bone alkaline phosphatase (BAP); osteocalcin]. RESULTS: The trial closed early after accrual of 31 patients, due to a pre-specified futility rule. PFS at 24 weeks was 8 %, and median PFS was 18.6 weeks. The PSA response rate (≥ 30 % decline) was 10 %, and median PPFS was 5.0 weeks. Additionally, 18 % of men with unfavorable (≥ 5) CTCs at baseline converted to favorable (<5) CTCs with treatment. The proportion of men with declines in bone turnover markers was 32 % for uNTx, 21 % for CTx, 10 % for BAP, and 25 % for osteocalcin. In pharmacokinetic studies, median C max was 61 (range 16-129) ng/mL, and median AUC was 156 (35-348) ng h/mL. Common toxicities included hepatic derangements, myelosuppression, fatigue, nausea, and constipation. CONCLUSION: KX2-391 dosed at 40 mg twice-daily lacks antitumor activity in men with CRPC, but has modest effects on bone turnover markers. Because a C max of ≥142 ng/mL is required for tubulin polymerization inhibition (defined from preclinical studies), higher once-daily dosing will be used in future trials.
Assuntos
Acetamidas/uso terapêutico , Neoplasias Ósseas/secundário , Neoplasias da Próstata/tratamento farmacológico , Piridinas/uso terapêutico , Moduladores de Tubulina/uso terapêutico , Quinases da Família src/antagonistas & inibidores , Acetamidas/efeitos adversos , Acetamidas/farmacocinética , Administração Oral , Idoso , Idoso de 80 Anos ou mais , Osso e Ossos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Morfolinas , Células Neoplásicas Circulantes/efeitos dos fármacos , Orquiectomia , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Piridinas/efeitos adversos , Piridinas/farmacocinética , Moduladores de Tubulina/efeitos adversos , Moduladores de Tubulina/farmacocinéticaRESUMO
PURPOSE: To investigate the antitumor effects of targeting Src and tubulin in mucinous ovarian carcinoma. EXPERIMENTAL DESIGN: The in vitro and in vivo effects and molecular mechanisms of KX-01, which inhibits Src pathway and tubulin polymerization, were examined in mucinous ovarian cancer models. RESULTS: In vitro studies using RMUG-S and RMUG-L cell lines showed that KX-01 inhibited cell proliferation, induced apoptosis, arrested the cell cycle at the G2-M phase, and enhanced the cytotoxicity of oxaliplatin in the KX-01-sensitive cell line, RMUG-S. In vivo studies showed that KX-01 significantly decreased tumor burden in RMUG-S and RMUG-L mouse models relative to untreated controls, and the effects were greater when KX-01 was combined with oxaliplatin. KX-01 alone and in combination with oxaliplatin significantly inhibited tumor growth by reducing cell proliferation and inducing apoptosis in vivo. PTEN knock-in experiments in RMUG-L cells showed improved response to KX-01. Reverse phase protein array analysis showed that in addition to blocking downstream molecules of Src family kinases, KX-01 also activated acute stress-inducing molecules. CONCLUSION: Our results showed that targeting both the Src pathway and tubulin with KX-01 significantly inhibited tumor growth in preclinical mucinous ovarian cancer models, suggesting that this may be a promising therapeutic approach for patients with mucinous ovarian carcinoma.
Assuntos
Acetamidas/farmacologia , Adenocarcinoma Mucinoso/tratamento farmacológico , Antineoplásicos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Piridinas/farmacologia , Moduladores de Tubulina/farmacologia , Quinases da Família src/antagonistas & inibidores , Acetamidas/uso terapêutico , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patologia , Animais , Antineoplásicos/uso terapêutico , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Nus , Microtúbulos/metabolismo , Morfolinas , Compostos Organoplatínicos/farmacologia , Compostos Organoplatínicos/uso terapêutico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Oxaliplatina , PTEN Fosfo-Hidrolase/metabolismo , Multimerização Proteica , Piridinas/uso terapêutico , Moduladores de Tubulina/uso terapêutico , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/metabolismoAssuntos
Azetidinas/química , Benzilaminas/química , Química/métodos , Cristalografia por Raios X/métodos , Trombina/antagonistas & inibidores , Calorimetria/métodos , Química Farmacêutica/métodos , Cicloexanos/química , Ciclopentanos/química , Desenho de Fármacos , Humanos , Cinética , Ligantes , Modelos Químicos , Conformação Molecular , Termodinâmica , Trombina/químicaRESUMO
Ligand functional groups can modulate the contributions of one another to the ligand-protein binding thermodynamics, producing either positive or negative cooperativity. Data presented for four thermolysin phosphonamidate inhibitors demonstrate that the differential binding free energy and enthalpy caused by replacement of a H with a Me group, which binds in the well-hydrated S2' pocket, are more favorable in presence of a ligand carboxylate. The differential entropy is however less favorable. Dissection of these differential thermodynamic parameters, X-ray crystallography, and density-functional theory calculations suggest that these cooperativities are caused by variations in the thermodynamics of the complex hydration shell changes accompanying the HâMe replacement. Specifically, the COO(-) reduces both the enthalpic penalty and the entropic advantage of displacing water molecules from the S2' pocket and causes a subsequent acquisition of a more enthalpically, less entropically, favorable water network. This study contributes to understanding the important role water plays in ligand-protein binding.