RESUMO
Racecadotril (acetorphan) is a neutral endopeptidase (NEP) inhibitor with known antidiarrheal activity in animals and humans; however, in humans, it suffers from shortcomings that might be improved with newer drugs in this class that have progressed to the clinic for nonenteric disease indications. To identify potentially superior NEP inhibitors with immediate clinical utility for diarrhea treatment, we compared their efficacy and pharmacologic properties in a rat intestinal hypersecretion model. Racecadotril and seven other clinical-stage inhibitors of NEP were obtained or synthesized. Enzyme potency and specificity were compared using purified peptidases. Compounds were orally administered to rats before administration of castor oil to induce diarrhea. Stool weight was recorded over 4 hours. To assess other pharmacologic properties, select compounds were orally administered to normal or castor oil-treated rats, blood and tissue samples collected at multiple time points, and active compound concentrations determined by mass spectroscopy. NEP enzyme activity was measured in tissue homogenates. Three previously untested clinical NEP inhibitors delayed diarrhea onset and reduced total stool output, with little or no effect on intestinal motility assessed by the charcoal meal test. Each was shown to be a potent, highly specific inhibitor of NEP. Each exhibited greater suppression of NEP activity in intestinal and nonintestinal tissues than did racecadotril and sustained this inhibition longer. These results suggest that newer clinical-stage NEP inhibitors originally developed for other indications may be directly repositioned for treatment of acute secretory diarrhea and offer advantages over racecadotril, such as less frequent dosing and potentially improved efficacy.
Assuntos
Antidiarreicos/uso terapêutico , Diarreia/tratamento farmacológico , Endopeptidases/metabolismo , Inibidores de Proteases/uso terapêutico , Tiorfano/análogos & derivados , Animais , Óleo de Rícino , Carvão Vegetal/farmacologia , Diarreia/induzido quimicamente , Relação Dose-Resposta a Droga , Fezes , Motilidade Gastrointestinal/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Tiorfano/uso terapêuticoRESUMO
Given the rise of parasite resistance to all currently used antimalarial drugs, the identification of novel chemotypes with unique mechanisms of action is of paramount importance. Since Plasmodium expresses a number of aspartic proteases necessary for its survival, we have mined antimalarial datasets for drug-like aspartic protease inhibitors. This effort led to the identification of spiropiperidine hydantoins, bearing similarity to known inhibitors of the human aspartic protease ß-secretase (BACE), as new leads for antimalarial drug discovery. Spiropiperidine hydantoins have a dynamic structure-activity relationship profile with positions identified as being tolerant of a variety of substitution patterns as well as a key piperidine N-benzyl phenol pharmacophore. Lead compounds 4e (CWHM-123) and 12k (CWHM-505) are potent antimalarials with IC50 values against Plasmodium falciparum 3D7 of 0.310 µM and 0.099 µM, respectively, and the former features equivalent potency on the chloroquine-resistant Dd2 strain. Remarkably, these compounds do not inhibit human aspartic proteases BACE, cathepsins D and E, or Plasmodium plasmepsins II and IV despite their similarity to known BACE inhibitors. Although the current leads suffer from poor metabolic stability, they do fit into a drug-like chemical property space and provide a new class of potent antimalarial agents for further study.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Hidantoínas/química , Hidantoínas/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/metabolismo , Antimaláricos/farmacocinética , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Descoberta de Drogas , Humanos , Hidantoínas/metabolismo , Hidantoínas/farmacocinética , Malária Falciparum/parasitologia , Camundongos , Microssomos Hepáticos/metabolismo , Piperidinas/química , Piperidinas/metabolismo , Piperidinas/farmacocinética , Piperidinas/farmacologia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/metabolismo , Ratos , Compostos de Espiro/química , Compostos de Espiro/metabolismo , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologiaRESUMO
New verapamil analogues were synthesized and their inhibitory activities against Mycobacterium tuberculosis H37Rv determined in vitro alone and in combination with rifampicin (RIF). Some analogues showed comparable activity to verapamil and exhibited better synergies with RIF. Molecular docking studies of the binding sites of Rv1258c, a M. tuberculosis efflux protein previously implicated in intrinsic resistance to RIF, suggested a potential rationale for the superior synergistic interactions observed with some analogues.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Verapamil/análogos & derivados , Verapamil/farmacologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/síntese química , Proteínas de Bactérias/metabolismo , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Rifampina/química , Relação Estrutura-Atividade , Verapamil/síntese química , Verapamil/químicaRESUMO
Despite the curative potential of hematopoietic stem cell transplantation (HSCT), conditioning-associated toxicities preclude broader clinical application. Antibody-drug conjugates (ADCs) provide an attractive approach to HSCT conditioning that minimizes toxicity while retaining efficacy. Initial studies of ADC conditioning have largely focused on syngeneic HSCT. However, to treat acute leukemias or induce tolerance for solid organ transplantation, this approach must be expanded to allogeneic HSCT (allo-HSCT). Using murine allo-HSCT models, we show that pharmacologic Janus kinase 1/2 (JAK1/2) inhibition combined with CD45- or cKit-targeted ADCs enables robust multilineage alloengraftment. Strikingly, myeloid lineage donor chimerism exceeding 99% was achievable in fully MHC-mismatched HSCT using this approach. Mechanistic studies using the JAK1/2 inhibitor baricitinib revealed marked impairment of T and NK cell survival, proliferation, and effector function. NK cells were exquisitely sensitive to JAK1/2 inhibition due to interference with IL-15 signaling. Unlike irradiated mice, ADC-conditioned mice did not develop pathogenic graft-versus-host alloreactivity when challenged with mismatched T cells. Finally, the combination of ADCs and baricitinib balanced graft-versus-host disease and graft-versus-leukemia responses in delayed donor lymphocyte infusion models. Our allo-HSCT conditioning strategy exemplifies the promise of immunotherapy to improve the safety of HSCT for treating hematologic diseases.
Assuntos
Azetidinas/farmacologia , Transplante de Células-Tronco Hematopoéticas , Imunoconjugados/farmacologia , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Inibidores de Janus Quinases/farmacologia , Purinas/farmacologia , Pirazóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Aloenxertos , Animais , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Leucemia/efeitos dos fármacos , Efeito Enxerto vs Leucemia/genética , Efeito Enxerto vs Leucemia/imunologia , Interleucina-15/genética , Interleucina-15/imunologia , Janus Quinase 1/genética , Janus Quinase 1/imunologia , Janus Quinase 2/genética , Janus Quinase 2/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Potent, highly selective and orally-bioavailable MMP-13 inhibitors have been identified based upon a (pyridin-4-yl)-2H-tetrazole scaffold. Co-crystal structure analysis revealed that the inhibitors bind at the S(1)(') active site pocket and are not ligands for the catalytic zinc atom. Compound 29b demonstrated reduction of cartilage degradation biomarker (TIINE) levels associated with cartilage protection in a preclinical rat osteoarthritis model.
Assuntos
Inibidores de Metaloproteinases de Matriz , Osteoartrite/tratamento farmacológico , Ácidos Picolínicos/química , Inibidores de Proteases/química , Tetrazóis/química , Administração Oral , Animais , Sítios de Ligação , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Modelos Animais de Doenças , Descoberta de Drogas , Metaloproteinase 13 da Matriz/metabolismo , Ácidos Picolínicos/síntese química , Ácidos Picolínicos/farmacologia , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Ratos , Tetrazóis/síntese química , Tetrazóis/farmacologia , Zinco/químicaRESUMO
Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a five-day course of granulocyte colony stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor, plerixafor, is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was co-administered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization provided by the VLA4 inhibitor and CXCR2 agonist combination in mice compared to currently approved HSPC mobilization methods, it represents an exciting potential strategy for clinical development in the future.
Assuntos
Medula Óssea/metabolismo , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Integrina alfa4beta1 , Receptores de Interleucina-8B , Aloenxertos , Animais , Granulócitos/metabolismo , Integrina alfa4beta1/antagonistas & inibidores , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismoRESUMO
BACKGROUND & AIMS: Pancreatic stellate cells (PSCs) regulate the development of chronic pancreatitis (CP) and are activated by the cytokine transforming growth factor ß (TGFB). Integrins of the αv family promote TGFB signaling in mice, probably by interacting with the Arg-Gly-Asp (RGD) sequence of the TGFB latency-associated peptide, which frees TGFB to bind its cellular receptors. However, little is known about the role of integrins in the development of CP. We investigated the effects of small-molecule integrin inhibitors in a mouse model of CP. METHODS: We induced CP in C57BL/6 female mice by repeated cerulein administration. An active RGD peptidomimetic compound (Center for World Health and Medicine [CWHM]-12) was delivered by continuous infusion, starting 3 days before or 5 days after cerulein administration began. Pancreata were collected and parenchymal atrophy, fibrosis, and activation of PSCs were assessed by histologic, gene, and protein expression analyses. We measured CWHM-12 effects on activation of TGFB in co-culture assays in which rat PSC cells (large T immortalized cells [LTC-14]) activate expression of a TGFB-sensitive promoter in reporter cells. RESULTS: Pancreatic tissues of mice expressed messenger RNAs encoding subunits of RGD-binding integrins. Cerulein administration increased expression of these integrins, altered pancreatic cell morphology, and induced fibrosis. The integrin inhibitor CWHM-12 decreased acinar cell atrophy and loss, and substantially reduced fibrosis, activation of PSCs, and expression of genes regulated by TGFB. CWHM-12 also reduced established fibrosis in mice and blocked activation of TGFB in cultured cells. CONCLUSIONS: Based on studies of a mouse model of CP and cultured PSCs, integrins that bind RGD sequences activate PSCs and promote the development of pancreatic fibrogenesis in mice. Small-molecule antagonists of this interaction might be developed for treatment of pancreatic fibrotic diseases.
RESUMO
Matrix metalloproteinase-13 (MMP-13) is a zinc-dependent protease responsible for the cleavage of type II collagen, the major structural protein of articular cartilage. Degradation of this cartilage matrix leads to the development of osteoarthritis. We previously have described highly potent and selective carboxylic acid containing MMP-13 inhibitors; however, nephrotoxicity in preclinical toxicology species precluded development. The accumulation of compound in the kidneys mediated by human organic anion transporter 3 (hOAT3) was hypothesized as a contributing factor for the finding. Herein we report our efforts to optimize the MMP-13 potency and pharmacokinetic properties of non-carboxylic acid leads resulting in the identification of compound 43a lacking the previously observed preclinical toxicology at comparable exposures.
Assuntos
Metaloproteinase 13 da Matriz/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz/síntese química , Inibidores de Metaloproteinases de Matriz/farmacologia , Osteoartrite/tratamento farmacológico , Pirimidinas/síntese química , Pirimidinas/farmacologia , Tetrazóis/síntese química , Tetrazóis/farmacologia , Animais , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Colagenases/efeitos dos fármacos , Cães , Desenho de Fármacos , Humanos , Rim/metabolismo , Macaca fascicularis , Masculino , Inibidores de Metaloproteinases de Matriz/toxicidade , Modelos Moleculares , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Integrins expressed on endothelial cells modulate cell migration and survival during angiogenesis. Integrins expressed on carcinoma cells potentiate metastasis by facilitating invasion and movement across blood vessels. We describe the activities of two synthetic low-molecular-weight peptidomimetics of the ligand amino acid sequence arg-gly-asp (RGD) in integrin-based functional assays in vitro. We also evaluate efficacy and potential mechanisms of action in models of both spontaneous and experimental metastasis. Broad-spectrum potency against the family of alpha v subunit-containing integrins was observed, with significantly less potency against alpha5beta1 and alpha(IIb)beta3. Both endothelial and tumor cell migration mediated by alpha(v)beta3 was inhibited, whereas proliferation of endothelial cells but not tumor cells was diminished. Continuous infusion of compound by minipumps or oral administration twice daily significantly reduced metastatic tumor burden in the lungs of mice despite no reduction in growth of 435/HAL primary tumors, and only a slight reduction in tumor cells detected in circulating blood. Delaying treatment in this model until after extensive dissemination of tumor cells to the lungs had occurred, and after primary tumor resection, still produced significant efficacy. Conversely, administration of the agent for only the first 18 h after tumor-cell inoculation into the tail vein also resulted in decreased metastases observed after several weeks. These data suggest these compounds or their relatives have potential to interfere with both early and late steps of metastasis involving tumor and endothelial cell functions. Furthermore, the metastatic process can be effectively inhibited independently of primary tumor growth using integrin antagonists.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/patologia , Carcinoma Ductal/secundário , Dipeptídeos/uso terapêutico , Integrina alfaVbeta3/antagonistas & inibidores , Neoplasias Pulmonares/secundário , Proteínas de Neoplasias/antagonistas & inibidores , Compostos Orgânicos/uso terapêutico , Pirimidinas/uso terapêutico , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Carcinoma Ductal/tratamento farmacológico , Carcinoma Ductal/patologia , Carcinoma Ductal/prevenção & controle , Carcinoma Ductal/cirurgia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral/transplante , Movimento Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Dipeptídeos/administração & dosagem , Dipeptídeos/farmacologia , Esquema de Medicação , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Feminino , Humanos , Bombas de Infusão Implantáveis , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Células Neoplásicas Circulantes , Oligopeptídeos , Compostos Orgânicos/administração & dosagem , Compostos Orgânicos/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Many viruses and bacterial pathogens are capable of exploiting host cell surface integrins during their replication cycles. The ligands for many integrins contain an arginine-glycine-aspartic acid (RGD) amino acid sequence that is essential for protein-protein interaction. Human adenovirus particles contain this sequence in the penton base protein, and previous studies support a role for this RGD in integrin-dependent internalization of the virus by the cell. As synthetic peptidomimetics of RGD have been shown in other experimental systems to be antagonists of the activities of specific integrins both in vitro and in vivo, we sought to determine whether these small molecules are antagonists of adenovirus infection. Such compounds inhibited viral infection of cultured cells with similar rank order potency to that determined in assays utilizing purified extracellular matrix proteins as integrin ligands. The maximal level of inhibition achieved with the peptidomimetics was comparable to that of RGD-containing peptides, whereas no significant effects were apparent with an RGE-containing peptide. An engineered adenovirus having a mutated RGD sequence in the penton base was not susceptible to the inhibition. The results obtained with these synthetic antagonists, which have varied structures and potencies, suggest that integrins interact with adenoviral RGD in a manner similar to that of other protein ligands such as vitronectin. Furthermore, the results confirm the role of RGD in the replication cycle, and suggest peptidomimetic compounds may be useful antimicrobial agents in the treatment of a variety of diseases.
Assuntos
Adenovírus Humanos/efeitos dos fármacos , Integrinas/antagonistas & inibidores , Oligopeptídeos/química , Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Linhagem Celular , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Mutação , Oligopeptídeos/genética , Fenilpropionatos/síntese química , Fenilpropionatos/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
Given the threat of drug resistance, there is an acute need for new classes of antimalarial agents that act via a unique mechanism of action relative to currently used drugs. We have identified a set of druglike compounds within the Tres Cantos Anti-Malarial Set (TCAMS) which likely act via inhibition of a Plasmodium aspartic protease. Structure-activity relationship analysis and optimization of these aminohydantoins demonstrate that these compounds are potent nanomolar inhibitors of the Plasmodium aspartic proteases PM-II and PM-IV and likely one or more other Plasmodium aspartic proteases. Incorporation of a bulky group, such as a cyclohexyl group, on the aminohydantion N-3 position gives enhanced antimalarial potency while reducing inhibition of human aspartic proteases such as BACE. We have identified compound 8p (CWHM-117) as a promising lead for optimization as an antimalarial drug with a low molecular weight, modest lipophilicity, oral bioavailability, and in vivo antimalarial activity in mice.
RESUMO
Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding α(v) integrin subunit because various α(v)-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride-induced hepatic fibrosis, whereas global loss of ß3, ß5 or ß6 integrins or conditional loss of ß8 integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the α(v) integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of α(v)-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all α(v) integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.
Assuntos
Integrina alfaV/metabolismo , Nefropatias/genética , Rim/patologia , Cirrose Hepática/genética , Fibrose Pulmonar/genética , Animais , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Feminino , Fibrose/genética , Marcação de Genes , Integrina alfaV/genética , Rim/metabolismo , Nefropatias/patologia , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fibrose Pulmonar/patologia , Transdução de Sinais/fisiologiaRESUMO
The integrin alpha(v)beta(3), vitronectin receptor, is expressed in a number of cell types and has been shown to mediate adhesion of osteoclasts to bone matrix, vascular smooth muscle cell migration, and angiogenesis. We recently disclosed the discovery of a tripeptide Arg-Gly-Asp (RGD) mimic, which has been shown to be a potent inhibitor of the integrin alpha(v)beta(3) and has excellent anti-angiogenic properties including its suppression of tumor growth in animal models. In other investigations involving RGD mimics, only compounds containing the S-isomers of the beta-amino acids have been shown to be potent. We were surprised to find the potencies of analogs containing enantiomerically pure S-isomers of beta-amino acids which were only marginally better than the corresponding racemic mixtures. We therefore synthesized RGD mimics containing R-isomers of beta-amino acids and found them to be relatively potent inhibitors of alpha(v)beta(3). One of the compounds was examined in tumor models in mice and has been shown to significantly reduce the rate of growth and the size of tumors.