RESUMO
The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed "mouse kidney parvovirus" (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and establish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans.
Assuntos
Nefrite Intersticial/virologia , Parvovirus/isolamento & purificação , Parvovirus/patogenicidade , Animais , Austrália , Progressão da Doença , Feminino , Fibrose/patologia , Fibrose/virologia , Humanos , Rim/metabolismo , Rim/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nefrite Intersticial/fisiopatologia , América do Norte , Infecções por Parvoviridae/metabolismoRESUMO
Val-boroPro (Talabostat, PT-100), a nonselective inhibitor of post-proline cleaving serine proteases, stimulates mammalian immune systems through an unknown mechanism of action. Despite this lack of mechanistic understanding, Val-boroPro has attracted substantial interest as a potential anticancer agent, reaching phase 3 trials in humans. Here we show that Val-boroPro stimulates the immune system by triggering a proinflammatory form of cell death in monocytes and macrophages known as pyroptosis. We demonstrate that the inhibition of two serine proteases, DPP8 and DPP9, activates the pro-protein form of caspase-1 independent of the inflammasome adaptor ASC. Activated pro-caspase-1 does not efficiently process itself or IL-1ß but does cleave and activate gasdermin D to induce pyroptosis. Mice lacking caspase-1 do not show immune stimulation after treatment with Val-boroPro. Our data identify what is to our knowledge the first small molecule that induces pyroptosis and reveals a new checkpoint that controls the activation of the innate immune system.
Assuntos
Ácidos Borônicos/farmacologia , Caspase 1/metabolismo , Dipeptidases/antagonistas & inibidores , Dipeptídeos/farmacologia , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Leucócitos Mononucleares/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Piroptose/efeitos dos fármacos , Inibidores de Serina Proteinase/farmacologia , Animais , Ácidos Borônicos/química , Caspase 1/deficiência , Linhagem Celular , Dipeptidases/metabolismo , Dipeptídeos/química , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Relação Dose-Resposta a Droga , Humanos , Leucócitos Mononucleares/enzimologia , Leucócitos Mononucleares/patologia , Macrófagos/enzimologia , Macrófagos/patologia , Camundongos , Conformação Molecular , Inibidores de Serina Proteinase/química , Relação Estrutura-AtividadeRESUMO
The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met â« Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro â« Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Ácidos Borônicos/química , Complexos Multienzimáticos/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Oligopeptídeos/química , Biblioteca de Peptídeos , Inibidores de Serina Proteinase/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ácidos Borônicos/farmacologia , Relação Dose-Resposta a Droga , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mycobacterium tuberculosis/crescimento & desenvolvimento , Oligopeptídeos/farmacologia , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologiaRESUMO
The selectivity of an enzyme inhibitor is a key determinant of its usefulness as a tool compound or its safety as a drug. Yet selectivity is never assessed comprehensively in the early stages of the drug discovery process, and only rarely in the later stages, because technical limitations prohibit doing otherwise. Here, we report EnPlex, an efficient, high-throughput method for simultaneously assessing inhibitor potency and specificity, and pilot its application to 96 serine hydrolases. EnPlex analysis of widely used serine hydrolase inhibitors revealed numerous previously unrecognized off-target interactions, some of which may help to explain previously confounding adverse effects. In addition, EnPlex screening of a hydrolase-directed library of boronic acid- and nitrile-containing compounds provided structure-activity relationships in both potency and selectivity dimensions from which lead candidates could be more effectively prioritized. Follow-up of a series of dipeptidyl peptidase 4 inhibitors showed that EnPlex indeed predicted efficacy and safety in animal models. These results demonstrate the feasibility and value of high-throughput, superfamily-wide selectivity profiling and suggest that such profiling can be incorporated into the earliest stages of drug discovery.
Assuntos
Inibidores da Dipeptidil Peptidase IV/farmacologia , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Animais , Ácidos Borônicos/química , Ácidos Borônicos/farmacologia , Carbamatos/farmacologia , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Descoberta de Drogas , Feminino , Teste de Tolerância a Glucose , Glutamatos/farmacologia , Humanos , Lipopolissacarídeos/metabolismo , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Nitrilas/química , Oligopeptídeos/farmacologia , Piperazinas/farmacologia , Prolina/análogos & derivados , Prolina/farmacologia , Serina Proteases/metabolismo , Inibidores de Serina Proteinase/farmacologiaRESUMO
The overexpression of fibroblast activation protein-α (FAP) in solid cancers relative to levels in normal tissues has led to its recognition as a target for delivering agents directly to tumors. Radiolabeled quinoline-based FAP ligands have established clinical feasibility for tumor imaging, but their therapeutic potential is limited due to suboptimal tumor retention, which has prompted the search for alternative pharmacophores. One such pharmacophore is the boronic acid derivative N-(pyridine-4-carbonyl)-d-Ala-boroPro, a potent and selective FAP inhibitor (FAPI). In this study, the diagnostic and therapeutic (theranostic) potential of N-(pyridine-4-carbonyl)-d-Ala-boroPro-based metal-chelating DOTA-FAPIs was evaluated. Methods: Three DOTA-FAPIs, PNT6555, PNT6952, and PNT6522, were synthesized and characterized with respect to potency and selectivity toward soluble and cell membrane FAP; cellular uptake of the Lu-chelated analogs; biodistribution and pharmacokinetics in mice xenografted with human embryonic kidney cell-derived tumors expressing mouse FAP; the diagnostic potential of 68Ga-chelated DOTA-FAPIs by direct organ assay and small-animal PET; the antitumor activity of 177Lu-, 225Ac-, or 161Tb-chelated analogs using human embryonic kidney cell-derived tumors expressing mouse FAP; and the tumor-selective delivery of 177Lu-chelated DOTA-FAPIs via direct organ assay and SPECT. Results: DOTA-FAPIs and their natGa and natLu chelates exhibited potent inhibition of human and mouse sources of FAP and greatly reduced activity toward closely related prolyl endopeptidase and dipeptidyl peptidase 4. 68Ga-PNT6555 and 68Ga-PNT6952 showed rapid renal clearance and continuous accumulation in tumors, resulting in tumor-selective exposure at 60 min after administration. 177Lu-PNT6555 was distinguished from 177Lu-PNT6952 and 177Lu-PNT6522 by significantly higher tumor accumulation over 168 h. In therapeutic studies, all 3 177Lu-DOTA-FAPIs exhibited significant antitumor activity at well-tolerated doses, with 177Lu-PNT6555 producing the greatest tumor growth delay and animal survival. 225Ac-PNT6555 and 161Tb-PNT6555 were similarly efficacious, producing 80% and 100% survival at optimal doses, respectively. Conclusion: PNT6555 has potential for clinical translation as a theranostic agent in FAP-positive cancer.
Assuntos
Radioisótopos de Gálio , Tomografia por Emissão de Pósitrons , Humanos , Animais , Camundongos , Distribuição Tecidual , Linhagem Celular Tumoral , PiridinasRESUMO
The boroProline-based dipeptidyl boronic acids were among the first DPP-IV inhibitors identified, and remain the most potent known. We introduced various substitutions at the 4-position of the boroProline ring regioselectively and stereoselectively, and incorporated these aminoboronic acids into a series of 4-substituted boroPro-based dipeptides. Among these dipeptidyl boronic acids, Arg-(4S)-boroHyp (4q) was the most potent inhibitor of DPP-IV, DPP8 and DPP9, while (4S)-Hyp-(4R)-boroHyp (4o) exhibited the most selectivity for DPP-IV over DPP8 and DPP9.
Assuntos
Ácidos Borônicos/química , Ácidos Borônicos/farmacologia , Dipeptídeos/química , Dipeptídeos/farmacologia , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Prolina/química , Prolina/farmacologia , Ácidos Borônicos/síntese química , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/enzimologia , Dipeptídeos/síntese química , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Humanos , Concentração Inibidora 50 , Prolina/síntese químicaRESUMO
The mRNA expression of the dipeptidyl peptidase 4 (DPP4) gene family is highly upregulated in human hepatocellular carcinoma (HCC) and is associated with poor survival in HCC patients. Compounds that inhibit the DPP4 enzyme family, such as talabostat and ARI-4175, can mediate tumour regression by immune-mediated mechanisms that are believed to include NLRP1 activation. This study investigated the expression and activity of the DPP4 family during the development of HCC and evaluated the efficacy of ARI-4175 in the treatment of early HCC in mice. This first report on this enzyme family in HCC-bearing mice showed DPP9 upregulation in HCC, whereas intrahepatic DPP8/9 and DPP4 enzyme activity levels decreased with age. We demonstrated that ARI-4175 significantly lowered the total number of macroscopic liver nodules in these mice. In addition, ARI-4175 increased intrahepatic inflammatory cell infiltration, including CD8+ T cell numbers, into the HCC-bearing livers. Furthermore, ARI-4175 activated a critical component of the inflammasome pathway, caspase-1, in these HCC-bearing livers. This is the first evidence of caspase-1 activation by a pan-DPP inhibitor in the liver. Our data suggest that targeting the DPP4 enzyme family may be a novel and effective approach to promote anti-tumour immunity in HCC via caspase-1 activation.
RESUMO
OBJECTIVE: Fibroblast Activation Protein (FAP), an enzyme structurally related to dipeptidyl peptidase-4 (DPP-4), has garnered interest as a potential metabolic drug target due to its ability to cleave and inactivate FGF-21 as well as other peptide substrates. Here we investigated the metabolic importance of FAP for control of body weight and glucose homeostasis in regular chow-fed and high fat diet-fed mice. METHODS: FAP enzyme activity was transiently attenuated using a highly-specific inhibitor CPD60 and permanently ablated by genetic inactivation of the mouse Fap gene. We also assessed the FAP-dependence of CPD60 and talabostat (Val-boroPro), a chemical inhibitor reportedly targeting both FAP and dipeptidyl peptidase-4 RESULTS: CPD60 robustly inhibited plasma FAP activity with no effect on DPP-4 activity. Fap gene disruption was confirmed by assessment of genomic DNA, and loss of FAP enzyme activity in plasma and tissues. CPD60 did not improve lipid tolerance but modestly improved acute oral and intraperitoneal glucose tolerance in a FAP-dependent manner. Genetic inactivation of Fap did not improve glucose or lipid tolerance nor confer resistance to weight gain in male or female Fap-/- mice fed regular chow or high-fat diets. Moreover, talabostat markedly improved glucose homeostasis in a FAP- and FGF-21-independent, DPP-4 dependent manner. CONCLUSION: Although pharmacological FAP inhibition improves glucose tolerance, the absence of a metabolic phenotype in Fap-/-mice suggest that endogenous FAP is dispensable for the regulation of murine glucose homeostasis and body weight. These findings highlight the importance of characterizing the specificity and actions of FAP inhibitors in different species and raise important questions about the feasibility of mouse models for targeting FAP as a treatment for diabetes and related metabolic disorders.
Assuntos
Dipeptidil Peptidase 4/metabolismo , Gelatinases/metabolismo , Glucose/metabolismo , Proteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal/fisiologia , Diabetes Mellitus/tratamento farmacológico , Dieta Hiperlipídica , Dipeptidil Peptidase 4/sangue , Inibidores da Dipeptidil Peptidase IV/administração & dosagem , Endopeptidases , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Gelatinases/fisiologia , Peptídeo 1 Semelhante ao Glucagon/sangue , Homeostase/fisiologia , Insulina/metabolismo , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Serina Endopeptidases/fisiologia , Aumento de PesoRESUMO
We describe here the epimerization-free synthesis and characterization of a new class of conformationally constrained lactam aminoboronic acid inhibitors of dipeptidyl peptidase IV (DPP IV; E.C. 3.4.14.5). These compounds have the advantage that they cannot undergo the pH-dependent cyclization prevalent in most dipeptidyl boronic acids that attenuates their potency at physiological pH. For example, D-3-amino-1-[L-1-boronic-ethyl]-pyrrolidine-2-one (amino-D-lactam-L-boroAla), one of the best lactam inhibitors of DPP IV, is several orders of magnitude less potent than L-Ala-L-boroPro, as measured by Ki values (2.3 nM vs 30 pM, respectively). At physiological pH, however, it is actually more potent than L-Ala-L-boroPro, as measured by IC50 values (4.2 nM vs 1400 nM), owing to the absence of the potency-attenuating cyclization. In an interesting and at first sight surprising reversal of the relationship between stereochemistry and potency observed with the conformationally unrestrained Xaa-boroPro class of inhibitors, the L-L diastereomers of the lactams are orders of magnitude less effective than the D-L lactams. However, this interesting reversal and the unexpected potency of the D-L lactams as DPP IV inhibitors can be understood in structural terms, which is explained and discussed here.
Assuntos
Alanina/análogos & derivados , Alanina/síntese química , Ácidos Bóricos/síntese química , Ácidos Borônicos/síntese química , Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV , Lactamas/síntese química , Peptídeos/química , Pirrolidinonas/síntese química , Alanina/química , Biomimética , Ácidos Bóricos/química , Ácidos Borônicos/química , Humanos , Concentração de Íons de Hidrogênio , Lactamas/química , Modelos Moleculares , Pirrolidinonas/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting its enzymatic activity.
Assuntos
Antígenos de Neoplasias/fisiologia , Biomarcadores Tumorais/fisiologia , Fibroblastos/metabolismo , Neoplasias/enzimologia , Serina Endopeptidases/fisiologia , Alanina/química , Animais , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Sítios de Ligação , Biomarcadores Tumorais/metabolismo , Western Blotting , Domínio Catalítico , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , DNA Complementar/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Endopeptidases , Citometria de Fluxo , Gelatinases , Humanos , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Cinética , Proteínas de Membrana , Camundongos , Microscopia de Fluorescência , Modelos Moleculares , Transplante de Neoplasias , Serina/química , Serina Endopeptidases/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
FGF-21 is a key regulator of metabolism and potential drug candidate for the treatment of type II diabetes and other metabolic disorders. However, the half-life of active, circulating, human FGF-21 has recently been shown to be limited in mice and monkeys by a proteolytic cleavage between P171 and S172. Here, we show that fibroblast activation protein is the enzyme responsible for this proteolysis by demonstrating that purified FAP cleaves human FGF-21 at this site in vitro, and that an FAP-specific inhibitor, ARI-3099, blocks the activity in mouse, monkey and human plasma and prolongs the half-life of circulating human FGF-21 in mice. Mouse FGF-21, however, lacks the FAP cleavage site and is not cleaved by FAP. These findings indicate FAP may function in the regulation of metabolism and that FAP inhibitors may prove useful in the treatment of diabetes and metabolic disorders in humans, but pre-clinical proof of concept studies in rodents will be problematic.
Assuntos
Fatores de Crescimento de Fibroblastos , Gelatinases/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Serina Endopeptidases/metabolismo , Animais , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Endopeptidases , Fatores de Crescimento de Fibroblastos/farmacocinética , Fatores de Crescimento de Fibroblastos/farmacologia , Humanos , Macaca fascicularis , Camundongos , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologia , Especificidade da EspécieRESUMO
Sphingosine kinase 1 (SK1) is a signaling enzyme that catalyzes the formation of sphingosine-1-phosphate. Overexpression of SK1 is causally associated with breast cancer progression and resistance to therapy. SK1 inhibitors are currently being investigated as promising breast cancer therapies. Two major transcriptional isoforms, SK143 kDa and SK151 kDa, have been identified; however, the 51 kDa variant is predominant in breast cancer cells. No studies have investigated the protein-protein interactions of the 51 kDa isoform and whether the two SK1 isoforms differ significantly in their interactions. Seeking an understanding of the regulation and role of SK1, we used a triple-labeling stable isotope labeling by amino acids in cell culture-based approach to identify SK1-interacting proteins common and unique to both isoforms. Of approximately 850 quantified proteins in SK1 immunoprecipitates, a high-confidence list of 30 protein interactions with each SK1 isoform was generated via a meta-analysis of multiple experimental replicates. Many of the novel identified SK1 interaction partners such as supervillin, drebrin, and the myristoylated alanine-rich C-kinase substrate-related protein supported and highlighted previously implicated roles of SK1 in breast cancer cell migration, adhesion, and cytoskeletal remodeling. Of these interactions, several were found to be exclusive to the 43 kDa isoform of SK1, including the protein phosphatase 2A, a previously identified SK1-interacting protein. Other proteins such as allograft inflammatory factor 1-like protein, the latent-transforming growth factor ß-binding protein, and dipeptidyl peptidase 2 were found to associate exclusively with the 51 kDa isoform of SK1. In this report, we have identified common and isoform-specific SK1-interacting partners that provide insight into the molecular mechanisms that drive SK1-mediated oncogenicity.
Assuntos
Neoplasias da Mama/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Ligação ao Cálcio , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Feminino , Humanos , Proteínas de Ligação a TGF-beta Latente/metabolismo , Lisofosfolipídeos/metabolismo , Células MCF-7 , Proteínas dos Microfilamentos , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
Recent studies have suggested that pan inhibitors of dipeptidyl peptidase-4 activity and/or structure homologs (DASH), including ARI-4175, can mediate tumor regression by immune-mediated mechanisms. This study assessed the potential of combining ARI-4175 with cancer vaccines. We evaluated ARI-4175's effect on immunogenic modulation, ability to sensitize tumor cells to antigen-specific CTL killing, effect on immune-cell subsets and function, and antitumor activity in 2 tumor models, both as a monotherapy and in combination with a recombinant viral or dendritic cell (DC)-based tumor-cell vaccine. ARI-4175's effects on the growth, surface phenotype, and antigen-specific CTL-mediated lysis of murine and human carcinoma cell lines were assessed in vitro. In vivo, C57BL-6 mice were treated orally with ARI-4175, after which splenocytes were assessed by flow cytometry and functional assays. Antitumor studies were performed in murine models of colon carcinoma (MC38-CEA(+) in CEA-transgenic C57BL-6 mice) and rhabdomyosarcoma (M3-9-M in C57BL-6 mice). Mice received oral ARI-4175 alone or in combination with a vaccine consisting of recombinant vaccinia/fowlpox CEA-TRICOM (colon model) or a DC-based tumor-cell vaccine (rhabdomyosarcoma model). Exposure to ARI-4175 had no effect on the proliferation or viability of carcinoma cells in vitro; however, it did alter tumor phenotype, making murine and human tumor cells more sensitive to antigen-specific CTL killing. Assessment of immune-cell subsets and function indicated that ARI-4175 increased levels of natural killer cells and DCs. Detrimental immune effects, including reduced T effector cells and increased immunosuppressive cells (Tregs, MDSCs), were normalized when treatment stopped, suggesting that scheduling is critical when combining this agent with vaccine. As a monotherapy, ARI-4175 had potent antitumor activity in both tumor models, and had even greater effects when combined with a vaccine (either DC-based or poxviral vector based). These findings provide the rationale for the combined use of cancer immunotherapy with DASH enzyme inhibitors such as ARI-4175.
Assuntos
Adjuvantes Imunológicos/farmacologia , Compostos de Boro/farmacologia , Vacinas Anticâncer/imunologia , Dipeptídeos/farmacologia , Linfócitos T Citotóxicos/imunologia , Administração Oral , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/prevenção & controle , Inibidores da Dipeptidil Peptidase IV/farmacologia , Feminino , Humanos , Subpopulações de Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transplante de Neoplasias , Rabdomiossarcoma/prevenção & controleRESUMO
Fibroblast activation protein (FAP) is a serine protease selectively expressed on reactive stromal fibroblasts of epithelial carcinomas. It is widely believed to play a role in tumor invasion and metastasis and therefore to represent a potential new drug target for cancer. Investigation into its biological function, however, has been hampered by the current unavailability of selective inhibitors. The challenge has been in identifying inhibitors that are selective for FAP over both the dipeptidyl peptidases (DPPs), with which it shares exopeptidase specificity, and prolyl oligopeptidase (PREP), with which it shares endopeptidase specificity. Here, we report the first potent FAP inhibitor with selectivity over both the DPPs and PREP, N-(pyridine-4-carbonyl)-d-Ala-boroPro (ARI-3099, 6). We also report a similarly potent and selective PREP inhibitor, N-(pyridine-3-carbonyl)-Val-boroPro (ARI-3531, 22). Both are boronic acid based inhibitors, demonstrating that high selectivity can be achieved using this electrophile. The inhibitors are stable, easy to synthesize, and should prove to be useful in helping to elucidate the biological functions of these two unique and interesting enzymes, as well as their potential as drug targets.
Assuntos
Descoberta de Drogas , Gelatinases/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Sequência de Aminoácidos , Animais , Ligação Competitiva , Ácidos Borônicos/química , Ácidos Borônicos/metabolismo , Ácidos Borônicos/farmacologia , Endopeptidases , Gelatinases/química , Gelatinases/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Prolina/metabolismo , Prolil Oligopeptidases , Serina Endopeptidases/química , Inibidores de Serina Proteinase/metabolismo , Especificidade por SubstratoRESUMO
Multimodality therapy consisting of surgery, chemotherapy, and radiation will fail in approximately 40% of patients with pediatric sarcomas and result in substantial long-term morbidity in those who are cured. Immunotherapeutic regimens for the treatment of solid tumors typically generate antigen-specific responses too weak to overcome considerable tumor burden and tumor suppressive mechanisms and are in need of adjuvant assistance. Previous work suggests that inhibitors of DASH (dipeptidyl peptidase IV activity and/or structural homologs) enzymes can mediate tumor regression by immune-mediated mechanisms. Herein, we demonstrate that the DASH inhibitor, ARI-4175, can induce regression and eradication of well-established solid tumors, both as a single agent and as an adjuvant to a dendritic cell (DC) vaccine and adoptive cell therapy (ACT) in mice implanted with the M3-9-M rhabdomyosarcoma cell line. Treatment with effective doses of ARI-4175 correlated with recruitment of myeloid (CD11b) cells, particularly myeloid DCs, to secondary lymphoid tissues and with reduced frequency of intratumoral monocytic (CD11bLy6-CLy6-G) myeloid-derived suppressor cells. In immunocompetent mice, combining ARI-4175 with a DC vaccine or ACT with tumor-primed T cells produced significant improvements in tumor responses against well-established M3-9-M tumors. In M3-9-M-bearing immunodeficient (Rag1) mice, ACT combined with ARI-4175 produced greater tumor responses and significantly improved survival compared with either treatment alone. These studies warrant the clinical investigation of ARI-4175 for treatment of sarcomas and other malignancies, particularly as an adjuvant to tumor vaccines and ACT.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Compostos de Boro/uso terapêutico , Células Dendríticas/imunologia , Dipeptídeos/uso terapêutico , Inibidores da Dipeptidil Peptidase IV/administração & dosagem , Rabdomiossarcoma/terapia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Compostos de Boro/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Terapia Combinada , Células Dendríticas/transplante , Dipeptídeos/administração & dosagem , Inibidores da Dipeptidil Peptidase IV/efeitos adversos , Feminino , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Rabdomiossarcoma/imunologia , Linfócitos T/transplante , Carga Tumoral/efeitos dos fármacosRESUMO
Bioactive peptides have evolved to optimally fulfill specific biological functions, a fact which has long attracted attention for their use as therapeutic agents. While there have been some recent commercial successes fostered in part by advances in large-scale peptide synthesis, development of peptides as therapeutic agents has been significantly impeded by their inherent susceptibility to protease degradation in the bloodstream. Here we report that incorporation of specially designed amino acid analogues at the P1' position, directly C-terminal of the enzyme cleavage site, renders peptides, including glucagon-like peptide-1 (7-36) amide (GLP-1) and six other examples, highly resistant to serine protease degradation without significant alteration of their biological activity. We demonstrate the applicability of the method to a variety of proteases, including dipeptidyl peptidase IV (DPP IV), dipeptidyl peptidase 8 (DPP8), fibroblast activation protein α (FAPα), α-lytic protease (αLP), trypsin, and chymotrypsin. In summary, the "P1' modification" represents a simple, general, and highly adaptable method of generating enzymatically stable peptide-based therapeutics.
Assuntos
Dipeptidil Peptidase 4/metabolismo , Peptídeos/farmacologia , Inibidores de Serina Proteinase/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was â¼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.
RESUMO
OBJECTIVE: Synthetic class A amphipathic helical peptide analogs of apolipoprotein-AI (apoAI; with varied phenylalanine residues) are emerging therapeutic approaches under investigation for atherosclerosis. Utilizing retroinverso sequencing, we designed reverse-D4F (Rev-D4F) peptide with 18 d-amino acids containing 4 phenylalanine residues and reverse order that allows the side chain residues to be of exact alignment and superimposable to those of the parent l-amino acid peptide. This study examined the effect of Rev-D4F on atherosclerosis in apolipoprotein E (apoE)-null mice and the underlying mechanisms. MATERIALS/METHODS: ApoE-null mice were fed a chow diet and administered water (control), Rev-D4F, or L4F mimetic peptides (0.4 mg/mL, equivalent to 1.6 mg/d) orally in drinking water for 6 weeks. Aortic root atherosclerotic lesion area, lesion macrophage content, and the ability of plasma high-density lipoprotein (HDL) to influence monocyte chemotaxis were measured. RESULTS: Rev-D4F significantly decreased aortic sinus atherosclerotic lesion area and lesion macrophage content without affecting plasma total and HDL-cholesterol levels in apoE-null mice. The HDL from Rev-D4F-treated mice showed enhanced anti-inflammatory monocyte chemotactic activity, while low-density lipoprotein (LDL) exhibited reduced proinflammatory activity. In in vitro studies, Rev-D4F inhibited LDL oxidation, endothelial cell vascular cell adhesion molecule 1 (VCAM-1), and monocyte chemotactic factor 1 (MCP-1) expression, and monocyte adhesion to aortic endothelial cells. CONCLUSIONS: The Rev-D4F inhibits atherosclerosis by inhibiting endothelial inflammatory/oxidative events and improving HDL function. The data suggest that Rev-D4F may be an effective apoAI mimetic peptide for further development in preventing atherosclerosis.
Assuntos
Apolipoproteína A-I/farmacologia , Apolipoproteínas E/metabolismo , Aterosclerose/prevenção & controle , Peptídeos/farmacologia , Animais , Aorta/citologia , Apolipoproteínas E/genética , Peso Corporal/efeitos dos fármacos , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Técnicas de Cocultura , Células Endoteliais , Feminino , Humanos , Lipídeos/sangue , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos KnockoutRESUMO
Val-boroPro, 1, is a potent, but relatively nonspecific inhibitor of the prolyl peptidases. It has antihyperglycemic activity from inhibition of DPPIV but also striking anticancer activity and a toxicity for which the mechanisms are unknown. 1 cyclizes at physiological pH, which attenuates its inhibitory potency >100-fold, which is a "soft drug" effect. Here we show that this phenomenon can be exploited to create prodrugs with unique properties and potential for selective in vivo targeting. Enzyme-mediated release delivers 1 to the target in the active form at physiological pH; cyclization attenuates systemic pharmacological effects from subsequent diffusion. This "pro-soft" design is demonstrated with a construct activated by and targeted to DPPIV, including in vivo results showing improved antihyperglycemic activity and reduced toxicity relative to 1. Pro-soft derivatives of 1 can help to illuminate the mechanisms underlying the three biological activities, or to help localize 1 at a tumor and thereby lead to improved anticancer agents with reduced toxicity. The design concept can also be applied to a variety of other boronic acid inhibitors.
Assuntos
Ácidos Borônicos/farmacologia , Dipeptídeos/farmacologia , Inibidores da Dipeptidil Peptidase IV/farmacologia , Pró-Fármacos/metabolismo , Serina Proteases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Animais , Ácidos Borônicos/química , Ácidos Borônicos/toxicidade , Ciclização , Dipeptídeos/química , Dipeptídeos/toxicidade , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/química , Inibidores da Dipeptidil Peptidase IV/toxicidade , Masculino , Camundongos , Pró-Fármacos/toxicidade , Ratos , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/toxicidade , Especificidade por Substrato , Fatores de TempoRESUMO
Bortezomib, a dipeptidyl boronic acid and potent inhibitor of the 26S proteasome, is remarkably effective against multiple myeloma (MM) but not against solid tumors. Dose-limiting adverse effects from "on target" inhibition of the proteasome in normal cells and tissues appear to be a key obstacle. Achieving efficacy against solid tumors therefore is likely to require making the inhibitor more selective for tumor tissue over normal tissues. The simplest strategy that might provide such tissue specificity would be to employ a tumor specific protease to release an inhibitor from a larger, noninhibitory structure. However, such release would necessarily generate an inhibitor with a free N-terminal amino group, raising a key question: Can short peptide boronic acids with N-terminal amino groups have the requisite properties to serve as warheads in prodrugs? Here we show that dipeptides of boroLeu, the smallest plausible candidates for the task, can indeed be sufficiently potent, cell-penetrating, cytotoxic, and stable to degradation by cellular peptidases to serve in this capacity.