RESUMEN
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.
Asunto(s)
Radioisótopos de Galio , Tomografía de Emisión de Positrones , Humanos , Animales , Ratones , Distribución Tisular , Línea Celular Tumoral , PiridinasRESUMEN
Val-boroPro (PT-100, Talabostat) induces powerful anti-tumor immune responses in syngeneic cancer models, but its mechanism of action has not yet been established. Val-boroPro is a non-selective inhibitor of post-proline-cleaving serine proteases, and the inhibition of the highly related cytosolic serine proteases Dpp8 and Dpp9 (Dpp8/9) by Val-boroPro was recently demonstrated to trigger an immunostimulatory form of programmed cell death known as pyroptosis selectively in monocytes and macrophages. Here we show that Dpp8/9 inhibition activates the inflammasome sensor protein Nlrp1b, which in turn activates pro-caspase-1 to mediate pyroptosis. This work reveals a previously unrecognized mechanism for activating an innate immune pattern recognition receptor and suggests that Dpp8/9 serve as an intracellular checkpoint to restrain Nlrp1b and the innate immune system.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Dipeptidasas/metabolismo , Inflamasomas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/química , Ácidos Borónicos/química , Ácidos Borónicos/metabolismo , Ácidos Borónicos/farmacología , Caspasa 1/metabolismo , Dipeptidasas/antagonistas & inhibidores , Dipéptidos/química , Dipéptidos/metabolismo , Dipéptidos/farmacología , Femenino , Células HEK293 , Humanos , Inmunidad Innata/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Piroptosis/efectos de los fármacos , Células RAW 264.7RESUMEN
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.
Asunto(s)
Ácidos Borónicos/farmacología , Caspasa 1/metabolismo , Dipeptidasas/antagonistas & inhibidores , Dipéptidos/farmacología , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/antagonistas & inhibidores , Leucocitos Mononucleares/efectos de los fármacos , Macrófagos/efectos de los fármacos , Piroptosis/efectos de los fármacos , Inhibidores de Serina Proteinasa/farmacología , Animales , Ácidos Borónicos/química , Caspasa 1/deficiencia , Línea Celular , Dipeptidasas/metabolismo , Dipéptidos/química , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Leucocitos Mononucleares/enzimología , Leucocitos Mononucleares/patología , Macrófagos/enzimología , Macrófagos/patología , Ratones , Conformación Molecular , Inhibidores de Serina Proteinasa/química , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Animales , Ácidos Borónicos/química , Ácidos Borónicos/farmacología , Carbamatos/farmacología , Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Descubrimiento de Drogas , Femenino , Prueba de Tolerancia a la Glucosa , Glutamatos/farmacología , Humanos , Lipopolisacáridos/metabolismo , Macaca fascicularis , Masculino , Ratones Endogámicos C57BL , Nitrilos/química , Oligopéptidos/farmacología , Piperazinas/farmacología , Prolina/análogos & derivados , Prolina/farmacología , Serina Proteasas/metabolismo , Inhibidores de Serina Proteinasa/farmacologíaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Descubrimiento de Drogas , Gelatinasas/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Secuencia de Aminoácidos , Animales , Unión Competitiva , Ácidos Borónicos/química , Ácidos Borónicos/metabolismo , Ácidos Borónicos/farmacología , Endopeptidasas , Gelatinasas/química , Gelatinasas/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Prolina/metabolismo , Prolil Oligopeptidasas , Serina Endopeptidasas/química , Inhibidores de Serina Proteinasa/metabolismo , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Ácidos Borónicos/química , Ácidos Borónicos/farmacología , Dipéptidos/química , Dipéptidos/farmacología , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/antagonistas & inhibidores , Prolina/química , Prolina/farmacología , Ácidos Borónicos/síntesis química , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/enzimología , Dipéptidos/síntesis química , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Humanos , Concentración 50 Inhibidora , Prolina/síntesis químicaRESUMEN
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.
Asunto(s)
Antineoplásicos/síntesis química , Ácidos Borónicos/síntesis química , Dipéptidos/síntesis química , Profármacos/síntesis química , Inhibidores de Proteasoma , Aminopeptidasas/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Ácidos Borónicos/química , Ácidos Borónicos/farmacología , Línea Celular , Línea Celular Tumoral , Ciclización , Dipéptidos/química , Dipéptidos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Profármacos/química , Profármacos/farmacología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Ácidos Borónicos/farmacología , Dipéptidos/farmacología , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Profármacos/metabolismo , Serina Proteasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Animales , Ácidos Borónicos/química , Ácidos Borónicos/toxicidad , Ciclización , Dipéptidos/química , Dipéptidos/toxicidad , Dipeptidil Peptidasa 4/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV/química , Inhibidores de la Dipeptidil-Peptidasa IV/toxicidad , Masculino , Ratones , Profármacos/toxicidad , Ratas , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/toxicidad , Especificidad por Sustrato , Factores de TiempoRESUMEN
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.