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.
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-1beta 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.
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.
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 approximately 20- 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.
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.
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.
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.
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.
