Inhibitor Report for: Allo-Ile-isoindoline

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 discovery of the DP4-related enzymes DP8 and DP9 raised controversial discussion regarding the physiological and pathophysiological function of distinct members of the DP4 family. Particularly with regard to their potential relevance in regulating immune functions, it is of interest to know which role the subcellular distribution of the enzymes play. Synthetic substrates as well as low molecular weight inhibitors are widely used as tools, but little is yet known regarding their features in cell experiments, such as their plasma membrane penetration capacity. The fluorogenic substrates Gly-Pro-AMC or (Ala-Pro)(2)-R110 predominantly detect plasma membrane-bound activities of viable cells (less than 0.1% of fluorochromes R110 or AMC inside viable cells after 1 h incubation). Additionally, the selective and non-selective DP8/9 inhibitors allo-Ile-isoindoline and Lys[Z(NO(2))]-pyrrolidide were found to be incapable of passing the plasma membrane easily. This suggests that previously reported cellular effects are not due to inhibition of the cytosolic enzymes DP8 or DP9. Moreover, our enzymatic studies with viable cells provided evidence that DP8 and/or DP9 are also present on the surface of immune cells under certain circumstances and could gain relevance particularly in the absence of DP4 expression. In summary, in cells which do express DP4 on the surface, this archetypical member of the DP4 family is the most relevant peptidase in the regulation of cellular functions.

To obtain selective and potent inhibitors of dipeptidyl peptidases 8 and 9, we synthesized a series of substituted isoindolines as modified analogs of allo-Ile-isoindoline, the reference DPP8/9 inhibitor. The influence of phenyl substituents and different P2 residues on the inhibitors' affinity toward other DPPs and more specifically, their potential to discriminate between DPP8 and DPP9 will be discussed. Within this series compound 8j was shown to be a potent and selective inhibitor of DPP8/9 with low activity toward DPP II.

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 discovery of the DP4-related enzymes DP8 and DP9 raised controversial discussion regarding the physiological and pathophysiological function of distinct members of the DP4 family. Particularly with regard to their potential relevance in regulating immune functions, it is of interest to know which role the subcellular distribution of the enzymes play. Synthetic substrates as well as low molecular weight inhibitors are widely used as tools, but little is yet known regarding their features in cell experiments, such as their plasma membrane penetration capacity. The fluorogenic substrates Gly-Pro-AMC or (Ala-Pro)(2)-R110 predominantly detect plasma membrane-bound activities of viable cells (less than 0.1% of fluorochromes R110 or AMC inside viable cells after 1 h incubation). Additionally, the selective and non-selective DP8/9 inhibitors allo-Ile-isoindoline and Lys[Z(NO(2))]-pyrrolidide were found to be incapable of passing the plasma membrane easily. This suggests that previously reported cellular effects are not due to inhibition of the cytosolic enzymes DP8 or DP9. Moreover, our enzymatic studies with viable cells provided evidence that DP8 and/or DP9 are also present on the surface of immune cells under certain circumstances and could gain relevance particularly in the absence of DP4 expression. In summary, in cells which do express DP4 on the surface, this archetypical member of the DP4 family is the most relevant peptidase in the regulation of cellular functions.

To obtain selective and potent inhibitors of dipeptidyl peptidases 8 and 9, we synthesized a series of substituted isoindolines as modified analogs of allo-Ile-isoindoline, the reference DPP8/9 inhibitor. The influence of phenyl substituents and different P2 residues on the inhibitors' affinity toward other DPPs and more specifically, their potential to discriminate between DPP8 and DPP9 will be discussed. Within this series compound 8j was shown to be a potent and selective inhibitor of DPP8/9 with low activity toward DPP II.

Dipeptide derivatives bearing various P2 residues and pyrrolidine derivatives as P1 mimics were evaluated in order to identify lead structures for the development of DPP8 and DPP9 inhibitors. Structure-activity-relationship data obtained in this way led to the preparation of a series of alpha-aminoacyl ((2S, 4S)-4-azido-2-cyanopyrrolidines). These compounds were shown to be nanomolar DPP8/9 inhibitors with modest overall selectivity toward DPP IV and DPP II.

The proline-specific dipeptidyl peptidases (DPPs) are emerging as a protease family with important roles in the regulation of signaling by peptide hormones. Inhibitors of DPPs have an intriguing, therapeutic potential, with clinical efficacy seen in patients with diabetes. Until now, only recombinant forms of DPP8 and DPP9 have been characterized. Their enzymatic activities have not been demonstrated in or purified from any natural source. Using several selective DPP inhibitors, we show that DPP activity, attributable to DPP8/9 is present in human PBMC. All leukocyte types tested (lymphocytes, monocytes, Jurkat, and U937 cells) were shown to contain similar DPP8/9-specific activities, and DPPII- and DPPIV-specific activities varied considerably. The results were confirmed by DPPIV/CD26 immunocapture experiments. Subcellular fractionation localized the preponderance of DPP8/9 activity to the cytosol and DPPIV in the membrane fractions. Using Jurkat cell cytosol as a source, a 30-fold, enriched DPP preparation was obtained, which had enzymatic characteristics closely related to the ones of DPP8 and/or -9, including inhibition by allo-Ile-isoindoline and affinity for immobilized Lys-isoindoline.