Valentine J. Klimkowski

Metabolic-intermediate complex formation with cytochrome P450: theoretical studies in elucidating the reaction pathway for the generation of reactive nitroso intermediate.

Mechanism‐based inhibition (MBI) of cytochrome P450 (CYP) can lead to drug–drug interactions and often to toxicity. Some aliphatic and aromatic amines can undergo biotransformation reactions to form reactive metabolites such as nitrosoalkanes, leading to MBI of CYPs. It has been proposed that the nitrosoalkanes coordinate with the heme iron, forming metabolic‐intermediate complex (MIC), resulting in the quasi‐irreversible inhibition of CYPs. Limited mechanistic details regarding the formation of reactive nitroso intermediate and its coordination with heme‐iron have been reported. A quantum chemical analysis was performed to elucidate potential reaction pathways for the generation of nitroso intermediate and the formation of MIC. Elucidation of the energy profile along the reaction path, identification of three‐dimensional structures of reactive intermediates and transition states, as well as charge and spin density analyses, were performed using the density functional B3LYP method. The study was performed using Cpd I [iron (IV‐oxo] heme porphine with SH− as the axial ligand) to represent the catalytic domain of CYP, simulating the biotransformation process. Three pathways: (i) N‐oxidation followed by proton shuttle, (ii) N‐oxidation followed by 1,2‐H shift, and (iii) H‐abstraction followed by rebound mechanism, were studied. It was observed that the proton shuttle pathway was more favorable over the whole reaction leading to reactive nitroso intermediate. This study revealed that the MIC formation from a primary amine is a favorable exothermic process, involving eight different steps and preferably takes place on the doublet spin surface of Cpd I. The rate‐determining step was identified to be the first N‐oxidation of primary amine.

Dual Exosite-binding Inhibitors of Insulin-degrading Enzyme Challenge Its Role as the Primary Mediator of Insulin Clearance in Vivo.

Background: Insulin-degrading enzyme (IDE) is the best characterized catabolic enzyme implicated in insulin proteolysis. Results: Newly discovered dual exosite IDE inhibitors do not significantly affect insulin action or clearance. Conclusion: IDE catabolism does not appear to be the primary mechanism of insulin clearance in vivo. Significance: These IDE inhibitors will enable broader investigation of IDE function. Insulin-degrading enzyme (IDE, insulysin) is the best characterized catabolic enzyme implicated in proteolysis of insulin. Recently, a peptide inhibitor of IDE has been shown to affect levels of insulin, amylin, and glucagon in vivo. However, IDE−/− mice display variable phenotypes relating to fasting plasma insulin levels, glucose tolerance, and insulin sensitivity depending on the cohort and age of animals. Here, we interrogated the importance of IDE-mediated catabolism on insulin clearance in vivo. Using a structure-based design, we linked two newly identified ligands binding at unique IDE exosites together to construct a potent series of novel inhibitors. These compounds do not interact with the catalytic zinc of the protease. Because one of these inhibitors (NTE-1) was determined to have pharmacokinetic properties sufficient to sustain plasma levels >50 times its IDE IC50 value, studies in rodents were conducted. In oral glucose tolerance tests with diet-induced obese mice, NTE-1 treatment improved the glucose excursion. Yet in insulin tolerance tests and euglycemic clamp experiments, NTE-1 did not enhance insulin action or increase plasma insulin levels. Importantly, IDE inhibition with NTE-1 did result in elevated plasma amylin levels, suggesting the in vivo role of IDE action on amylin may be more significant than an effect on insulin. Furthermore, using the inhibitors described in this report, we demonstrate that in HEK cells IDE has little impact on insulin clearance. In total, evidence from our studies supports a minimal role for IDE in insulin metabolism in vivo and suggests IDE may be more important in helping regulate amylin clearance.

The design of potent, selective, non-covalent, peptide thrombin inhibitors utilizing imidazole as a S1 binding element

Modeling of neutral or mildly basic functional groups in the S1 site of thrombin led to the targeting of imidazole as a S1 binding element and correctly predicted the optimal chain length for connecting this group with the S2 and S3 binding elements. Derivatives of 4-(3-aminopropyl)-imidazole can be selective inhibitors of thrombin demonstrating potent anticoagulant activity.

Identification of potent and selective hydantoin inhibitors of aggrecanase-1 and aggrecanase-2 that are efficacious in both chemical and surgical models of osteoarthritis.

A disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and ADAMTS-5 are zinc metalloproteases commonly referred to as aggrecanase-1 and aggrecanase-2, respectively. These enzymes are involved in the degradation of aggrecan, a key component of cartilage. Inhibitors of these enzymes could be potential osteoarthritis (OA) therapies. A series of hydantoin inhibitors of ADAMTS-4 and ADAMTS-5 were identified from a screening campaign and optimized through structure-based drug design to give hydantoin 13. Hydantoin 13 had excellent selectivity over other zinc metalloproteases such as TACE, MMP2, MMP3, MMP13, and MMP14. The compound also produced efficacy in both a chemically induced and surgical model of OA in rats.

Dibasic benzo[b]thiophene derivatives as a novel class of active site directed thrombin inhibitors: 2. Sidechain optimization and demonstration of in vivo efficacy.

Potent, subnanomolar thrombin inhibitors 4, 5, and 6 are developed through side chain optimization of novel, benzo[b]thiophene-based small organic entities 2 and 3 and through SAR additivity studies of the new structural elements identified. X-ray crystallographic studies of 4b-thrombin complex revealed a hydrophobic and an electrostatic interaction of these new elements with thrombin at the S2 and S3 binding sites. In vitro and in vivo pharmacological studies showed that 4, 5, and 6 are potent anticoagulants in human plasma with demonstrated antithrombotic efficacy in a rat model of thrombosis.