Sulphostin

The structure of sulphostin (1), a novel dipeptidyl peptidase IV (DPP-IV) inhibitor, is consisted of three key functional groups, including a characteristic amino(sulfoamino)phosphinyl group, on a piperidine ring. To examine the relationship between its structure and the inhibitory activity against DPP-IV, various analogues were synthesized and their activities were investigated. These results indicated that all of the functional groups on the piperidine ring were crucial to the DPP-IV inhibitory activity of sulphostin, and that the sulfonic acid group, which constructed the amino(sulfoamino)phosphinyl group, contributed to the stability of the compound. Moreover, those functional groups should be adjoined on the piperidine ring for the inhibitory activity. The size of the nitrogen-containing heterocyclic ring including piperidine appeared to scarcely affect the activity. In the present study, the sulfonic acid-deficient five-membered ring analogue 27a showed the strongest inhibitory activity (IC50=11 nM).

Scoring changes in enzyme or pathway performance by their effect on growth behavior is a widely applied strategy for identifying improved biocatalysts. While in directed evolution this strategy is powerful in removing non-functional catalysts in selections, measuring subtle differences in growth behavior remains difficult at high throughput, as it is difficult to focus metabolic control on only one or a few enzymatic steps over the entire process of growth-based discrimination. Here, we demonstrate successful miniaturization of a growth-based directed enzyme evolution process. For cultivation of library clones we employed optically clear gel-like microcarriers of nanoliter volume (NLRs) as reaction vessels and used fluorescence-assisted particle sorting to estimate the growth behavior of each of the gel-embedded clones in a highly parallelized fashion. We demonstrate that the growth behavior correlates with the desired improvements in enzyme performance and that we can fine-tune selection stringency by including an antimetabolite in the assay. As a model enzyme reaction, we improve the racemization of ornithine, a possible starting block for the large-scale synthesis of sulphostin, by a broad-spectrum amino acid racemase and confirm the discriminatory power by showing that even moderately improved enzyme variants can be readily identified.

Dipeptidyl peptidase 4 (DPP-4), a substrate-specific serine protease, has been validated as a promising drug target for the treatment of type 2 diabetes. DPP-4 inhibitors significantly lowered blood glucose levels in patients with type 2 diabetes without common body weight gain, hypoglycemia and gastrointestinal disturbance side effects. Therefore, DPP-4 inhibitors attracted more and more attention. In particular, non-peptidomimetic DPP-4 inhibitors have been a focus of research and development and made great progress in recent years, which resulted in the discovery of a wide variety of potent non-peptidomimetic DPP-4 inhibitors. Some of them, such as sitagliptin, alogliptin and linagliptin have already been used as marketed drugs, while others have been into clinical trials. Based on the core structural features of non-peptidomimetic DPP-4 inhibitors, seven types were classified in the article. For each type, we focused on the description of strategies for design and optimization, together with a discussion on concluded structure-activity relationships (SAR). In addition, the contribution of specific substituents to the inhibition of DPP-4 was summarized. Selectivity towards the inhibition of DPP-4 over dipeptidyl peptidase 8 (DPP-8) and dipeptidyl peptidase 9 (DPP-9) was also presented.