Cyclic tripeptide-based potent human SIRT7 inhibitors

In the current study, two cyclic tripeptides respectively harboring a thiourea-type and a carboxamide-type of catalytic mechanism-based sirtuin inhibitory warheads as the central residue were found to behave as potent (low μM level) inhibitors against the tRNA-activated human SIRT7 deacetylase activity. Despite exhibiting a potent pan-inhibition against the deacylase activities of the five tested human sirtuins (i.e. SIRT1/2/3/6/7), these two compounds represent the first examples of potent SIRT7 inhibitors ever identified thus far, and their identification could facilitate the future development of more potent and selective SIRT7 inhibitors.     

Determination of the pH dependence,substrate specificity,and turnovers of alternative substrates for human ornithine aminotransferase

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and occurs predominantly in patients with underlying chronic liver diseases. Over the past decade, human ornithine aminotransferase (hOAT), which is an enzyme that catalyzes the metabolic conversion of ornithine into an intermediate for proline or glutamate synthesis, has been found to be overexpressed in HCC cells. hOAT has since emerged as a promising target for novel anticancer therapies, especially for the ongoing rational design effort to discover mechanism-based inactivators (MBIs). Despite the significance of hOAT in human metabolism and its clinical potential as a drug target against HCC, there are significant knowledge deficits with regard to its catalytic mechanism and structural characteristics. Ongoing MBI design efforts require in-depth knowledge of the enzyme active site, in particular, pKa values of potential nucleophiles and residues necessary for the molecular recognition of ligands. Here, we conducted a study detailing the fundamental active-site properties of hOAT using stopped-flow spectrophotometry and X-ray crystallography. Our results quantitatively revealed the pH dependence of the multistep reaction mechanism and illuminated the roles of ornithine α-amino and δ-amino groups in substrate recognition and in facilitating catalytic turnover. These findings provided insights of the catalytic mechanism that could benefit the rational design of MBIs against hOAT. In addition, substrate recognition and turnover of several fragment-sized alternative substrates of hOATs, which could serve as structural templates for MBI design, were also elucidated.     

Engineering protonation conformation of l-aspartate-α-decarboxylase to relieve mechanism-based inactivation

Mechanism-based inactivation of l -aspartate-α-decarboxylase (PanD), which leads to irreversible modification of active site, is a major challenge in the efficient production of β-alanine from L -aspartic acid. In this study, a semi-rational strategy that combined conformational dynamics and structural alignment was applied to increase the catalytic stability of Bacillus subtilis PanD (BsPanD). Using site-saturation and C-terminal deletion, the variant Q5 (BsPanD^{I46V/I88M/K104S/I126*}) was generated. The catalytic half-life and the total turnover number (TTN) of Q5 were 3.48-fold and 2.52-fold higher, respectively, compared with that of the parent Q0. The reasons for the differences were the prolonged distance d1 between the phenolic group of Tyr58 and pyruvoyl group of Ser25 (4.9 Å in Q0 vs. 5.5 Å in Q5), an increased difficulty for incorrect protonation to occur, and the decreased flexibility of residues in regions A, B, and C, thereby enhancing the probability of correct protonation. Variant Q5, coupled with l -aspartase (AspA) in a 15-L bioreactor, generated a linear cascade system using fumaric acid as a substrate, yielding 118.6 g/L β-alanine with a product/catalyst (P/C) ratio of 5.9 g/g and a conversion > 99%. These results showed that reshaping the protonation conformation of PanD can efficiently relieve mechanism-based inactivation and boost catalytic stability.     

Development of a Target cell-Biologics-Effector cell (TBE) complex-based cell killing model to characterize target cell depletion by T cell redirecting bispecific agents

T-cell redirecting bispecific antibodies (bsAbs) or antibody-derived agents that combine tumor antigen recognition with CD3-mediated T cell recruitment are highly potent tumor-killing molecules. Despite the tremendous progress achieved in the last decade, development of such bsAbs still faces many challenges. This work aimed to develop a mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) modeling framework that can be used to assist the development of T-cell redirecting bsAbs. A Target cell-Biologics-Effector cell (TBE) complex-based cell killing model was developed using in vitro and in vivo data, which incorporates information on binding affinities of bsAbs to CD3 and target receptors, expression levels of CD3 and target receptors, concentrations of effector and target cells, as well as respective physiological parameters. This TBE model can simultaneously evaluate the effect of multiple system-specific and drug-specific factors on the T-cell redirecting bsAb exposure–response relationship on a physiological basis; it reasonably captured multiple reported in vitro cytotoxicity data, and successfully predicted the effect of some key factors on in vitro cytotoxicity assays and the efficacious dose of blinatumomab in humans. The mechanistic nature of this model uniquely positions it as a knowledge-based platform that can be readily expanded to guide target selection, drug design, candidate selection and clinical dosing regimen projection, and thus support the overall discovery and development of T-cell redirecting bsAbs.     

Reactions Between Dipeptidyl Peptidase Iv and Diacyl Hydroxylamines: Mechanistic Investigations

Abstract

Kinetics of inactivation of dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) by N-peptidyl-O-(4-nitrobenzoyl) hydroxylamines and their enzyme-catalyzed hydrolysis were followed using independent monitoring methods, all giving similar efficiency ratios of K_cat/K_Inact

Different temperature dependences of the DP IV-inactivation and enzyme-catalyzed hydrolysis provide evidence of independent rate determining steps for both reactions. Activation parameters of inactivation are similar to those of spontaneous decomposition of the compounds, suggesting a mechanistic relationship.

Investigation of DP IV-inactivation, DP IV-catalyzed hydrolysis of N-Ala-Pro-O-Bz(4-NO₂) and the decomposition of the suicide substrate in H₂O and D₂O gave solvent isotope effects of 4.65, 2.54 and 1.02, respectively. A proton inventory of the inactivation reaction indicates involvement of more than one proton in the formation or breakdown of its transition state. The linear proton inventory found for the hydrolytic reaction is consistent with one proton transition in the rate determining step and resembles the rate limiting deacylation of Ala-Pro-DP IV. The hypothetical reaction model now locates splitting in both reactions prior to formation of a covalent intermediate during the catalytic cycle.     

Histone deacetylases 1 and 2 regulate DNA replication and DNA repair: potential targets for genome stability-mechanism-based therapeutics for a subset of cancers

Histone deacetylases 1 and 2 (HDAC1,2) belong to the class I HDAC family, which are targeted by the FDA-approved small molecule HDAC inhibitors currently used in cancer therapy. HDAC1,2 are recruited to DNA break sites during DNA repair and to chromatin around forks during DNA replication. Cancer cells use DNA repair and DNA replication as survival mechanisms and to evade chemotherapy-induced cytotoxicity. Hence, it is vital to understand how HDAC1,2 function during the genome maintenance processes (DNA replication and DNA repair) in order to gain insights into the mode-of-action of HDAC inhibitors in cancer therapeutics. The first-in-class HDAC1,2-selective inhibitors and Hdac1,2 conditional knockout systems greatly facilitated dissecting the precise mechanisms by which HDAC1,2 control genome stability in normal and cancer cells. In this perspective, I summarize the findings on the mechanistic functions of class I HDACs, specifically, HDAC1,2 in genome maintenance, unanswered questions for future investigations and views on how this knowledge could be harnessed for better-targeted cancer therapeutics for a subset of cancers.     

Towards the development of novel antibiotics: synthesis and evaluation of a mechanism-based inhibitor of Kdo8P synthase

The design and two synthetic pathways to aminophosphonate 4 which mimics the ionic and steric properties of putative oxocarbenium intermediate 3 in the Kdo8P synthase-catalyzed reaction are reported. It was found that 4 is a slow-binding, most potent inhibitor of the enzyme yet tested, with a Ki value of 0.4 microM.     

氟代糖在糖苷酶研究中的应用

近年来,氟代糖应用于糖苷酶反应研究,显示出越来越重要的作用。氟代糖可以作为糖苷酶及其突变酶的水解底物研究酶学性质;氟代糖抑制剂可以标记糖苷酶催化中心,鉴定亲核体氨基酸。尤为重要的是,氟代糖可作为糖苷酶的糖基供体来合成糖类。糖苷酶突变后,可生成糖苷合成酶和硫代糖苷合成酶,可以用与正常底物构型相反的氟代糖作为糖基供体高效合成糖类,收率一般为60%~90%,有的可达100%。糖苷酶及其突变酶以氟代糖为底物高效合成糖类的研究,必将促进生物学、糖生物学和纳米生物材料的发展。     

Adenosine Analogues as Inhibitors or Inactivators of S-Adenosylhomocysteine Hydrolase from Bovine Pancreas

Abstract

The ability of some substrate-analogues to inhibit or to inactivate S-adenosylhomocysteine hydrolase (SAHase) purified from bovine pancreas was investigated. Our results confirm that 3-deazaarysteromicin (DZAry) is a more potent competitive inhibitor than 3-deazaadenosine (DZA), while nebularine (purine riboside), contrary to previous reports, showed an uncompetitive inhibition. Moreover, 2-chloroadenosine and 2′-deoxyadenosine were found to be irreversible inactivators of SAHase with increasing potency, respectively. K_i values found for these drugs were of the same order of magnitude as those reported for SAHases from other mammalian tissues. The SAHase substrate-analogues studied are believed to act as antineoplastic and/or antiviral agents. It is conceivable to postulate that their therapeutic effects could be, at least in part, attributable to inhibition or even to inactivation of SAHase which, in turn, causes a reduction in S-adenosylmethionine-dependent methylation reactions.