Novel internally quenched substrate of the trypsin-like subunit of 20S eukaryotic proteasome

This article describes the synthesis, using combinatorial chemistry, of internally quenched substrates of the trypsin-like subunit of human 20S proteasome. Such substrates were optimized in both the nonprime and prime regions of the peptide chain. Two were selected as the most susceptible for proteasomal proteolysis with excellent kinetic parameters: (i) ABZ-Val-Val-Ser-Arg-Ser-Leu-Gly-Tyr(3-NO2)-NH2 (kcat/KM = 934,000 M(-1) s(-1)) and (ii) ABZ-Val-Val-Ser-GNF-Ala-Met-Gly-Tyr(3-NO2)-NH2 (kcat/KM = 1,980,000 M(-1) s(-1)). Both compounds were efficiently hydrolyzed by the 20S proteasome at picomolar concentrations, demonstrating significant selectivity over other proteasome entities

Atomic resolution crystal structure of HV-BBI protease inhibitor from amphibian skin in complex with bovine trypsin

Protease inhibitors of the Bowman-Birk (BBI) family are commonly found in plants and animals where they play a protective role against invading pathogens. Here, we report an atomic resolution (1Å) crystal structure of a peptide inhibitor isolated from a skin secretion of a Chinese bamboo odorous frog Huia versabilis (HV-BBI) in complex with trypsin. HV-BBI shares significant similarities in sequence with a previously described inhibitor from a diskless-fingered odorous frog Odorrana graham (ORB). However, the latter is characterized by more than a 16,000 fold higher K$_i$ against trypsin than HV-BBI. Comparative analysis of trypsin cocrystal structures of HV-BBI and ORB and additionally that of Sunflower Trypsin Inhibitor (SFTI-1) together with accessory information on the affinities of inhibitor variants allowed us to pinpoint the inhibitor moiety responsible for the observed large difference in activity and also to define the extent of modifications permissible within the common protease-binding loop scaffold of BBI inhibitors. We suggest that modifications outside of the inhibitory loop permit the evolution of specificity toward different enzymes characterized by trypsin-like specificity. Proteins 2015; 83:582–589. © 2014 Wiley Periodicals, Inc

Design, Synthesis, and Antitumor Evaluation of an Opioid Growth Factor Bioconjugate Targeting Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) presents a formidable challenge with high lethality and limited effective drug treatments. Its heightened metastatic potential further complicates the prognosis. Owing to the significant toxicity of current chemotherapeutics, compounds like [Met5]-enkephalin, known as opioid growth factor (OGF), have emerged in oncology clinical trials. OGF, an endogenous peptide interacting with the OGF receptor (OGFr), plays a crucial role in inhibiting cell proliferation across various cancer types. This in vitro study explores the potential anticancer efficacy of a newly synthesized OGF bioconjugate in synergy with the classic chemotherapeutic agent, gemcitabine (OGF-Gem). The study delves into assessing the impact of the OGF-Gem conjugate on cell proliferation inhibition, cell cycle regulation, the induction of cellular senescence, and apoptosis. Furthermore, the antimetastatic potential of the OGF-Gem conjugate was demonstrated through evaluations using blood platelets and AsPC-1 cells with a light aggregometer. In summary, this article demonstrates the cytotoxic impact of the innovative OGF-Gem conjugate on pancreatic cancer cells in both 2D and 3D models. We highlight the potential of both the OGF-Gem conjugate and OGF alone in effectively inhibiting the ex vivo pancreatic tumor cell-induced platelet aggregation (TCIPA) process, a phenomenon not observed with Gem alone. Furthermore, the confirmed hemocompatibility of OGF-Gem with platelets reinforces its promising potential. We anticipate that this conjugation strategy will open avenues for the development of potent anticancer agents