Positional Scanning Identifies the Molecular Determinants of a High Affinity Multi-Leucine Inhibitor for Furin and PACE4

The proprotein convertase family of enzymes includes seven endoproteases with significant redundancy in their cleavage activity. We previously described the peptide Ac-LLLLRVK-Amba that displays potent inhibitory effects on both PACE4 and prostate cancer cell lines proliferation. Herein, the molecular determinants for PACE4 and furin inhibition were investigated by positional scanning using peptide libraries that substituted its leucine core with each natural amino acid. We determined that the incorporation of basic amino acids led to analogues with improved inhibitory potency toward both enzymes, whereas negatively charged residues significantly reduced it. All the remaining amino acids were in general well tolerated, with the exemption of the P6 position. However, not all of the potent PACE4 inhibitors displayed antiproliferative activity. The best analogues were obtained by the incorporation of the Ile residue at the P5 and P6 positions. These substitutions led to inhibitors with increased PACE4 selectivity and potent antiproliferative effects

Table_1_A drink equals how many cigarettes? Equating mortality risks from alcohol and tobacco use in Canada.DOCX

ObjectiveTo quantify and communicate risk equivalencies for alcohol-and tobacco-attributable mortality by comparing per standard drinks consumed to per number of cigarettes smoked in Canada.MethodsAlcohol-and tobacco-attributable premature deaths (≤75 years of age) and years of life lost (YLL) were estimated using a lifetime risk modeling approach. Alcohol-attributable death statistics were obtained from the 2023 Canadian Guidance on Alcohol and Health data source. Tobacco-attributable death statistics were derived from the Mortality Population Risk Tool (MPoRT) model.ResultsThe risk of alcohol use on premature death and YLL increased non-linearly with the number of drinks consumed, while the risk for tobacco use on these two measures increased linearly with the number of cigarettes smoked. Males who consumed 5 drinks/day—a standard drink contains 13.45 grams of alcohol in Canada—had an equivalent risk as smoking 4.9 cigarettes/day (when modeling for premature death) and 5.1 cigarettes/day (when modeling for YLL). Females who consumed 5 drinks/day experienced an equivalent risk as smoking 4.2 cigarettes/day for premature deaths and YLL. At all levels of alcohol consumption females and males who consumed ConclusionRisk equivalencies comparing alcohol use to tobacco use could help people who drink improve their knowledge and understanding of the mortality risks associated with increased number of drinks consumed per day.</p

Design, Synthesis, and Structure–Activity Relationship Studies of a Potent PACE4 Inhibitor

PACE4 plays an important role in the progression of prostate cancer and is an attractive target for the development of novel inhibitor-based tumor therapies. We previously reported the design and synthesis of a novel, potent, and relatively selective PACE4 inhibitor known as a Multi-Leu (ML) peptide. In the present work, we examined the ML peptide through detailed structure–activity relationship studies. A variety of ML-peptide analogues modified at the P8–P5 positions with leucine isomers (Nle, DLeu, and DNle) or substituted at the P1 position with arginine mimetics were tested for their inhibitory activity, specificity, stability, and antiproliferative effect. By incorporating d isomers at the P8 position or a decarboxylated arginine mimetic, we obtained analogues with an improved stability profile and excellent antiproliferative properties. The DLeu or DNle residue also has improved specificity toward PACE4, whereas specificity was reduced for a peptide modified with the arginine mimetic, such as 4-amidinobenzylamide

The Multi-Leu Peptide Inhibitor Discriminates Between PACE4 and Furin And Exhibits Antiproliferative Effects On Prostate Cancer Cells

The proprotein convertases (PCs) play an important role in protein precursor activation through processing at paired basic residues. However, significant substrate cleavage redundancy has been reported between PCs. The question remains whether specific PC inhibitors can be designed. This study describes the identification of the sequence LLLLRVKR, named Multi-Leu (ML)-peptide, that displayed a 20-fold selectivity on PACE4 over furin, two enzymes with similar structural characteristics. We have previously demonstrated that PACE4 plays an important role in prostate cancer and could be a druggable target. The present study demonstrates that the ML-peptide significantly reduced the proliferation of DU145 and LNCaP prostate cancer-derived cell lines and induced G₀/G₁ cell cycle arrest. However, the ML-peptide must enter the cell to inhibit proliferation. It is concluded that peptide-based inhibitors can yield specific PC inhibitors and that the ML-peptide is an important lead compound that could potentially have applications in prostate cancer