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  • Dose Response Studies Against Puromycin Sensitive Aminopepti

    2024-06-14

    Dose–Response Studies Against Puromycin–Sensitive Aminopeptidase (PSA). PSA is a ubiquitous metallopeptidase encoded by the NPEPPS gene with subcellular distribution in the cytosol and nucleus [49]. Comprising of a 919 amino how to do molarity sequence, it has a broad substrate specificity, and is responsible for the release of N–terminal amino acids from a wide array of peptides, amides, and arylamides. The inhibition of PSA in a biochemical assay was studied and dose response curves obtained for all the analogs (Table 1, and Fig. 3). First, PSA was cloned from cDNA, and purified in the lab (see Experimental Section). Assays were performed at room temperature in a buffer of 25 mM Tris, pH 7.5. The reaction was started with the addition of alanine–4–methoxy–2–naphthylamide (Ala–4–MNA) substrate. The accumulation of 4–MNA was measured by exciting at 340 nm and reading the fluorescence at 425 nm at room temperature for 30 min. The effects of steric constraints in the structural manifold were explored. Compared to compound 8, the presence of extra aromatic rings in compounds 9 and 10 led to an enhancement of their potency in impeding PSA activity. Notably, the diphenylmethyl substituent (9) enhanced the anti–PSA activity to nanomolar levels. Further introduction of additional steric bulk to the molecular structure led to a decrease in potency. The trityl compound 10 exhibited a 204–fold loss in potency as compared to 9. These results suggest the requirement of an optimum size of steric bulk in this class of inhibitors of aminopeptidases. As compared to the L–isomeric analogs, the corresponding D–isomers of mono–, di–, and tri– phenyl ring containing derivatives (11, 12, and 13, respectively) did not display desirable anti–PSA potency. Among the non–nucleoside compounds, while bestatin (2) displayed IC50 value of 3.5 μM, tosedostat (3) inhibited PSA with submicromolar concentration. The synthesized non–nucleoside derivative 17 did not exhibit effective inhibition of PSA. Even though the comparison of the aminopropanol fragment in 17, with the corresponding 3′– to 5′–hydroxy fragment of the parent compound, is rendered tenuous due to the lack of structural constraints in 17, the diminished potency for this compound (53 μM) as compared to the corresponding nucleoside compound 9 (0.007 μM) strongly indicates that advantageous anti–aminopeptidase properties are induced by the presence of nucleosidic motif in these inhibitors of aminopeptidases. Anti–aminopeptidase Enzyme Studies Against Aminopeptidase N (APN). Apart from PSA, Aminopeptidase N (APN) is another enzyme relevant to this study. In reference to its subcellular distribution, APN is also known as membrane aminopeptidase. Encoded by the ANPEP gene, it exists as a homodimer that requires one Zn2+ cofactor per subunit. This broad–specificity aminopeptidase displays preference for the cleavage of neutral and basic amino acids from the N–terminal of the peptides. Dose–response curves were obtained for selected APN–active compounds (Fig. 4). Compound 9 displayed the best anti–APN effect at 10 μM (92.5%). Submicromolar activity was recorded for several compounds including 9 and 10. The lack of APN inhibition by non–nucleoside compound 17 underscored the relevance of the presence of the nucleoside motif for effective inhibition of aminopeptidases. In comparison with the inhibition of PSA, the synthesized derivatives were generally found to be less potent inhibitors of APN. Toxicity–profile Studies. Since the design rationale of synthesized molecules was based on impeding the action of aminopeptidases, without inducing toxicity towards normal cells, we sought to study the ability of synthesized derivatives in inhibiting protein synthesis. These analogs were tested for the inhibition of translation and post–transcriptional modification of GFP derived from the control plasmid included in the 1–Step Human In Vitro Protein Expression Kit from Thermo Scientific. The prepared analogs were tested at 25 μM with puromycin included as a negative control. Preincubation of the compounds at 30 °C for 30 min was followed by measuring the GFP production at the same temperature for 2 h by using a Molecular Devices SpectraMax i3 spectrophotometer with excitation at 482 nm, and recording emission at 518 nm. Notably, some of the most active compounds against PSA and/or APN (compounds 8 through 13) did not affect protein synthesis, affording better toxicity–profiles for these derivatives. Tosedostat (3) displayed 35% inhibition of protein synthesis at 25 μM concentration.