geldanamycin mg br Material and methods br Results br Discus

Material and methods
Results
Discussion Aminopeptidase N is a transmembrane protease present in a wide variety of human tissues and cell types (endothelial, epithelial, fibroblast, leukocyte). APN expression is dysregulated in inflammatory diseases as well as in solid and hematologic tumors [12]. Therefore, inhibitors of APN may be effective against cancer and inflammation [12], [31], [32]. Chelating zinc ions through a ZBG is the most common mode of action of neutral aminopeptidase inhibitors [13], [28], [29], [30], [33], [34], [35], [36], [37], [38]. Most of these inhibitors display a competitive inhibition mode of action. Here, we identified three non-competitive inhibitors (α > 1) of human and porcine neutral aminopeptidases; these ligands do not contain a ZBG in their 3D structure. The Ki values of these new compounds were similar to those reported for natural APN inhibitors [12], [29]. Molecular docking simulations predicted that the inhibitors bind to a site distinct from that occupied by the substrate in the human APN active site (Fig. 3). This is consistent with the non-competitive inhibition profile obtained in the kinetic assays. The analysis of hAPN:Inhibitor complexes showed that interactions span from S1 to S5′ sub-sites, without Zn ion coordination. This uncommon mechanism of inhibition is similar to that described for geldanamycin mg MMV666023, an inhibitor of the neutral M1 aminopeptidase of Plasmodium falciparum[39]. The newly identified inhibitors share with compound MMV666023 a high hydrophobicity and a lack of typical ZBG [39]. The lack of ZBG may diminish inhibitor interactions with other metalloenzymes, and may improve pharmacokinetics and oral bioavailability of APN inhibitors [39]. Additional experiments, including site-directed mutagenesis and crystallography studies, are needed to confirm the binding mode of the hAPN:Inhibitor complexes. To check the selectivity of the inhibitor we tested its activity over porcine APA. APN and APA form the same interactions with the main chain of the N-terminal residue (P1 residue) of their ligands, suggesting that the two enzymes share a common catalytic mechanism despite their modest sequence similarity [2], [40]. However, the S1 pocket that accommodates the P1 side chain of ligands differs markedly between APN and APA, consistent with the different substrate specificities of the two enzymes. Residues Phe-896 and Ala-351 of the S1 pocket of APN favor the accommodation of bulky hydrophobic side chains [2]. Conversely, the S1 pocket of APA is well suited to accommodate the side chains of acidic residues; the carboxylate side chain of bound glutamate forms a strong salt bridge with Arg-887 and a hydrogen bond with Thr-356. These energetically favorable interactions are consistent with high APA activity on substrates with a P1 glutamate [40]. Therefore, we did not expect selectivity of the identified inhibitors for APN over APA because we predicted these ligands target several conserved residues. Preliminary kinetics experiments confirmed that the new compounds also inhibited pAPA with IC50 values similar to those detected for pAPN. However, BTB11079, JFD00064, and BTB07018 displayed a competitive inhibition mode that differed from that vs pAPN. This may result from an accommodation of the inhibitors at the active site of pAPA that differs from the binding mode predicted for APN. Additional experiments should clarify the selectivity of the newly identified inhibitors against other aminopeptidases of the M1 family, and may identify dual inhibitions of biomedical relevance [13]. Based on the sequence and structural analyses, we predicted that targeting the APN residues Ala-351, Arg-442, Ala-474, Phe-896 and Asn-900 might be useful for improving the selectivity of the identified inhibitors. The inhibition of APN activity by two of the compounds in rat brain, kidney, or liver suggests their rapid absorption and wide bio-distribution. This finding is relevant due to the implication of APN in physio- and patho-physiological processes in these tissues [41], [42], [43], [44], [45], [46]. Many hydrophobic drugs reversibly bind to Site 1 of human serum albumin (HSA), an association that improves the pharmacokinetics properties of therapeutic compounds [47]. Since mammalian serum albumins share more than 70% of sequence identity and have similar functions [48], the effective delivery of JFD00064 and BTB07018 to rat tissues may have been facilitated by their reversible binding to rat serum albumin hydrophobic sites. Moreover, HSA delivery systems have been extensively studied for treating brain disease since HSA can pass through the blood brain barrier [48]; interestingly, compounds JFD00064 and BTB07018 inhibited APN activity in the brain of rats, indicating these molecules crossed the blood-brain barrier. Further experiments are needed to confirm the interaction of these ligands with serum albumin.