Serine-Carboxyl Peptidases, Sedolisins: From Discovery to Evolution.

Sedolisin is a proteolytic enzyme, listed in the peptidase database MEROPS as a founding member of clan SB, family S53. This enzyme, although active at low pH, was originally shown not to be inhibited by an aspartic peptidase specific inhibitor, S-PI (pepstatin Ac). In this Perspective, the S53 family is described from the moment of original identification to evolution. The representative enzymes of the family are sedolisin, kumamolisin, and TPP-1. They exhibit the following unique features. (1) The fold of the molecule is similar to that of subtilisin, but the catalytic residues consist of a triad, Ser/Glu/Asp, that is unlike the Ser/His/Asp triad of subtilisin. (2) The molecule is expressed as a pro-form composed of the amino-terminal prosegment and the active domain. Additionally, some members of this family have an additional, carboxy-terminal prosegment. (3) Their optimum pH for activity is in the acidic region, not in the neutral to alkaline region where subtilisin is active. (4) Their distribution in nature is very broad across the three kingdoms of life. (5) Some of these enzymes from fungi and bacteria are pathogens to plants. (6) Some of them have significant potential applications for industry. (7) The lack of a TPP-1 gene in human brain is the cause of incurable juvenile neuronal ceroid lipofuscinosis (Batten's disease).

Molecular Docking-Based Screening for Novel Inhibitors of the Human Immunodeficiency Virus Type 1 Protease that Effectively Reduce the Viral Replication in Human Cells.

Therapeutic pressure by protease inhibitors (PIs) contributes to accumulation of mutations in the HIV type 1 (HIV-1) protease (PR) leading to development of drug resistance with subsequent therapy failure. Current PIs target the active site of PR in a competitive manner. Identification of molecules that exploit non-active site mechanisms of inhibition is essential to overcome resistance to current PIs. Potential non-active site HIV-1 protease (PR) inhibitors (PI) were identified by in silico screening of almost 140,000 molecules targeting the hinge region of PR. Inhibitory activity of best docking compounds was tested in an in vitro PR inhibition biochemical assay. Five compounds inhibited PR from multiple HIV-1 sub-types in vitro and reduced replicative capacity by PI-sensitive or multi-PI resistant HIV-1 variants in human cells ex vivo. Antiviral activity was boosted when combined with Ritonavir, potentially diminishing development of drug resistance, while providing effective treatment for drug resistant HIV-1 variants.

Fast-Acting Small Molecules Targeting Malarial Aspartyl Proteases, Plasmepsins, Inhibit Malaria Infection at Multiple Life Stages.

The eradication of malaria remains challenging due to the complex life cycle of Plasmodium and the rapid emergence of drug-resistant forms of Plasmodium falciparum and Plasmodium vivax. New, effective, and inexpensive antimalarials against multiple life stages of the parasite are urgently needed to combat the spread of malaria. Here, we synthesized a set of novel hydroxyethylamines and investigated their activities in vitro and in vivo. All of the compounds tested had an inhibitory effect on the blood stage of P. falciparum at submicromolar concentrations, with the best showing 50% inhibitory concentrations (IC50) of around 500 nM against drug-resistant P. falciparum parasites. These compounds showed inhibitory actions against plasmepsins, a family of malarial aspartyl proteases, and exhibited a marked killing effect on blood stage Plasmodium. In chloroquine-resistant Plasmodium berghei and P. berghei ANKA infected mouse models, treating mice with both compounds led to a significant decrease in blood parasite load. Importantly, two of the compounds displayed an inhibitory effect on the gametocyte stages (III-V) of P. falciparum in culture and the liver-stage infection of P. berghei both in in vitro and in vivo. Altogether, our findings suggest that fast-acting hydroxyethylamine-phthalimide analogs targeting multiple life stages of the parasite could be a valuable chemical lead for the development of novel antimalarial drugs.

Antiplasmodial activity of hydroxyethylamine analogs: Synthesis, biological activity and structure activity relationship of plasmepsin inhibitors.

Malaria, particularly in endemic countries remains a threat to the human health and is the leading the cause of mortality in the tropical and sub-tropical areas. Herein, we explored new C2 symmetric hydroxyethylamine analogs as the potential inhibitors of Plasmodium falciparum (P. falciparum; 3D7) in in-vitro cultures. All the listed compounds were also evaluated against crucial drug targets, plasmepsin II (Plm II) and IV (Plm IV), enzymes found in the digestive vacuole of the P. falciparum. Analog 10f showed inhibitory activities against both the enzymes Plm II and Plm IV (Ki, 1.93 ±â€¯0.29 µM for Plm II; Ki, 1.99 ±â€¯0.05 µM for Plm IV). Among all these analogs, compounds 10g selectively inhibited the activity of Plm IV (Ki, 0.84 ±â€¯0.08 µM). In the in vitro screening assay, the growth inhibition of P. falciparum by both the analogs (IC50, 2.27 ±â€¯0.95 µM for 10f; IC50, 3.11 ±â€¯0.65 µM for 10g) displayed marked killing effect. A significant growth inhibition of the P. falciparum was displayed by analog 12c with IC50 value of 1.35 ±â€¯0.85 µM, however, it did not show inhibitory activity against either Plms. The hemolytic assay suggested that the active compounds selectively inhibit the growth of the parasite. Further, potent analogs (10f and 12c) were evaluated for their cytotoxicity towards mammalian HepG2 and vero cells. The selectivity index (SI) values were noticed greater than 10 for both the analogs that suggested their poor toxicity. The present study indicates these analogs as putative lead structures and could serve as crucial for the development of new drug molecules.

Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c).

The Mycobacterium tuberculosis (Mtb) serine protease Hip1 (hydrolase important for pathogenesis; Rv2224c) promotes tuberculosis (TB) pathogenesis by impairing host immune responses through proteolysis of a protein substrate, Mtb GroEL2. The cell surface localization of Hip1 and its immunomodulatory functions make Hip1 a good drug target for new adjunctive immune therapies for TB. Here, we report the crystal structure of Hip1 to a resolution of 2.6 Å and the kinetic studies of the enzyme against model substrates and the protein GroEL2. The structure shows a two-domain protein, one of which contains the catalytic residues that are the signature of a serine protease. Surprisingly, a threonine is located within the active site close enough to hydrogen bond with the catalytic residues Asp463 and His490. Mutation of this residue, Thr466, to alanine established its importance for function. Our studies provide insights into the structure of a member of a novel family of proteases. Knowledge of the Hip1 structure will aid in designing inhibitors that could block Hip1 activity.

Exploring the scope of new arylamino alcohol derivatives: Synthesis, antimalarial evaluation, toxicological studies, and target exploration.

Synthesis of new 1-aryl-3-substituted propanol derivatives followed by structure-activity relationship, in silico drug-likeness, cytotoxicity, genotoxicity, in silico metabolism, in silico pharmacophore modeling, and in vivo studies led to the identification of compounds 22 and 23 with significant in vitro antiplasmodial activity against drug sensitive (D6 IC50 ≤ 0.19 µM) and multidrug resistant (FCR-3 IC50 ≤ 0.40 µM and C235 IC50 ≤ 0.28 µM) strains of Plasmodium falciparum. Adequate selectivity index and absence of genotoxicity was also observed. Notably, compound 22 displays excellent parasitemia reduction (98 ± 1%), and complete cure with all treated mice surviving through the entire period with no signs of toxicity. One important factor is the agreement between in vitro potency and in vivo studies. Target exploration was performed; this chemotype series exhibits an alternative antimalarial mechanism.

Effects of Hinge-region Natural Polymorphisms on Human Immunodeficiency Virus-Type 1 Protease Structure, Dynamics, and Drug Pressure Evolution.

Multidrug resistance to current Food and Drug Administration-approved HIV-1 protease (PR) inhibitors drives the need to understand the fundamental mechanisms of how drug pressure-selected mutations, which are oftentimes natural polymorphisms, elicit their effect on enzyme function and resistance. Here, the impacts of the hinge-region natural polymorphism at residue 35, glutamate to aspartate (E35D), alone and in conjunction with residue 57, arginine to lysine (R57K), are characterized with the goal of understanding how altered salt bridge interactions between the hinge and flap regions are associated with changes in structure, motional dynamics, conformational sampling, kinetic parameters, and inhibitor affinity. The combined results reveal that the single E35D substitution leads to diminished salt bridge interactions between residues 35 and 57 and gives rise to the stabilization of open-like conformational states with overall increased backbone dynamics. In HIV-1 PR constructs where sites 35 and 57 are both mutated (e.g. E35D and R57K), x-ray structures reveal an altered network of interactions that replace the salt bridge thus stabilizing the structural integrity between the flap and hinge regions. Despite the altered conformational sampling and dynamics when the salt bridge is disrupted, enzyme kinetic parameters and inhibition constants are similar to those obtained for subtype B PR. Results demonstrate that these hinge-region natural polymorphisms, which may arise as drug pressure secondary mutations, alter protein dynamics and the conformational landscape, which are important thermodynamic parameters to consider for development of inhibitors that target for non-subtype B PR.

Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei.

The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria. Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites. Plasmepsins residing in the food vacuole (FV) of the parasite hydrolyze hemoglobin in red blood cells. In this study, we cloned PbPM4, the FV plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature enzyme from genomic DNA. We over-expressed this PbPM4 zymogen as inclusion bodies (IB) in Escherichia coli, and purified the protein following in vitro IB refolding. Auto-maturation of the PbPM4 zymogen to mature enzyme was carried out at pH 4.5, 5.0, and 5.5. Interestingly, we found that the PbPM4 zymogen exhibited catalytic activity regardless of the presence of the pro-segment. We determined the optimal catalytic conditions for PbPM4 and studied enzyme kinetics on substrates and inhibitors of aspartic proteinases. Using combinatorial chemistry-based peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1', S2' and S3'. Based on these results, we designed and synthesized a selective peptidomimetic compound and tested its inhibition of PbPM4, seven FV plasmepsins from human malaria parasites, and human cathepsin D (hcatD). We showed that this compound exhibited a >10-fold selectivity to PbPM4 and human malaria parasite plasmepsin 4 orthologs versus hcatD. Data from this study furthesr our understanding of enzymatic characteristics of the plasmepsin family and provides leads for anti-malarial drug design.

Hydroxyethylamine Based Phthalimides as New Class of Plasmepsin Hits: Design, Synthesis and Antimalarial Evaluation.

A novel class of phthalimides functionalized with privileged scaffolds was designed, synthesized and evaluated as potential inhibitors of plasmepsin 2 (Ki: 0.99 ± 0.1 µM for 6u) and plasmepsin 4 (Ki: 3.3 ± 0.3 µM for 6t), enzymes found in the digestive vacuole of the plasmodium parasite and considered as crucial drug targets. Three compounds were identified as potential candidates for further development. The listed compounds were also assayed for their antimalarial efficacy against chloroquine (CQ) sensitive strain (3D7) of Plasmodium falciparum. Assay of twenty seven hydroxyethylamine derivatives revealed four (5e, 6j, 6o and 6s) as strongly active, which were further evaluated against CQ resistant strain (7GB) of P. falciparum. Compound 5e possessing the piperidinopiperidine moiety exhibited promising antimalarial activity with an IC50 of 1.16 ± 0.04 µM. Further, compounds 5e, 6j, 6o and 6s exhibited low cytotoxic effect on MCF-7 cell line. Compound 6s possessing C2 symmetry was identified as the least cytotoxic with significant antimalarial activity (IC50: 1.30 ± 0.03 µM). The combined presence of hydroxyethylamine and cyclic amines (piperazines and piperidines) was observed as crucial for the activity. The current studies suggest that hydroxyethylamine based molecules act as potent antimalarial agent and may be helpful in drug development.