Mycobacterium tuberculosis (Mtb), the bacterium responsible for tuberculosis, is the leading cause of death due to a single infectious agent. Given the alarming increase in drug-resistant cases, therapeutic agents targeting novel Mtb drug targets are urgently needed. Hip1, a serine protease required for Mtb survival in macrophages and tolerance to various antibiotics, has been identified as an attractive therapeutic target. In the current study, we describe the design and synthesis of highly potent (pM range Ki) peptidomimetic α-keto ester inhibitors of Hip1. We also report the first two X-ray cocrystal structures of Hip1 bound to these compounds and describe the binding interactions in the active site of recombinant Hip1. Finally, we show that these compounds effectively reduce the intracellular bacillary burden in a macrophage model of Mtb infection.

The 2.6 Å crystal structure of the apo form of Hip1 (hydrolase important for pathogenesis) has been previously reported. However, very little is known about the active site architecture of this M. tuberculosis (Mtb), serine hydrolase drug target. To begin mapping the active site of Hip1, we cocrystallized Hip1 with the irreversible serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF). We chose AEBSF for cocrystallization with Hip1 since the similar inhibitor, phenylmethylsulfonyl fluoride (PMSF), interestingly exhibited no activity against Hip1. We obtained crystals that diffracted to 2.1 Å but to our bewilderment, we did not observe any electron density for the inhibitor in the omit map for the Hip1-AEBSF complex. Rather, in the active site, dehydroalanine (dAla) was found to occupy the expected position of the catalytic Ser228, thus yielding anhydrohip1. Here we present a comparative analysis of the crystal structures of anhydrohip1 and Hip1 and provide a mechanism for the conversion of the enzyme to the anhydro-form through reaction with AEBSF. With the aid of molecular docking, we propose an explanation for the differential inhibition of Hip1 by AEBSF and PMSF. We also present a preliminary definition of the S1 and S2 pockets of the protease's active site and propose a mechanism for a ligand-induced conformational change within the S2 pocket. Finally, we expand upon the previous demarcation of the putative lipid binding pocket in the α-domain of the enzyme. We believe that this detailed analysis of the structures of anhydrohip1 and Hip1 provides valuable information useful for the structure-based drug design of novel Hip1-directed Mtb therapeutics.

Crystal structures of the serine-carboxyl proteinase from Pseudomonas sp. 101 (PSCP), complexed with a number of inhibitors, have been solved and refined at high- to atomic-level resolution. All of these inhibitors (tyrostatin, pseudo-tyrostatin, AcIPF, AcIAF, and chymostatin, as well as previously studied iodotyrostatin and pseudo-iodotyrostatin) make covalent bonds to the active site Ser287 through their aldehyde moieties, while their side chains occupy subsites S1-S4 of the enzyme. The mode of binding of the inhibitors is almost identical for their P1 and P2 side chains, while significant differences are observed for P3 and P4 (if present). Kinetic parameters for the binding of these nanomolar inhibitors to PSCP have been established and correlated with the observed mode of binding. The preferences of this enzyme for a larger side chain in P2 as well as Tyr or Phe in P1 are explained by the size, shape, and characteristics of the S2 and S1 regions of the protein structure, respectively. Networks of hydrogen bonds involving glutamic and aspartic acids have been analyzed for the atomic-resolution structure of the native enzyme. PSCP contains a calcium-binding site that consists of Asp328, Asp348, three amide carbonyl groups, and a water molecule, in almost perfect octahedral coordination. The presence of Ca(2+) cation is necessary for the activity of the enzyme.

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.

HIV-1 protease (PR) serves as an important drug target because its inhibition generates immature non-infectious virions. However, drug-pressure selected mutations, along with natural polymorphisms, can lead to drug resistance to HIV-1 protease inhibitors (PIs), which change the drug binding configuration and equilibrium protease conformational sampling. As was suggested in our previous study, (1) a new protease conformation, curled-open, was identified in constructs carrying natural polymorphisms including E35D, and consequently, these constructs displayed higher protein backbone dynamics compared with wild-type subtype B construct characterized by nuclear magnetic resonance (NMR) spectroscopy. In the present work, we discuss the impact of the single E35D natural polymorphism on generating a minor protease conformation (curled-open), and consequently, influencing the protein structure and dynamics. The combined results from Double electron-electron resonance (DEER), NMR spectroscopy and X-ray crystallography structures provide a possible structural mechanism for altered conformational dynamics. References: 1. Huang, X., M. D. Britto, J. L. Kear, B. D. Christopher, J. R. Rocca, C. Simmerling, R. McKenna, M. Bieri, P. R. Gooley, B. M. Dunn, and G. E. Fanucci. 2014. The role of select subtype polymorphisms on HIV-1 protease conformational sampling and dynamics. J Biol Chem 289:17203 - 17214.

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.

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.

Three series of short peptide-based compounds were synthesized, which upon evaluation against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum in vitro, produced IC50 values ranging between 1.4-4.7 mu g mL(-1). Importantly, higher antimalarial activity against the drug-resistant strain of P. falciparum (W2) was observed for the tested peptides, indicating their potential in the treatment of drug-resistant malaria parasites. The lack of cytotoxicity in all tested peptides provides evidence of their safety profile. The selected peptides were evaluated in an enzymatic inhibitory assay against plasmepsin II, a potential target for antiplasmodial activity, also indicated from the results of the molecular docking studies.

HIV drug resistance continues to emerge; consequently, there is an urgent need to develop next generation antiretroviral therapeutics.1 Here we report on the structural and kinetic effects of an HIV protease drug resistant variant with the double mutations Gly48Thr and Leu89Met (PRG48T/L89M), without the stabilizing mutations Gln7Lys, Leu33Ile, and Leu63Ile. Kinetic analyses reveal that PRG48T/L89M and PRWT share nearly identical Michaelis-Menten parameters; however, PRG48T/L89M exhibits weaker binding for IDV (41-fold), SQV (18-fold), APV (15-fold), and NFV (9-fold) relative to PRWT. A 1.9 Å resolution crystal structure was solved for PRG48T/L89M bound with saquinavir (PRG48T/L89M-SQV) and compared to the crystal structure of PRWT bound with saquinavir (PRWT-SQV). PRG48T/L89M-SQV has an enlarged active site resulting in the loss of a hydrogen bond in the S3 subsite from Gly48 to P3 of SQV, as well as less favorable hydrophobic packing interactions between P1 Phe of SQV and the S1 subsite. PRG48T/L89M-SQV assumes a more open conformation relative to PRWT-SQV, as illustrated by the downward displacement of the fulcrum and elbows and weaker interatomic flap interactions. We also show that the Leu89Met mutation disrupts the hydrophobic sliding mechanism by causing a redistribution of van der Waals interactions in the hydrophobic core in PRG48T/L89M-SQV. Our mechanism for PRG48T/L89M-SQV drug resistance proposes that a defective hydrophobic sliding mechanism results in modified conformational dynamics of the protease. As a consequence, the protease is unable to achieve a fully closed conformation that results in an expanded active site and weaker inhibitor binding.

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.

Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.

Specificity counts: A template-based approach to protease inhibitors is presented using a core macrocycle that presents a generic β-strand template for binding to protease active sites. This is then specifically functionalized at P2 , and the C and N termini to give inhibitors of calpain 2, 20S proteasome, and HIV-1 protease.