Cyclophilins (Cyp) are proteins that catalyze the interconversion of trans/cis isomers of proline belonging to the peptidyl-prolyl isomerases family (PPIase). In addition to their PPIase activity, Cyps have diverse biological roles and have been implicated in a number of different diseases such as HIV-1 and HCV. Although several Cyp inhibitors have been reported in the literature, none are able to inhibit with high specificity various Cyp isoforms. To facilitate the development of isoform-specific Cyp ligands, we have pursued detailed studies of Cyp dynamics and ligand binding thermodynamics using molecular simulations, biophysical assays and protein X-ray crystallography. Research efforts were focussed on the identification of novel Cyp inhibitors using X-ray crystallographic studies and Surface Plasmon Resonance (SPR) experiments on fragments from an in-house bespoke library of small compounds. These biophysical studies revealed a number of fragments that are able to bind to diverse Cyp isoforms with high micromolar – low millimolar activity. To further examine the binding of these fragments to cyclophilins, identify interactions with the proteins and explain specificity trends from SPR and X-ray results, molecular dynamics (MD) simulations and free energy calculations were pursued. Models of apo and holo Cyps in complex with fragments that we had experimentally tested were set up using the Amber, AmberTools and FESetup software. Free energy calculations were performed using the thermodynamic integration (TI) technique with the Sire/OpenMM software. The results were analysed with custom scripts. Correlations between computed and measured binding energies, and calculated and observed binding modes were analysed to help develop guidelines for the development of isoform specific cyclophilin ligands. A detailed comparison of the merits and drawbacks of the experimental and computational techniques used in this work has also been made, and strategies for effective combination of the methodologies in structure-based projects are outlined.

The enzyme nitrogenase catalyzes the energy-dependent reduction of dinitrogen to ammonia via biological nitrogen fixation. Nitrogenase is composed of two metalloproteins known as the molybdenum-iron (MoFe) protein and the iron (Fe) protein. The Fe protein, a 60-kDa dimer of the product of the nifH gene, contains a single 4Fe-4S cluster and two Mg-ATP-binding sites, one at each subunit. The Fe protein is the obligate electron donor to the MoFe protein. To date, no other mutual protein has shown to substitute Fe protein in biological fixation, and the NifH is functional only in the presence of the nifessory protein NifM. Interestingly, the protochlorophyllide reductase (ChlL) encoded by the chlL gene of Chlamydomonas reinhardtii shows significant homology and structural similarity with NifH. Previously, our laboratory has shown that the ChlL can substitute the Fe protein in the functioning nitrogenase only in the absence of NifM. We have also shown that the NifM is a PPIase and the Pro-258 located in the C-terminus of NifH is one of the substrates for NifM. Since the least structural homology exists between NifH and ChlL at the C-terminal region, we hypothesized that we can generate a NifM-independent NifH-ChlL chimeric protein by replacing the C-terminus of NifH (that spans the substrate of PPIase) with that of ChlL. To test this idea we created a chimeric construct by replacing the NifH C-terminal region (residues 248-291) with the ChlL C-terminal region (residues 240-294). The chimeric gene was then transformed into the nifM- Azotobacter vinelandii strain AV98. While the wild type nifH could not render a Nif+ phenotype to the nifM- AV98, the chimera could impart Nif+ phenotype to this nifM- strain. This result demonstrated that the NifH-ChlL chimeric protein is NifM-independent.

The aim of this study was to identify and characterise peptidyl-prolyl cis-trans isomerases (PPIases) from the bacterium Burkholderia pseudomallei, the causative agent of the disease melioidosis. The longer term goal was to assess their potential as vaccine candidates or antimicrobial targets. Using bioinformatic approaches, six putative FK506-binding proteins (FKBPs) proteins and three putative parvulin proteins were identified in B. pseudomallei. Of these, six were expressed and purified as recombinant proteins. The purified proteins were used to immunise BALB/c mice, with some providing protection against a subsequent B. pseudomallei infection. These proteins could therefore be proposed as potential vaccine candidates. Homologues of Mip or SurA, which are associated with virulence in other bacterial species, were identified in B. pseudomallei and closely related B. thailandensis. Recombinant Mip or SurA homologues from B. pseudomallei were shown to have characteristic PPIase enzyme activity. To evaluate the role of the Mip homologue from B. pseudomallei in virulence, an unmarked deletion mutant was constructed. The mutant had reduced intracellular survival; defects in putative virulence mechanisms and attenuated virulence in mice. To assess the role of a SurA homologue, closely related B. thailandensis was used as a model organism, with deletion of the gene resulting in defects in intracellular infection, outer membrane integrity and virulence. This indicates that PPIases from B. pseudomallei and B. thailandensis represent novel virulence determinants and potential antimicrobial targets for therapeutics against melioidosis.

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An important role of Pin1 is to catalyze the cis-trans isomerization of pSer/Thr-Pro bonds; as such, it plays an important role in many cellular events through the effects of conformational change on the function of its biological substrates, including Cdc25, c-Jun, and p53. The expression of Pin1 correlates with cyclin D1 levels, which contributes to cancer cell transformation. Overexpression of Pin1 promotes tumor growth, while its inhibition causes tumor cell apoptosis. Because Pin1 is overexpressed in many human cancer tissues, including breast, prostate, and lung cancer tissues, it plays an important role in oncogenesis, making its study vital for the development of anti-cancer agents. Many inhibitors have been discovered for Pin1, including 1) several classes of designed inhibitors such as alkene isosteres, non-peptidic, small molecular Pin1 inhibitors, and indanyl ketones, and 2) several natural products such as juglone, pepticinnamin E analogues, PiB and its derivatives obtained from a library screen. These Pin1 inhibitors show promise in the development of novel diagnostic and therapeutic anticancer drugs due to their ability to block cell cycle progression. In order to develop potent Pin1 inhibitors, the concept of transition-state analogues was used for the design of three classes of compounds: ketoamide, ketone, and reduced amide analogues. Specifically, a convergent synthesis of α-ketoamide inhibitors of Pin1 was developed. An α-hydroxyorthothioester derivative of Ser was reacted directly with an aminyl synthon. The reaction was catalyzed by HgO and HgCl2 to form an α-hydroxyamide. Hydrolysis and coupling were combined in one step in 80% yield. Two diastereomers of a phospho-Ser-Pro α-ketoamide analogue were synthesized. The resulting IC50 values of 100 µM and 200 µM were surprisingly weak for the Pin1 peptidyl-prolyl isomerase. Diastereomeric ketones were synthesized by coupling cyclohexenyl lithium to the serine Weinreb amide, via the Michael addition of a carboxylate synthon. The IC50 values of the two ketone diastereomers were determined to be 260 μM and 61 μM, respectively. Five reduced amide inhibitors for Pin1 were synthesized through a selective reduction using borane. The most potent inhibitor was found to be Fmocâ pSerâ Ψ[CH2N]-Proâ tryptamine, which had an IC50 value of 6.3 µM. This represents a 4.5-fold better inhibition for Pin1 than a comparable cis-amide alkene isostere. The co-crystal structure of Acâ pSerâ Ψ[CH2N]-Proâ tryptamine bound to Pin1 was determined to 1.76 Ã resolution. Towards understanding the two proposed mechanisms of Pin1 catalysis, nucleophilic-additition mechanism and twisted-amide mechanism, three classes of Pin1 inhibitors (ketoamide, ketone, and reduced amide analogues) involving a total of nine compounds were synthesized and evaluated. The weak inhibitory activities of ketoamide and ketone analogues do not support the nucleophilic-addition mechanism, while the twisted-amide mechanism of Pin1 catalysis is promising based on the reduced amide inhibitors with good potencies. / Ph. D.