High-Activity Mutants of Butyrylcholinesterase for Cocaine Hydrolysis and Method of Generating the Same

A novel computational method and generation of mutant butyrylcholinesterase for cocaine hydrolysis is provided. The method includes molecular modeling a possible BChE mutant and conducting molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical calculations thereby providing a screening method of possible BChE mutants by predicting which mutant will lead to a more stable transition state for a rate determining step. Site-directed mutagenesis, protein expression, and protein activity is conducted for mutants determined computationally as being good candidates for possible BChE mutants, i.e., ones predicted to have higher catalytic efficiency as compared with wild-type BChE. In addition, mutants A199S/A328W/Y332G, A199S/F227A/A328W/Y332G, A199S/S287G/A328W/Y332G, A199S/F227A/S287G/A328W/Y332G, and A199S/F227A/S287G/A328W/E441D all have enhanced catalytic efficiency for (−)-cocaine compared with wild-type BChE

High-Activity Mutants of Butyrylcholinesterase for Cocaine Hydrolysis and Method of Generating the Same

A novel computational method and generation of mutant butyrylcholinesterase for cocaine hydrolysis is provided. The method includes molecular modeling a possible BChE mutant and conducting molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical calculations thereby providing a screening method of possible BChE mutants by predicting which mutant will lead to a more stable transition state for a rate determining step. Site-directed mutagenesis, protein expression, and protein activity is conducted for mutants determined computationally as being good candidates for possible BChE mutants, i.e., ones predicted to have higher catalytic efficiency as compared with wild-type BChE. In addition, mutants A199S/A328W/Y332G, A199S/F227A/A328W/Y332G, A199S/S287G/A328W/Y332G, A199S/F227A/S287G/A328W/Y332G, and A199S/F227A/S287G/A328W/E441D all have enhanced catalytic efficiency for (−)-cocaine compared with wild-type BChE

High-Activity Mutants of Butyrylcholinesterase for Cocaine Hydrolysis and Method of Generating the Same

A novel computational method and generation of mutant butyrylcholinesterase for cocaine hydrolysis is provided. The method includes molecular modeling a possible BChE mutant and conducting molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical calculations thereby providing a screening method of possible BChE mutants by predicting which mutant will lead to a more stable transition state for a rate determining step. Site-directed mutagenesis, protein expression, and protein activity is conducted for mutants determined computationally as being good candidates for possible BChE mutants, i.e., ones predicted to have higher catalytic efficiency as compared with wild-type BChE. In addition, mutants A199S/A328W/Y332G, A199S/F227A/A328W/Y332G, A199S/S287G/A328W/Y332G, A199S/F227A/S287G/A328W/Y332G, and A199S/F227A/S287G/A328W/E441D all have enhanced catalytic efficiency for (−)-cocaine compared with wild-type BChE

Computational chemistry studies of subtypes B and South African C HIV proteases.

Master of Medical Science in Pharmacy. University of KwaZulu-Natal, Durban, 2016.HIV/AIDs is a prevalent disease infecting millions of people throughout the world. Although a lot of improvement has been achieved over the year in regard to the reduction of AIDs related deaths, a huge task lies ahead as the HIV/AIDs global epidemic keeps spreading annually. It is therefore paramount to discover and develop more and efficient drug inhibitors against HIV. The HIV protease (HIV PR) is a C2-symmentric homodimer and consisting of 99-amino acids in each monomer and because of the important role it plays in the HIV mutation, it became a major HIV drug target for the past three decades. It is on this basis that various effective antiretroviral protease inhibitors have been designed and approved for application in HIV therapy.The HIV subtype B strain is prominent in Europe and North America and is the most researched virus. The majority of the antiretroviral drugs were designed and tested against HIV subtype B. However, non-subtype B strains of the HIV virus makes up most of these infections in Southern and Eastern Africa, which are highly affected regions in the world. In South Africa, subtype C HIV-1 is the dominant strain and little research has been done regarding drug design for this subtype or testing of the effectiveness of the HIV approved antiretroviral drugs against these non-subtype B strains. Two potentially devastating mutations of subtype C-SA HIV PR were recently reported by our group. These were designated I36T↑T and L38L↑N↑L HIV PR. The I36T↑T PR mutant includes an extra amino acid, the mutation occurs at position 36 (isoleucine to threonine) and is followed by an insertion at the second threonine indicated by the upward arrow. The L38L↑N↑L PR mutant involves two amino acids insertions that is completely different from the usual 99-amino acids HIV PR, as well as five point mutations occur at the E35D, I36G, N37S, M46L and D60E. The two insertions occur at position 38 (asparagine and leucine) indicated by the two upward arrows. Therefore, the I36T↑T and L38L↑N↑L mutations consist of 100 and 101-amino acids in each monomer of the proteases respectively.In this thesis, a hybrid computational model (QM: MM) using the ONIOM approach was followed. The selected FDA inhibitors were complexed with the various proteases in the active pocket interacting with Asp 25/25' catalytic residues using the same pose in the subtype B PR as a reference X-ray structure. The HIV PR inhibitors and Asp 25/25' were treated at a high-level with quantum mechanics (QM) theory using B3LYP/6-31G(d), and the remaining HIV PR residues were considered at a low layer using molecular mechanics (MM) with the AMBER force field. This method was applied to calculate the binding free interaction energies of the selected FDA approved HIV PR drugs complexed to the HIV protease enzyme. The aim was to create and test this computational model that will reflect the experimental binding energies against subtype B, C-SA HIV PR and also a mutant from the subtype C-SA PR designated L38L↑N↑L HIV PR. The calculated binding free interaction energies results from the subtype B follow a satisfactory trend with the experimental data. However, the C-SA HIV PR inhibitor―enzyme complexes showed some discrepancies and this was ascribed to the simplified computational model that omitted water in the active site of the enzyme. The calculated binding free interaction energies for L38L↑N↑L PR as well as experimental results, showed reduced binding affinities for all the selected FDA approved inhibitors in comparison with the subtype C-SA HIV PR. The deviation could be as a result of the insertion and mutation of the subtype C HIV-1 PR that is expected to have a significant effect in altering either the binding affinity of the HIV PR inhibitors and or characteristics of the parent protease. The computational model used in this research will be improved by introducing water into the active pocket of the Asp 25/25' catalytic residues that will be treated at least at semi-empirical level. Optimization of the different ONIOM levels will be attempted in order to accurately predict activities of new potential HIV PR inhibitors

High-Activity Mutants of Butyrylcholinesterase for Cocaine Hydrolysis and Method of Generating the Same

A novel computational method and generation of mutant butyrylcholinesterase for cocaine hydrolysis is provided. The method includes molecular modeling a possible BChE mutant and conducting molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical calculations thereby providing a screening method of possible BChE mutants by predicting which mutant will lead to a more stable transition state for a rate determining step. Site-directed mutagenesis, protein expression, and protein activity is conducted for mutants determined computationally as being good candidates for possible BChE mutants, i.e., ones predicted to have higher catalytic efficiency as compared with wild-type BChE. In addition, mutants A199S/A328W/Y332G, A199S/F227A/A328W/Y332G, A199S/S287G/A328W/Y332G, A199S/F227A/S287G/A328W/Y332G, and A199S/F227A/S287G/A328W/E441D all have enhanced catalytic efficiency for (−)-cocaine compared with wild-type BChE

Computational studies of mycobacterium tuberculosis L, d-transpeptidase2.

Masters Degree. University of KwaZulu-Natal, Durban.Tuberculosis (TB) is still one of the most highly elusive lethal transmittable diseases to eradicate and persist to be a major threat to public health due to emergence of drug resistance. Drug-resistant is steadily increasing worldwide, therefore, there is an urgent need for development of improved efficacious antibiotics and novel drug targets to successfully contain the disease. Peptidoglycan layer (PG) is the major attribute in bacterial cell envelope and is essential for protection and growth in all bacterial species including Mycobacterium tuberculosis(Mtb). The biosynthesis pathway for PG is extremely intricate and involves numerous interconnected metabolites such as N-acetylmuramic(NAM) acid and N-acetylglucosamine(NAG), that are required during transpeptidation. These two sugar molecules are linked together by a β (1-4) glycosidic bond and the NAM attaches 3-5 amino acid peptide stems. Consequently, the peptidoglycan strands are cross linked by transpeptidases, namely D, D- and L, D-transpeptidases, forming crucial 4→3 and 3→3 cross-linkages respectively. Both D, D- and L, D-transpeptidases need to be inhibited concomitantly to eradicate the bacterium. L, D-transpeptidase 2 (LdtMt2) is one of the paralogs that is essential for Mtb growth, cell morphology and virulence during the chronic stage of the disease. This paralog has major influence in drug resistance and persistence of tuberculosis. The traditional β-lactam family of antibiotics have been reported to be effective against Mtb following the inactivation of β-lactamases (BlaC) known to rapidly hydrolyze the core β-lactam ring. The classic penicillins inhibit D, D-transpeptidases, while L, D transpeptidases are blocked by carbapenems. Despite several studies in this field, to the best of our knowledge, limited attention has been paid to the inhibition mechanism of LdtMt2 using carbapenem derivatives. In this regard, we need to explore reliable alternative strategies that are most cost-effective in terms of investigating the interactions of FDA approved carbapenems against Mtb L, D-Transpeptidases and study the role of explicit water molecule confined in the active site. As a result, computational chemistry has provided the possibility to sightsee and investigate this problem with relatively cost effective computational techniques. In this thesis, we applied a hybrid quantum mechanics and molecular mechanics techniques (QM:MM), Our own N-layered Integrated molecular Orbital and Molecular Mechanics (ONIOM) approach, to investigate the binding interaction energies of carbapenems (biapenem, imipenem, meropenem and tebipenem) against L, D-transpeptidase 2. Furthermore, the role of explicit water molecule confined in the active site was also explored using the same hybrid method to ascertain the nature of binding interaction energies of carbapenems against LdtMt2. In all the investigated carbapenem─LdtMt2 complexes, the carbapenems and the catalytic active site residues of LdtMt2 (Cys205, His187, Ser188, His203 and Asn207) were treated at QM (B3LYP/6-31+G(d)) level of theory whereas the remaining part of the complexes were treated at MM level (AMBER force field). The explicit water molecules near the carbapenems were considered and treated at QM as well. The obtained findings of Gibbs free energy (G), enthalpy (H) and entropy (S) for all studied complexes showed that the carbapenems exhibit reasonable binding interactions towards LdtMt2. This can be attributed by the structural dissimilarities of the carbapenems side chain which significantly induce conformational changes in the LdtMt2. In comparison, the binding free energy calculations of the model system with explicit water molecule yielded significant binding interaction energies. The QTAIM and NBO results confirmed the nature of binding free energies that the topological properties of atoms in molecules and the delocalization of electrons are from a bonding to antibonding orbitals in hydrogen bond interactions and this has enhanced the stability of carbapenem―LdtMt2 complexes. We believe that molecular insight of the carbapenems binding to LdtMt2 and the role of explicit solvent will enable us to understand the inhibition mechanisms