Faculty of Applied Sciences

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Computational and micro-analytical techniques to study the in vitro and in silico models of novel therapeutic drugs
(2016) Gumede, Njabulo Joyfull; Bisetty, Krishna; Sagrado, Sagrado
In drug discovery and development projects, metabolism of new chemical entities (NCEs) is a major contributing factor for the withdrawal of drug candidates, a major concern for other chemical industries where chemical-biological interactions are involved. NCEs interact with a target macro-molecule to stimulate a pharmacological or toxic response, known as pharmacodynamics (PD) effect or through the Adsorption, Distribution, Metabolism, and Excretion (ADME) process, triggered when a bio-macromolecule interacts with a therapeutic drug. Therefore, the drug discovery process is important because 75% of diseases known to human kind are not all cured by therapeutics currently available in the market. This is attributed to the lack of knowledge of the function of targets and their therapeutic use in order to design therapeutics that would trigger their pharmacological responses. Accordingly, the focus of this work is to develop cost saving strategies for medicinal chemists involved with drug discovery projects. Therefore, studying the synergy between in silico and in vitro approaches maybe useful in the discovery of novel therapeutic compounds and their biological activities. In this work, in silico methods such as structure-based and ligand-based approaches were used in the design of the pharmacophore model, database screening and flexible docking methods. Specifically, this work is presented by the following case studies: The first involved molecular docking studies to predict the binding modes of catechin enantiomer to human serum albumin (HSA) interaction; the second involved the use of docking methods to predict the binding affinities and enantioselectivity of the interaction of warfarin enantiomers to HSA. the third case study involved a combined computational strategy in order to generate information on a diverse set of steroidal and non-steroidal CYP17A1 inhibitors obtained from literature with known experimental IC50 values. Finally, the fourth case study involved the prediction of the site of metabolisms (SOMs) of probe substrates to Cytochrome P450 metabolic enzymes CYP 3A4, 2D6, and 2C9 making use of P450 module from Schrödinger suite for ADME/Tox prediction. The results of case study I were promising as they were able to provide clues to the factors that drive the synergy between experimental kinetic parameters and computational thermodynamics parameters to explain the interaction between drug enantiomers and thetarget protein. These parameters were correlated/converted and used to estimate the pseudo enantioselectivity of catechin enantiomer to HSA. This approach of combining docking methodology with docking post-processing methods such as MM-GBSA proved to be vital in estimating the correct pseudo binding affinities of a protein-ligand complexes. The enantioselectivity for enantiomers of catechin to HSA were 1,60 and 1,25 for site I and site II respectively. The results of case study II validates and verifies the preparation of ligands and accounting for tautomers at physiological pH, as well as conformational changes prior to and during docking with a flexible protein. The log KS = 5.43 and log KR = 5.34 for warfarin enantiomer-HSA interaction and the enantioselectivity (ES = KS/KR) of 1.23 were close to the experimental results and hence referred to as experimental-like affinity constants which validated and verified their applicability to predict protein-ligand binding affinities. In case study III, a 3D-QSAR pharmacophore model was developed by using 98 known CYP17A1 inhibitors from the literature with known experimental IC50 values. The starting compounds were diverse which included steroidal and non-steroidal inhibitors. The resulting pharmacophore models were trained with 69 molecules and 19 test set ligands. The best pharmacophore models were selected based on the regression coefficient for a best fit model with R2 (ranging from 0.85-0.99) & Q2 (ranging from 0.80-0.99) for both the training and test sets respectively, using Partial Least Squares (PLS) regression. On the other hand, the best pharmacophore model selected was further used for a database screening of novel inhibitors and the prediction of their CYP17A1 inhibition. The hits obtained from the database searches were further subjected to a virtual screening workflow docked to CYP17A1 enzyme in order to predict the binding mode and their binding affinities. The resulting poses from the virtual screening workflow were subjected to Induced Fit Docking workflow to account for protein flexibility during docking. The resulting docking poses were examined and ranked ordered according to the docking scores (a measure of affinity). Finally, the resulting hits designed from an updated model from case study III were further synthesized in an external organic chemistry laboratory and the synthetic protocols as well as spectroscopic data for structure elucidation forms part of the provisional patent specification. A provisional patent specification has been filed (RSA Pat. Appln. 2015/ 07849). The case studies performed in this thesis have enabled the discovery of non-steroidal CYP17A1 inhibitors.
Evaluation of enantioresolution of (±)-catechin using electrokinetic chromatography and molecular docking
(SRP, 2012) Sabela, Myalowenkosi Innocent; Singh, Parvesh; Gumede, Njabulo Joyfull; Bisetty, Krishna; Sagrado, Sagrado
This study involves the enantioresolution of (±) catechin with the highly sulphated beta cyclodextrin (HS-β-CD) as a chiral selector using capillary electrophoresis (CE). The purpose of this study was to be tter understand enantioresolution amongst host-guest interactions. Furthermore, molecular docking was carried out to elucidate the mechanism of the enantioselective separations of (±) catechin enantiomers obtained in Electrokinetic chroma tography (EKC). A large difference in the interaction energies observed between the two enantiomers represents significant enantiodifferentiation. Our results also suggest that the host-guest interactions between the phenyl ring of the ligand and the open cavity of the HS-β-CD are due mainly to hydrophobic interactions. Interestingly, the stronger interactions observed with (+)-catechin is consistent with the elution order observed in the CE experiments.
Harmonization of internal quality tasks in analytical laboratories case studies : water analysis methods using polarographic and voltammetric techniques
(2008) Gumede, Njabulo Joyfull; Bisetty, Krishna; Redhi, Gyanasivan Govindsamy
In this work, a holistic approach to validate analytical methods was assessed by virtue of Monte Carlo simulations. This approach involves a statement of the methodsÃ¢ s scope (i.e. analytes, matrices and concentration levels) and requisites (internal or external); selection of the methodÃ¢ s (fit-for-purpose) features; pre-validation and validation of the intermediate accuracy and its assessment by means of Monte Carlo simulations. Validation of the other methodÃ¢ s features and a validity statement in terms of a Ã¢ fit-for-purposeÃ¢ decision making, harmonized validation-control-uncertainty statistics and short-term routine work with the aim of proposing virtually Ã¢ ready-to-useÃ¢ methods. The protocol could be transferred to other methods. The main aim is to harmonize the work to be done by research teams and routine laboratories assuming that different aims, strategies and practical viewpoints exist. As a result, the recommended protocol should be seen as a starting point. It is necessary to propose definitive (harmonized) protocols that must be established by international normalisation/accreditation entities. The Quality Assurance (Method verification and Internal Quality Control, IQC) limits, as well as sample uncertainty were estimated consistently with the validated accuracy statistics i.e. E U (E) and RSDi + U (RSDi). Two case studies were used to assess Monte Carlo simulation as a tool for method validation in analytical laboratories, the first involves an indirect polarographic method for determining nitrate in waste water and the second involves a direct determination of heavy metals in sea water by differential pulse anodic stripping voltammetry, as an example of the application of the protocol. In this sense the uncertainty obtained could be used for decision making purposes as it is very tempting to use uncertainty as a commercial argument and in this work it has been shown that the smaller the uncertainty, the better the measurement of the instrument or the laboratoryÃ¢ s reputation.

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