Combined Quantum/Classical Studies of the Mechanism of G6PD in the Oxidative Pentose Phosphate Pathway

Abstract

The glucose-6-phosphate dehydrogenase (G6PD) enzyme is a vital enzyme of pentose phosphate pathway involved in the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, as well as in reducing NADP+

to NADPH. The enzyme acts as a double end sword where its deficiency leads to a condition commonly known as G6PD deficiency causing hemolytic anemia neonatal jaundice and kernicterus. On the other hand, over expression of G6PD leads to increased cellular growth and carcinogenesis. Therefore; detailed understanding of induction and inhibition mechanism of G6PD enzyme is required. Induction may bring about increased enzymatic activity leading to enhanced production of cellular machinery and enhanced cellular growth resulting in tumorigenesis; conversely inhibition mechanism leads to deficiency of varying degree leading to several clinical manifestations. This thesis aims to advance our mechanistic and molecular level understanding of G6PD enzyme. Computational enzymology has become an emerging field in recent years for investigation of enzyme activity since it allows the calculation of the energies and structures of short-lived intermediates and transition states. Different chemical pathways can be studied using computational enzymology, and their validity can be determined by thoroughly evaluating predicted energy barriers. Molecular level picture provides physical basis of structure and function of protein/enzyme. During the chemical process, a change in electronic structure of species at reactive site of enzyme may occur. Cluster modeling approach is one of the powerful tool in computational enzymology used for modeling the active site of enzyme and investigation of reaction mechanisms. Mechanism of G6PD enzyme and associated reaction energetics have been explored in this thesis by employing cluster modeling approach using M06-2X functional. Reaction energetics have been compared to get an insight in to the catalytic role of Asp246- residue in the active site with neutral, protonated and deprotonated Histidine (His309). Proton abstraction from His309 by Asp246-

adopts a multistep sequential mechanism with a low energy barrier

indicating its ultimate role in catalysis. Proteins structures remain dynamic to accommodate substrate binding giving rise to changes in the local and global dynamics of enzyme during the course of reaction. Understanding the dynamical behavior of protein is critical in grasping the functionality and protein stability. Several

Abstract

xv biochemical experimental techniques such as structural, biochemical, kinetic and spectroscopic characterization have advanced in solving the mystery of molecular mechanisms considerably. However; employing molecular dynamics simulations in understanding the dynamical behavior of protein and its effect on functionality by using experimentally solved crystal structures is a good starting point which has been employed in this thesis. Location of mutation in three-dimensional structure of protein is critical in determining the shape and structural integrity of enzyme. In G6PD dimer interface and structural NADP+

binding site has been known for structural integrity of enzyme since long. Dynamical properties for wildtype G6PD and three of its mutants have been compared to explore the effect of location of mutation on enzyme structure with respect to structural NADP+

binding site.

Enzyme catalyzed reactions occur in complex environment consisting of several rearrangements in addition to bond making and breaking. QC/MM methods with ab-initio quantum mechanics with MD simulation are the powerful and accurate methods to identify the reaction mechanism and associated dynamical changes of enzyme catalyzed reaction which require large computational time. Nevertheless to reduce the computational cost; Semi-empirical methods are popular for

understanding reaction site mechanism at atomistic level with associated dynamics. Semi- empirical QC/MM methods have been implemented in this thesis to explore the reaction

mechanism of G6PD in the presence of surrounding environment. The most important moieties proposed in reaction mechanism including nicotinamide ring atoms of NADP+, all G6P atoms, imidazole ring of catalytic and activating His309 and amine group of Lys145 were included in QC region whereas a mobile and fixed region was setup around catalytic center. Catalytic mechanism validated proton abstraction role of Asp246 as observed in cluster modeling approach. Computer aided drug discovery is a breakthrough in the field of drug development which have expedited this lengthy process in various aspects. The discovery and development of novel therapeutic agents in oncology has resulted in considerably improved survival rates for many types of cancer. G6PD plays a significant role in cellular proliferation in various forms of cancers. Among the various cancer treatment regimes, inhibition of G6PD to stop production of cancer cells remained under debate since long. Several steroid inhibitors formulated against G6PD have been shown to improve anticancer treatment and effective against drug resistance. However, similar to several anticancer drugs, requirement of higher concentration of effective dose of drug

Abstract

xvi reduces the efficacy of treatment. Information regarding inhibitory site of G6PD is unclear in literature. The steroid inhibitors have been reported to un-competitively inhibit G6PD elucidating allosteric nature of these inhibitors. Therefore, an attempt has been made to identify the potent inhibitory binding site of G6PD for effective inhibitor binding. Moreover, optimal features for G6PD inhibition have been formulated using ligand based pharmacophore modeling approach. Our developed a pharmacophore model based on docking studies and preferred inhibitory site was complementary to the receptor site. Our developed model can help to design better drug targets for future rational drug design. The results obtained from mechanistic and dynamics studies can give an insight in to further development of inhibitory molecules with better targeted binding and inhibition of G6PD for development of antitumor therapies.