STUDIES ON FARNESYL DIPHOSPHATE SYNTHASE AND MEVALONATE KINASE ENZYMES OF HELICOVERPA ARMIGERA

Abstract

Helicoverpa armigera (Lepidoptera: Noctuidae), commonly referred to as cotton bollworm, ranks among the world's top hundred invasive species. This devastating herbivore insect has been recorded to damage a wide variety of plant species, including cotton, some of which are valuable crops. Due to its wide range of hosts, short generation time, high fecundity, migration activity, and high tolerance to many insecticides, effective pest control has become tricky and cumbersome. In addition, management of this pest is further constrained due to the emergence of resistance towards conventional management tools such as chemical pesticides, as well as genetically modified transgenic crops expressing insecticidal toxins, for instance, Bt (Bacillus thuringiensis) cotton. Therefore, alternative management methods are an urgent need. The morphogenetic and gonadotropic development in insects is regulated by the Juvenile Hormone (JH). Since JH is found solely in insects, the enzyme involved in its biosynthesis is categorically an attractive target for the development of selective insecticides. JH is principally a lipophilic sesqui-terpenoid produced by corpora allata, a pair of endocrine glands located behind the insect skull and released into the hemolymph. Recent studies have shown that JH is engaged in a myriad of physiological processes in insects, including caste differentiation, diapause, reproduction, and several other physiological functions. Juvenile hormones are prominently present during larval stages to facilitate the development of the larva and orchestrate progression from one larval stage to the next, leading to metamorphosis. During pre-metamorphic larval stadium, any decline in larval JH titer can potentially cause precocious metamorphosis, resulting in ill-developed adults that are either unviable or impotent. The biosynthesis of JH proceeds through the mevalonate pathway (MVP). Apart from JH biosynthesis, the MVP also takes part in other processes in insects, such as pheromone production and defensive secretions. The first step in the MVP is the condensation of acetyl-coenzyme A (acetyl-CoA) to acetoacetyl-CoA using the enzyme thiolase. It further proceeds with the biosynthesis of mevalonate with the help of enzymes 3- Hydroxy-3-methyl glutaryl-CoA (HMG-CoA) synthase and HMG-CoA reductase. ATP dependant phosphorylation of mevalonate is catalyzed by mevalonate kinase to form Mevalonate-5-P which is further converted to mevalonate-5-PP by phosphomevalonate kinase. Isopentenyl diphosphate (IPP), a five-carbon isoprene unit is synthesized after a decarboxylation reaction catalyzed by diphosphomevalonate decarboxylase. IPP isomerase catalyzes the transformation of IPP into its isomer dimethylallyl diphosphate (DMAPP). Thereafter, farnesyl diphosphate synthase (FPPS) catalyzes the two steps head-to-tail condensation reactions, first IPP with DMAPP where DMAPP acts as a chain initiator, and second IPP with intermediate geranyl pyrophosphate (GPP) forming farnesyl diphosphate (FPP), the precursor of JH. Farnesyl diphosphate is eventually converted to JH with the help of additional enzymatic steps specific to the synthesis of JH. Farnesyl diphosphate synthase (EC 2.5.1.10) is an MVP enzyme that is one of this pathway's most essential and regulatory enzymes. Hundreds of crystal structures of FPPS have been reported to date because, in humans, FPPS is a promising drug target for the treatment or prevention of diseases like osteoporosis and Paget’s disease. Also, many human parasites such as Leishmania and Trypanosoma are targeted by inhibiting their FPPS enzymes. There are two binding sites, allylic and homoallylic, present in the active site of this enzyme. While IPP binds to the homoallylic binding site, DMAPP or GPP binds to the allylic binding site. Although FPPS homologs are encoded by the genomes of most living organisms, those of some insects, including Lepidoptera, exhibiting taxon-specific structural characteristics, may be explored to identify and develop selective inhibitors with insecticidal properties. Previous studies have reported that the silencing of JH pathway genes leads to hindrance in the normal process of metamorphosis in H. armigera. The functional study of Helicoverpa armigera- FPPS (HaFPPS) using RNAi in H. armigera revealed that the knockdown of HaFPPS triggered a decrease in JH titer along with a negative effect on the transcription levels of other genes in the JH pathway. Subsequently, disturbances in the larval moulting phenomenon were noticed. Hence, it was inferred that in JH biosynthesis, HaFPPS played an invaluable role and was essential for controlling the transcription of JH pathway genes. Owing to its crucial function in the biosynthesis of JH, FPPS was studied as a potential bio-rational target point for insecticide production against H. armigera in the present study. Mevalonate kinase (EC 2.7.1.36) belonging to MVP is another essential enzyme for the synthesis of JH precursor FPP. Mevalonate kinase (MK) catalyzes the nucleophilic C5 anion of mevalonate on the ᵧ-phosphate of ATP forming mevalonate-5-phosphate. MK belongs to the superfamily of GHMP kinases which are sugar kinases namely galactokinases, homoserine kinases, mevalonate kinases, and phosphomevalonate kinases. In humans, MK is mainly associated with cholesterol biosynthesis which is a potent metabolic target in cancer therapy. It has been observed that MK plays an important role as a regulatory enzyme of MVP in prokaryotes and eukaryotes. Inhibition of MK by FPP and GPP has been observed to inhibit the synthesis of IPP hence limiting the production of FPP by feedback inhibition. Also, the inhibition of MK resulted in decreased cell viability of the protozoan parasite Leishmania. Additionally, MK has been observed to play an important role in reducing oxidative stress and preventing peroxidation of cellular lipids in Leishmania. Hence, the mevalonate kinase enzyme may also act as a potential bio-rational target for designing novel insecticidal agents against H. armigera. The present work is intended to identify phytochemical molecules that may potentially inhibit the activity of farnesyl diphosphate synthase and mevalonate kinase from Helicoverpa armigera, the important biosynthetic enzymes of JH, for effective and eco-friendly intervention in the control of this pest. The objectives of the present work are as follows: I. In-silico screening of potential phytochemical inhibitors targeting farnesyl diphosphate synthase of Helicoverpa armigera. II. Gene cloning, protein expression, purification, and characterization of recombinant farnesyl diphosphate synthase of Helicoverpa armigera. III. Evaluation of binding affinities of selected inhibitor (chlorogenic acid) with HaFPPS and insect feeding bioassay. IV. Structure-based screening of natural inhibitors against Mevalonate kinase of Helicoverpa armigera (HaMK). Chapter 1 begins with an introduction and a review of the literature related to each objective. It discusses the life cycle and economic importance of H. armigera. Also explains biosynthesis and the role of JH in the life cycle of insects. Further describes the rationale behind selecting two crucial enzymes (Farnesyl diphosphate synthase and mevalonate kinase) involved in the biosynthesis of JH as a potential target for insecticide development. Moreover, it discusses the significance of employing natural compounds for developing environment-friendly insect growth regulator(s). Chapter 2 deals with the in-silico approach to identify the small molecule inhibitor(s) of HaFPPS to develop bioinsecticide against H. armigera. We have identified three phytochemical compounds against the 3-D modelled structure of HaFPPS from the virtual screening workflow, i.e., mitraphylline, chlorogenic acid, and llagate, that may serve as candidates for potential insecticide(s). Various molecular descriptors and toxicity properties were predicted showing that the selected compounds fulfilled the drug-like properties as per Lipinski’s rule of five and were safer from a biohazard perspective. Collectively, the molecular dynamics simulation and MMPBSA results confirmed that the identified compounds lead to the formation of stable HaFPPS-inhibitor(s) complexes. Chlorogenic acid is the most efficient among the three ligands as it formed the maximum number of hydrogen bonds in the entire simulation run and it had the highest AutoDock Vina and MMPBSA binding energy. Hence, chlorogenic acid was selected for further study. Chapter 3 deals with gene cloning, protein expression, purification, and characterization of recombinant farnesyl diphosphate synthase of H. armigera. We have cloned the HaFPPS genes into a histidine-tagged expression vector (pET28c). Then the confirmed clone of pET28c-HaFPPS was transformed into Rosetta-2 Gami (DE3) E. coli cells. Further, the recombinant protein expression was optimized and purified to homogeneity using Ni-NTA affinity chromatography. Western blotting was done to confirm the identity of purified proteins as His-tagged fusion proteins. Next, we characterized the HaFPPS protein. Oligomeric state characterization by using gel filtration chromatography indicated that HaFPPS is a homodimer. Circular dichroism spectra of HaFPPS undergo significant changes when the pH of the solution deviates from its native pH range of 8 to 9 and the melting temperature was found to be 45.5°C. Antioxidant activity of HaFPPS using TEAC assay shows that HaFPPS may play a role in redox maintenance in the insect whose inhibition may cause redox imbalance. Chapter 4 describes the evaluation of binding affinity using biophysical techniques of a selected inhibitor (chlorogenic acid) with HaFPPS and insect-feeding bioassay. Using fluorescence spectroscopy, Isothermal Titration Calorimetry (ITC), Differential Scanning Calorimetry (DSC), and Circular dichroism (CD) it has been found that chlorogenic acid binds well to HaFPPS. Further, in-vivo insect bioassay demonstrated that chlorogenic acid reduces H. armigera larval weight gain, concluding that chlorogenic acid is the appropriate natural insecticide also because it leaves no residue or hazard to the user and ecosystem. Chapter 5 describes molecular cloning and bioinformatic screening of natural inhibitors against the mevalonate kinase enzyme of H. armigera (HaMK) for the development of bioinsecticides. Genes encoding mevalonate kinase enzyme was amplified from the cDNA of H. armigera and cloned into the pET-28c vector. Three natural compounds from the virtual screening workflow, i.e., magnolol, aloe-emodin, and niazirin may serve as candidates for potential insecticide(s). Finally, MD simulation and MMPBSA analysis confirmed that the identified compounds bind at the active site of HaMK protein and lead to the formation of stable HaMK-inhibitor(s) complexes. Thus, the selected novel compounds may pave the way for further bioinsecticide development against H. armigera.