Dissertations / Theses on the topic 'Enzymes - Antimalarials'

Histone deacetylases (HDACs) are recognised as potential drug targets for many diseases including cancer, inflammatory diseases and some parasitic diseases including malaria. In eukaryotic cells, these enzymes play an important role in transcriptional regulation through modification of chromatin structure. Inhibitors of mammalian HDAC enzymes including trichostain A and apicidin are active against P. falciparum parasites, however these compounds are not selective for malaria parasites versus normal cell lines. The aims of this study were to examine the antimalarial potential of new hydroxamate-based HDAC inhibitors and to investigate a P. falciparum HDAC, PfHDAC1, as a potential new antimalarial drug target.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Science
Griffith Health
Full Text

Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.

Malaria continues to be an enormous health-threat in the developing world and the emergence of drug resistance has further compounded the problem. The parasite-specific enzyme, 1-deoxY-D-xylulose-S-phosphate reductoisomerase (DXR), has recently been validated as a promising antimalarial drug target. The present study comprises a combination of synthetic, physical organic, computer modelling and bioassay techniques directed towards the development of novel DXR inhibitors. A range of 2-heteroarylamino-2-oxoethyl- and 2- heteroarylamino-2-oxopropyl phosphonate esters and their corresponding phosphonic acid salts have been synthesised as analogues of the highly active DXR inhibitor, fosmidomycin. Treatment of the heteroarylamino precursors with chloroacetyl chloride or chloropropionyl chloride afforded chloroamide intermediates, Arbuzov reactions of which led to the corresponding diethyl phosphonate esters. Hydrolysis of the esters has been effected using bromotrimethylsilane. Twenty-four new compounds have been prepared and fully characterised using elemental (HRMS or combustion) and spectroscopic (1- and 2-D NMR and IR) analysis. A 31p NMR kinetic study has been carried out on the two-step silylation reaction involved in the hydrolysis of the phosphonate esters and has provided activation parameters for the reaction. The kinetic analysis was refined using a computational method to give an improved fit with the experimental data. Saturation transfer difference (STD) NMR analysis, computer-simulated docking and enzyme inhibition assays have been used to evaluate the enzyme-binding and -inhibition potential of the synthesised ligands. Minimal to moderate inhibitory activity has been observed and several structure-activity relationships have been identified. In silica exploration of the DXR active site has revealed an additional binding pocket and information on the topology of the active site has led to the de novo design of a new series of potential ligands.
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Plasmodium falciparum 1–deoxy–D–xylulose–5 phosphatereductoisomerase (PfDXR) plays a role in isoprenoid biosynthesis in the malaria parasite and is absent in the human host, making this parasite enzyme an attractive target for antimalarial drug design. To characterize PfDXR, it is necessary to produce large quantities of the enzyme in a soluble and functional form. However, the over–production of malarial proteins in prokaryotic host systems often results in the formation of truncated proteins or insoluble protein aggregates. A heterologous expression system was developed for the production of active PfDXR using codon harmonization and tight control of expression in the presence of lac repressor. Yields of up to 2 mg/l of enzyme were reported using the optimised expression system, which is 8 to 10– fold greater than previously reported yields. The kinetic parameters Km, Vmax and kcat were determined for PfDXR; values reported in this study were consistent with those reported in the literature for other DXR enzymes. A three–dimensional model of the malarial drug target protein PfDXR was generated, and validated using structure–checking programs and protein docking studies. Structural and functional features unique to PfDXR were identified using the model and comparative sequence analyses with apicomplexan and non–apicomplexan DXR proteins. Residues Val44 and Asn45, essential for NADPH binding; and catalytic hatch residues Lys224 and Lys226, which are unique to the species of Plasmodium, were mutated to resemble those of E. coli DXR. Interestingly,these mutations resulted in significant reductions in substrate affinity, when compared to the unmutated PfDXR. Mutant enzymes PfDXR(VN43,44AG) and PfDXR(KK224,226NS) also demonstrated a decreased ability to turnover substrate by 4–fold and 2–fold respectively. This study indicates a difference in the role of the catalytic hatch of PfDXR with regards to the way in which it captures substrates. The study also highlights subtle differences in cofactor binding to PfDXR, compared with the well characterized EcDXR enzyme. The validated PfDXR model was also used to develop a novel efficient in silico screening method for potential tool compounds for use in the rational design of novel DXR inhibitors. Following in silico screening of 46 potential DXR inhibitors, a two–tiered in vitro screening approach was undertaken. DXR inhibition was assessed for the 46 novel compounds using an NADPH– ependant DXP enzyme inhibition assay and antimalarial potential was assessed using P.falciparum–infected erythrocyte growth assays. Select compounds were tested in human cells in order to determine whether they were toxic to the host. From the parallel in silico and in vitro drug screening, it was evident that only a single compound demonstrated reasonable potential binding to DXR (determined using in silico docking), inhibited DXR in vitro and inhibited P. falciparum growth, without being toxic to human cells. Its potential as a lead compound in antimalarial drug development is therefore feasible. Two outcomes were evident from this work. Firstly, analogues of known antimalarial natural products can be screened against malaria, which may then lead towards the rational design of novel compounds that are effective against a specific antimalarial drug target enzyme, such as PfDXR. Secondly, the rational design of novel compounds against a specific antimalarial drug target enzyme can be untaken by adopting a coupled in silico and in vitro approach to drug discovery.

Malaria is a lethal disease caused by the Plasmodium parasite. The current arsenal of antimalarial therapies targets the parasite, thereby selecting for mutant, resistant parasites. New antimalarials are desperately needed and a potential clue for a new therapeutic strategy has been provided by so-called "natural genetic antimalarials". Host genetic changes to red cell genes have offered millennia of stable protection to individuals living in endemic regions. By imitating natural resistance, this thesis proposes novel host-directed antimalarial pharmacologic therapies through the targeting of erythrocyte molecules required by the parasite for growth and survival. Work in this thesis investigated several enzymes from the haem biosynthetic pathway as potential targets for a host-directed therapy (HDT). Here, multiple experimental approaches were used to investigate and validate d-aminolevulinate dehydratase (ALAD), ferrochelatase (FECH) and uroporphyrinogen-III synthase (UROS) as targets for a novel host-directed antimalarial therapy. Firstly, it was demonstrated that host ALAD, FECH and UROS were localised in Plasmodium during intraerythrocytic growth. Moreover, the host enzymes were demonstrated to be required for normal parasite development as Plasmodium growth in vitro was impaired in UROS and FECH deficient red cells. This was shown using genetic models of human and mouse haem synthetic enzyme deficiency. Finally, the HDT strategy was validated with several inhibitors of ALAD and FECH demonstrating in vitro and in vivo anti-plasmodial activity. Host ALAD was specifically inhibited with succinylacetone (SA), a non-competitive irreversible ALAD inhibitor, demonstrating parasite growth inhibition in a P. Jalciparum in vitro assay with an IC\(_{50}\) of 2.5 μM. The antimalarial activity of SA was also demonstrated in vivo with SA treated mice demonstrating a significant reduction in P. chabaudi infection and increased survival compared to untreated controls. The competitive FECH inhibitor N-methylprotoporphyrin (NMPP) demonstrated anti-plasmodial activity in vitro with an IC\(_{50}\) of 25 nM, a figure comparable with many current antimalarials today. Griseofulvin, a second FECH inhibitor, is an antifungal agent, approved for use for over 50 years with an anti-FECH side effect, mediated through NMPP. Griseofulvin inhibited P. falciparum growth in an in vitro growth inhibition assay, with an IC\(_{50}\) between 10 and 50 μM on both chloroquine resistant and susceptible parasites. As griseofulvin is FDA and TGA-approved for human use, work in this thesis investigated parasite growth capacity in red cells from individuals taking pharmacologic doses of griseofulvin. It was demonstrated that griseofulvin concentrates in red cells and that parasites were unable to grow in red cells collected from human volunteers eight-hours after taking a clinically relevant dose of griseofulvin. Together, this data suggests that griseofulvin may be a useful antimalarial drug with a novel mode of action, potentially avoiding parasite resistance. Overall, work in this thesis has demonstrated that the parasite requires several host haem enzymes for growth and has provided proof-of-principle that targeting these enzymes as a HDT is a potentially effective antimalarial strategy. As griseofulvin is FDA and TGA approved for human use, it is quite possible that griseofulvin may be an "off the shelf' next generation antimalarial. The ultimate outcome from this work is a new generation of antimalarial therapies that may target the host and not the parasite, potentially limiting the development of drug resistance.

Human malaria, caused by four species of the intracellular protozoan parasite Plasmodium, is a major health and economic burden in the tropics where the disease is endemic. The biotindependent enzyme acetyl-CoA carboxylase catalyses the commitment step in de novo fatty acid biosynthesis in several organisms. Acetyl-CoA carboxylase is a target for anti-parasitic drug development due to its relevance in membrane biogenesis. This study describes the detection of acetyl-CoA carboxylase and the partial characterisation of the biotinoyl domain of the enzyme of the mouse malaria parasite, Plasmodium yoelii. Acetyl-CoA carboxylase mRNA was detected by RT-PCR performed on total RNA isolated from P. yoelii 17XL-infected mouse erythrocytes using primers designed from PY01695 ORF of the Plasmodb-published MALPY00458 gene of P. yoelii 17XNL. The RT-PCR was confirmed by sequencing and comparative analysis of the sequenced RT-PCR cDNA products. Northern blot analysis performed on total RNA using probes designed from a 1 kb region of the gene showed that the transcript was greater than the predicted 8.7 kb ORF. An immunogenic peptide corresponding to the P. yoelii theoretical acetyl-CoA carboxylase sequence was selected using epitope prediction and multiple sequence alignment algorithms. The immunogenic peptide was coupled to rabbit albumin carrier for immunisation in chickens and the affinity purified antibody titre was approximately 25 mg. The anti-peptide antibodies detected a 330 kD protein in P. yoelii lysate blot, which corresponds to the predicted size of the enzyme. The enzyme was also detected in situ by immunofluorescence microscopy using the anti-peptide antibodies. A 1 kb region of the P. yoelii acetyl-CoA carboxylase gene containing the biotinoyl domain was cloned and expressed in E. coli as 66 kD GST-tag and 45 kD His-tag protein. Both recombinant biotinoyl proteins were shown to contain bound biotin using peroxidaseconjugated avidin-biotin detection system. This suggested in vivo biotinylation of the recombinant P. yoelii biotinoyl protein, possibly by the E. coli biotin protein ligase. The Proscan™ and the NetPhos 2.0™ algorithms were used to predict protein kinase phosphorylation sites on the biotin carboxylase and the carboxyltransferase domains of the enzyme. The three-dimensional structure of the biotinoyl and the biotin carboxylase domains were predicted using the SWISS-MODEL™ homology modelling algorithm. Homology modelling revealed a similarity in the 3D conformation of the predicted P. yoelii biotinoyl domain and the E. coli biotinoyl protein with negligible root mean square deviation. The model also revealed the possibility of inhibiting P. yoelii and falciparum acetyl-CoA carboxylases with soraphen A based on the similarity in conformation with S. cerevisiae biotin carboxylase and the stereochemical properties of the residues predicted to interact with soraphen A. This study demonstrated that malaria parasite expresses acetyl-CoA carboxylase and, combined with data on other enzymes involved in fatty acid metabolism suggests that the parasite synthesizes fatty acids de novo. This enzyme could be a target for rational drug design.
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Malaria, caused by the parasite Plasmodium, continues to exact high global morbidity and mortality rate next only to tuberculosis. It causes 300-500 million clinical infections out of which more than a million people succumb to death annually. Worst affected are the children below 5 years of age in sub-Saharan Africa. Plasmodium is a protozoan parasite classified under the phylum Apicomplexa that also includes parasites such as Toxoplasma, Lankestrella, Eimeria and Cryptosporidium. Of the four species of Plasmodium affecting man viz., P. falciparum, P. vivax, P. ovale and P. malariae, Plasmodium falciparum is the deadliest as it causes cerebral malaria. The situation has worsened recently with the emergence of drug resistance in the parasite. Therefore, deciphering new pathways in the parasite for developing lead antimalarial compounds is the need of the hour. The discovery of the type II fatty acid biosynthesis pathway in Plasmodium falciparum has opened up new avenues for the design of new antimalarials as this pathway is different from the one in human hosts. Although many biochemical pathways such as the purine, pyrimidine and carbohydrate metabolic pathways, and the phospholipid, folate and heme biosynthetic pathways operate in the malaria parasite and are being investigated for their amenability as antimalarial therapeutic targets, no antimalarial of commercial use based on the direct intervention of these biochemical pathways has emerged so far. This is due to the fact that the structure and function of the targets of these drugs overlaps with that of the human host. A description of the parasite, its metabolic pathways, efforts to use these pathways for antimalarial drug discovery, inhibitors targeting these pathways, introduction to fatty acid biosynthesis pathway, discovery of type II fatty acid biosynthesis pathway in Plasmodium falciparum and prospects of developing lead compounds towards antimalarial drug discovery is given in Chapter 1 of the thesis. In the exploration of newly discovered type II fatty acid biosynthesis pathway of P. falciparum as a drug target for antimalarial drug discovery, one of the enzymes; β-hydroxyacyl- acyl carrier protein dehydratase (PfFabZ) was cloned and being characterized in the lab. The atomic structure of PfFabZ was not known till that point of time. Chapter 2 describes the homology modeled structure of PfFabZ and docking of the discovered inhibitors with this structure to provide a rationale for their inhibitory activity. Despite low sequence identity of ~ 21% with the closest available atomic structure then, E. coli FabA, a good model of PfFabZ could be built. A comparison of the modeled structure with recently determined crystal structure of PfFabZ is provided and design of new potential inhibitors is described. This study provides insights to further improve the inhibition of this enzyme. Enoyl acyl carrier protein reductase (ENR) is the most important enzyme in the type II fatty acid biosynthesis pathway. It has been proved as an important target for antibacterial as well as antimalarial drug discovery. The most effective drug against tuberculosis – Isoniazid targets this enzyme in M. tuberculosis. The well known antibacterial compound – Triclosan, a diphenyl ether, also targets this enzyme in P. falciparum. I designed a number of novel diphenyl ether compounds. Some of these compounds could be synthesized in the laboratory. Chapter 3 describes the design, docking studies and inhibitory activity of these novel diphenyl ether compounds against PfENR and E. coli ENR. Some of these compounds inhibit PfENR in nanomolar concentrations and EcENR in low micromolar concentrations, and many of them inhibit the growth of parasites in culture also. The structure activity relationship of these compounds is discussed that provides important insights into the activity of this class of compounds which is a step towards developing this class of compounds into an antimalarial and antibacterial candidate drugs. Components of the green tea extract and polyphenols are well known for their medicinal properties since ages. Recently they have been shown to inhibit components of the bacterial fatty acid biosynthesis pathway. Some selected tea catechins and polyphenols were tested in the laboratory for their inhibitory activity against PfENR. I conducted docking studies to find their probable binding sites in PfENR. On kinetic analysis of their inhibition, these compounds were found to be competitive with respect to the cofactor NADH. This has an implication that they could potentiate inhibition of PfENR by Triclosan in a fashion similar to that of NADH. As a model case, one of the tea catechins; EGCG ((-) Epigalocatechin gallate) was tested for this property. Indeed, in the presence of EGCG, the inhibition of PfENR improved from nanomolar to picomolar concentration of Triclosan.conducted molecular modeling studies and propose a model for the formation of a ternary complex consisting of EGCG, Triclosan and PfENR. Docking studies of these inhibitors and a model for the ternary complex is described in Chapter 4. Docking simulations show that these compounds indeed occupy NADH binding site. This study provides insights for further improvements in the usage of diphenyl ethers in conjugation or combination with tea catechins as possible antimalarial therapeutics. In search for new lead compounds against deadly diseases, in silico virtual screening and high throughput screening strategies are being adopted worldwide. While virtual screening needs a large amount of computation time and hardware, high throughput screening proves to be quite expensive. I adopted an intermediate approach, a combination of both these strategies and discovered compounds with a 2-thioxothiazolidin-4-one core moiety, commonly known as rhodanines as a novel class of inhibitors of PfENR with antimalarial properties. Chapter 5 describes the discovery of this class of compounds as inhibitors of PfENR. A small but diverse set of 382 compounds from a library of ~2,00,000 compounds was chosen for high throughput screening. The best compound gave an IC50 of 6.0 µM with many more in the higher micromolar range. The compound library was searched again for the compounds similar in structure with this best compound, virtual screening was conducted and 32 new compounds with better binding energies compared to the first lead and reasonable binding modes were tested. As a result, a new compound with an IC50 of 240 nM was discovered. Many more compounds gave IC50 values in 3-15 µM range. The best inhibitor was tested in red blood cell cultures of Plasmodium, it was found to inhibit the growth of the malaria parasite at an IC50 value of 0.75 µM. This study provides a new scaffold and lead compounds for further exploration towards antimalarial drug discovery. The summary of the results and conclusions of studies described in various chapters is given in Chapter 6. This chapter concludes the work described in the thesis. Cloning, over-expression and purification of PanD from M. tuberculosis, FabA and FabZ from E. coli are described in the Appendix.

The disease malaria afflicts more than a billion people and kills almost one to three million of them every year. Of the four species of Plasmodium affecting man viz., P. falciparum, P. vivax, P. ovale and P. malariae, Plasmodium falciparum is the deadliest as it causes cerebral malaria. The situation has become worse with the continuous emergence of drug resistance in the parasite. Therefore, improving existing drugs and deciphering new pathways for drug development are the need of the hour. The discovery of the type II fatty acid biosynthesis pathway in Plasmodium falciparum (Surolia and Surolia, 2001) has opened up new avenues for the development of new antimalarials as this pathway is entirely different from the human host in which type I pathway exists. Although many biochemical pathways such as the purine, pyrimidine and carbohydrate metabolic pathways, and the phospholipid, folate and heme biosynthetic pathways operate in the malaria parasite and are being investigated for their amenability as antimalarial therapeutic targets, no antimalarial of commercial use based on the direct use of these biochemical pathways as targets has emerged so far. This is due to the fact that the structure and function of the targets of these drugs overlaps with that of the human host. A description of such pathways forms the Chapter 1 of the thesis. This is followed by a description of the discovery and the importance of fatty acid biosynthesis pathway and the available literature on the various enzymes that are targets of potential antimalarials. Three isoforms are known for condensing enzymes - FabH which functions in initiation, and FabB and FabF which function in elongation. These isoforms differ in their biochemical properties and have unique roles to play in deciding the membrane composition of any organism. This aspect is also discussed in this chapter. Cloning and expression of -ketoacyl-ACP synthase, FabB/F from Plasmodium falciparum is described in Chapter 2. PfFabB/F is coded by the nuclear genome and is targeted to the apicoplast. The gene is coded by the locus MAL6P1.165 and the putative amino acid sequence of the protein exists in PlasmoDB. All apicoplast targeted proteins have a characteristic bipartite leader sequence consisting of a signal and a transit peptide sequence (Waller et al., 1998). Since the mature protein start site was not known and none of the software packages could predict the site, I aligned the PfFabB/F sequence with the sequences of other -ketoacyl-ACP synthases. On the basis of similarity with E. coli synthases and the mature protein start site of plant synthases, I cloned the first construct of PfFabB/F. The sequence was amplified by PCR and ligated in pET as well as pGEX vector. Expression in various hosts under different temperature and induction conditions could not solubilize the protein in significant quantities and most of the protein was found in inclusion bodies. Next I expressed the sequence with five more amino acids towards the N-terminal and expressed it as an N- terminal NusA fusion. The protein was purified by single step Ni-NTA affinity chromatography. Along with the full length protein (108 kDa), a truncated version of the protein was also obtained. The identity of the protein was confirmed by western blotting using anti-His antibody and anti-FabB/F antibody. In Chapter 3, the substrate specificity of PfFabB/F has been elucidated. PfFabB/F condenses malonyl-ACP with a range of acyl-ACPs. In vivo, acyl carrier protein (ACP) shuttles the acyl substrates between various enzymes of the fatty acid biosynthesis pathway. Enzymes of the pathway other than synthases can accept substrate analogs like acyl-CoA and acyl-NAC’s also in vitro. Acyl-ACPs are not very stable species and thus are not commercially available. Therefore, they have to be synthesized. Since malonyl-ACP could not be synthesized by chemical means, enzymatic synthesis of acyl-ACPs was done. Acyl-ACP synthetase (Aas) or holo-ACP synthase (ACPS) can be used for enzymatic synthesis. Aas is specific only for longer chain substrates; therefore, I decided to use holo-ACP synthase, an enzyme responsible for converting apo-ACP to holo-ACP in the presence of CoA in vivo (Lambalot and Walsch, 1995). When acyl-CoAs are supplied in place of CoA, acyl-ACP is produced. Malonyl-ACP and acyl-ACPs (C4-C16:1) were thus synthesized using holo-ACP synthase from E. coli. The reaction went to almost 95% completion, indicating broad substrate specificity of this enzyme. Bacterial or plant acyl-ACPs of different chain lengths can be resolved by Conformation Sensitive PAGE (Heath and Rock, 1995, Post- Beittenmiller et al., 1991). However, Pfacyl-ACPs synthesized using ACPS did not show any significant shift on CS-PAGE. Therefore I resorted to MALDI-TOF (Matrix Assisted Laser Desorption and Ionization- Time Of Flight) for monitoring the PfFabB/F condensation reactions. PfFabB/F condensed C4-C12-ACPs with malonyl-ACP to their corresponding -ketoacyl-ACP products, with C6, C8 and C10-ACPs being most readily elongated. C14-ACP was very sluggishly elongated, and C16 and C16:1-ACPs were not elongated at all. The condensation reaction was also followed by autoradiography using14C labeled malonyl-ACP, exploiting the clear mobility shift between malonyl-ACP and the other acyl-ACPs. The inhibitory effect of cerulenin, a known inhibitor of condensing enzymes was also checked. PfFabB/F also exhibited malonyl decarboxylase activity resulting in the production of acetyl-ACP in the absence of any significant condensation activity. All the enzymes of fatty acid synthesis pathway required to complete a cycle were assembled together for the in vitro reconstitution of Plasmodium fatty acid synthesis cycle which is described in Chapter 4. Earlier studies of Surolia & Surolia have shown that C12 and C14 fatty acids are the major constituents of Plasmodium lipids. One of my objectives was to determine the maximum length of the acyl ACP product that is synthesized when all the functionally active enzymes of fatty acid synthesis are put together (Kapoor et. al, 2001, Sharma et al., 2003, Karmodiya and Surolia, 2006). Condensing enzymes have a deterministic role in the fatty acid composition as they catalyze the only irreversible step in fatty acid biosynthesis. By analyzing products of the elongation cycle by mass spectrometry it was apparent that C14-ACP is the longest species formed. As already mentioned, PfFabB/F readily elongates C12-ACP but C14-ACP is weakly elongated. Thus the end product of the Plasmodium FAB pathway is influenced by the substrate specificity of PfFabB/F. This confirms the role of PfFabB/F as a decisive enzyme in determining the length of fatty acids synthesized. The inhibition of the cycle by cerulenin and triclosan is also described in this chapter. Chapter 5 describes the ability of the PffabB/F gene to complement for the mutation of condensing enzymes in CY244 cells (fabBtsfabF-, Yasuno et al., 2004). CY244 cells were transformed with pBAD alone or PfFabB/F cloned in pBAD vector (pBADPffabB/F) and the growth was monitored at non-permissive temperature. The product of PfFabB/F could rescue the growth of mutant cells at high temperature but only in the presence of oleic acid. FabB and FabF are the isoforms of condensing enzymes involved in elongation of the fatty acid synthesis cycle but they have a unique role to play (Garwin et al., 1980). FabB is responsible for unsaturated fatty acid synthesis, and fabB-mutants require oleic acid supplementation for growth. FabF is utilized in temperature regulation of membrane fluidity and E. coli FabF elevates the level of C18:1 or cis-vaccenic acid at lower growth temperature but FabF-mutants have no growth phenotype (Ulrich et al., 1983). Rescue of CY244 cells in the presence of oleic acid supplementation indicated that the PffabB/F gene behaves like FabF and not FabB. Analysis of the fatty acid composition of membrane lipids of CY244 cells transformed with pBAD vector or pBADPffabB/F by GC-MS demonstrated no elevated levels of cis-vaccenic acid in transformed cells. This observation is in agreement with the in vitro determined substrate specificity data which shows that PfFabB/F does not elongate C16:1ACP. The thesis ends with a summary of the findings in Chapter 6 in the context of FabB and FabF enzymes known from other sources. 2, 4, 4’-Trichloro-2’hydroxydiphenylether, commonly known as triclosan, has been used as a topical antibacterial agent for decades. I determined its efficacy in treating acute systemic bacterial infection in mouse model. Triclosan, as compared to other well known antibiotics, could extend the survival time of mice by 48 hours. This work is described in Appendix I. (Sharma et al., 2003)

Malaria, caused by Plasmodium, is one of the most devastating infectious diseases of the world in terms of mortality as well as morbidity (WHO, 2002). The development of resistance in the Plasmodium falciparum against the present antimalarials has made the situation very alarming (Trape et al., 2000). To combat this situation, new antimalarials as well as identification of new drug targets are urgently required. The discovery of the presence of type II fatty acid biosynthesis system in the malarial parasite has offered several promising new targets for this mission. This thesis describes the successful cloning of fabZ from Plasmodium falciparum, its expression in E. coli, single step affinity purification, kinetic characterization and most importantly discovery of two small molecule inhibitors (Sharma et al., 2003). The study was executed to gain insights into the structure and function of PfFabZ to get better understanding of the interactions with its substrate analogs, novel inhibitors and also acyl carrier protein (PfACP). The molecular details of the interactions of the two novel inhibitors were also determined. Lastly, the residues of PfFabZ important for the interaction with PfACP were successfully elucidated. Chapter 1 presents a brief review of the literature about the disease as well as the life cycle, biology and the metabolic pathways operational in malarial parasite, Plasmodium falciaparum. The discovery of type II FAS in P. falciparum and the aims and the scope of the thesis are also discussed. The quest of developing new antimalarials, study of the mechanism of actions of antimalarials such as quinine and its derivatives along with the major metabolic pathways (Purine, pyrimidine, phospholipids, carbohydrate metabolism, folate and heme biosynthesis pathways etc.) existing in P. falciparum are described in detail in this chapter. Origin and importance of apicoplast in P. falciaprum is also described in brief. For long, it was believed that Plasmodium spp. are incapable of de novo fatty acid synthesis but this view has undergone substantial revision due to the recent discovery of plant and bacterial type of fatty acid biosynthesis pathway in them (Surolia and Surolia, 2001). As this pathway is distinct from that of the human host it has accelerated the momentum for the discovery of new antimalarials (Surolia and Surolia, 2001). The Chapter also surveys the details of type II FAS in bacteria, particularly that of E. coli (Rock and Cronan, 1996). The dehydratase step which is the third step of fatty acid elongation cycle has been covered in considerable detail. Lastly, it focuses on the recent advancement in the understanding of fatty acid biosynthesis system in Plasmodium falciparum along with some inhibitors targeting the malarial FAS. As each enzyme of the Plasmodium FAS can serve as good antimalarial targets, my work focuses on the dehydratase step catalyzed by β-hydroxyacyl-ACP dehydratase (PfFabZ). Cloning, expression and kinetic characterization of PfFabZ forms the major content of Chapter 2. The PlasmoDB data base was searched for this gene and the mined out open reading frame contained sequence of the putative FabZ together with the bipartite leader polypeptide. Our aim was to clone the mature PfFabZ without the bipartite leader sequence. Amplification of the mature pffabZ using Plasmodium falciparum genomic DNA revealed the presence of an intron in the ORF and the gene was finally cloned by RT-PCR in pET-28a(+) vector. It was expressed with an N-terminal hexahistidine tag in BL-21(DE3) cells and purified to near homogeneity but the protein was insoluble and unstable. Truncation of 12 residues from the N-terminal end improved the stability and solubility of the protein by 3-5 fold. Truncated PfFabZ was used for all future experiments. FabZs from other sources are reported to be hexamer in solution but PfFabZ showed homodimeric arrangement in the conditions used for gel filtration as well as dynamic light scattering studies. Kinetics of PfFabZ was characterized using substrate analogs, β-hydroxybutryl-CoA (forward substrate) and Crotonoyl-CoA (reverse substrate). Both the forward and reverse reaction were thoroughly characterized by spectrophotometry and HPLC and the reverse reaction was found to be 7 times faster than the forward reaction. Km οf crotonoyl-CoA was calculated to be 86 µM and kcat/Km of 220 M-1s-1 whereas the Kmfor β-hydroxybutryl-CoA was found to be 199 µM and kcat/Kmof 80.2 M-1s-1. The kinetic data clearly indicates the higher affinity of PfFabZ for the reverse substrate. Chapter 3 describes the discovery of two small molecules inhibitors, NAS-21 and NAS-91 for PfFabZ, their detailed inhibition kinetics and their effect on the growth of Plasmodium falciparum in culture. These inhibitors were the first inhibitors to be reported for FabZ class of enzymes with an IC50 ranging below 15 µM. Both of them inhibited PfFabZ following competitive kinetics with respect to the substrates utilized for both the forward and reverse reactions. The inhibition data were analyzed by Lineweaver-Burk and Dixon plots and both inhibitors showed competitive inhibition kinetics with dissociation constant in submicromolar range. Binding constants for both the inhibitors were also determined by fluorescence titration method and were calculated to be 1.6 (± 0.04) X 106 M-1 for NAS-91 and 1.2 (± 0.03) X 106 M-1 for NAS-21. These inhibitors were checked on Plasmodium falciparum culture and both inhibited parasite growth with IC50 values of 7 µM and 100 µM for NAS-21 and NAS-91, respectively. They also inhibited the incorporation of [1,2-14C]-acetate in the fatty acids of the P. falciparum conforming the inhibition of fatty acid biosynthesis. FabZ class of enzymes are thought to contain His-Glu as a catalytic dyad. Based on the disparity in the arrangement of residues at the active site of the dimeric (Swarnamukhi et al., 2006) and hexameric forms of PfFabZ in the crystal structures (Kosteriva et al., 2005), we set out to elucidate the active site residues in PfFabZ which is described in Chapter 4. The role of each of the presumed active site residues His-133 and Glu-147 along with Arg-99 and His-98 were analyzed by chemical modification studies and site directed mutagenesis. Single and double mutants were prepared and the activity of the mutants was monitored by spectrophotometry and isothermal titration calorimetry (ITC). It was concluded that in PfFabZ, His-133 and Glu-147 makes the catalytic dyad, His-98 might be important in directing the substrate in correct orientation while Arg-99 is involved in maintaining the active site loop in proper orientation rather than taking direct part in catalysis. Chapter 4 also concludes that dimeric form of PfFabZ is inactive species and turns into active hexameric form in the presence of substrate. Chapter 5 describes the molecular details of NAS-21 and NAS-91 interactions with PfFabZ. The fact that both these compounds inhibited PfFabZ in competitive manner, prompted me to examine their interaction with the residues in the active site tunnel. Apart from the His-133 and Glu-147 catalytic dyad the only polar residue is His-98 and chemical modification and site directed mutagenesis studies were done to elucidate the interactions of these residues with NAS-21 and NAS-91. Both the inhibitors were able to protect the modification of histidines by DEPC in wild type PfFabZ, His-98-Ala mutant and His-133-Ala mutant but with differential strength, indicating that they do interact with histidines. The interaction of these inhibitors was further confirmed by determining the dissociation constants of wPfFabZ, His-98-Ala, His-133-Ala, His-98-Ala/His-133-Ala double mutant, Glu-147-Ala mutant by fluorescence titration method. The results obtained from chemical modification and fluorescence titration studies confirmed that NAS-21 interacts strongly with histidines, His-98 and His-133 but not with Glu-147. On the other hand NAS-91 interacts loosely with His-98 and His-133 but strongly with Glu-147. Chapter 5 concludes with the observation that both the inhibitors (NAS-21 and NAS-91) interact with the active site residues of PfFabZ, preventing the substrate to enter the active site tunnel. Acyl carrier protein (ACP) is a small acidic protein to which the acyl chain intermediates are tethered and shuttled from one enzyme to another for the completion of fatty acid elongation cycle. Whenever acyl carrier proteins are expressed in E. coli, they are present in three forms apo, holo and acyl-ACPs. Chapter 6 describes a novel method for the expression of histidine tagged PfACP in pure holo form, protocol for the cleavage of his-tag from PfACP by thrombin preparation of homogenous singly enriched ie PfACP [15N]-labeled or [13C]-labeled PfACP as well as doubly enriched [15N]-[13C] PfACP samples for its structure elucidation by NMR (Sharma et al., 2005). These studies also constituted reporting of a holo-ACP structure from any of the sources for the first time (Sharma, et. al. 2006). The purified pure holo-PfACP was further used for the interaction studies with PfFabZ. Earlier studies have shown that ACP interacts with FAS enzymes via helix II with conserved set of residues but the molecular details of the interactions are poorly known (Zhang, et. al., 2003). We have recently solved the NMR structure (Sharma, et. al., 2006) of PfACP and crystal structure of PfFabZ (Swarnamukhi, et. al., 2006). So, both the structures were docked using Cluspro server. Chapter 7 elucidates the roles of important residues on PfFabZ surface near the active site entry which are responsible for interacting with PfACP. The residues lining the active site entry were identified and mutated. The residues lining the active site tunnel of PfFabZ are Arg102, Lys104, Lys105, Lys123, Leu94, Phe95, Ala96, Gly97, Ile128, Ile145, Phe150 and Ala151. Charged residues were mutated to alanine and also to oppositely charged residues while the neutral residues were changed to charged residues. The interaction of PfFabZ mutants with PfACP was studied by ACP independent enzymatic assay and surface plasmon resonance (SPR) spectroscopy. It was concluded that PfFabZ and PfACP interaction is mainly governed by electrostatic interaction made by the charged residues (Lys104 being the most important residue) and is fine tuned by hydrophobic interactions. Chapter 8 summarizes the findings of the thesis. FabZ from Plasmodium falciparum was cloned and biochemically characterized. Two inhibitors for this enzyme were discovered and their molecular details of binding to PfFabZ were elucidated. The presence of catalytic dyad was confirmed and finally the residues of PfFabZ important for interaction with PfACP were elucidated. Appendix I describes the inhibition of PfENR (enoyl ACP reductase), the rate limiting and the fourth enzyme of the fatty acid elongation pathway by green tea extracts. Three tea catechins (EGCG, EGC and ECG) and two plant polyphenols (quercetin and buteine) were selected for the inhibition study. All the catechins inhibited PfENR potently with Ki values in nanomolar range. Among the five compounds studied, EGCG was found to be the best inhibitor. All of them blocked the NADH binding site showing competitive kinetics with respect to NADH and uncompetitive kinetics with crotonoyl-CoA, the substrate analog. Most importantly, the catechins potentiated the inhibition of PfENR by triclosan, a well known PfENR inhibitor. We also report that in the presence of tea catechins triclosan behaves as a slow-tight binding inhibitor of PfENR. The overall inhibition constant of triclosan in the presence of EGCG was calculated to be 2pM which is 50 times better than the earlier reported values with NAD+ (Kapoor, et. al., 2004).

Ph.D., Faculty of Science, University of the Witwatersrand, 2011
This thesis describes the application pteridine chemistry in various aspects of methodology development and natural product synthesis. The introductory chapter sets the scene by describing naturally occurring pteridines, their applications in biological systems, and recent synthetic strategies. Firstly, Sonogashira coupling reactions employing benzenesulfonyloxy-O-pteridine (27) and terminal alkynes to give various 6-substituted pteridines are described. This methodology allowed for the total synthesis of a natural occurring pteridine, Sepiapterin-C (46). Negishi coupling reactions involving benzenesulfonyloxy-O-pteridine (27) and various Znreagents are also reported. This methodology, representing the first Negishi coupling on a pteridine nucleus, allowed for the introduction of both aryl- and heteroaryl- substituents at the 6- position of the pteridine ring. The use of methanesulfonyloxy-O-pteridine (26) as a coupling partner is also described. Selective deprotection and hydrolysis of the formamidine protecting groups to give either the 6- substituted 2,4-diaminopterine or 2-amino-4-oxo-pteridine (pterin), is described. The synthesized structures are supported by NMR and mass spectral data and melting points where applicable. Novel compounds are verified by NMR spectroscopy, infrared and mass spectrometry.

The intraerythrocytic stage of the malaria parasite Plasmodium falciparum has an absolute requirement for vitamin B5 (also known as pantothenate) in order to survive. The parasite takes up extracellular pantothenate and subsequently converts it into coenzyme A (CoA) via a series of five universal enzymatic steps. The first enzyme of the pathway is pantothenate kinase (PanK), which phosphorylates pantothenate and commits the molecule to CoA biosynthesis. There are two putative PanK genes (designated Pfpank1 and Pfpank2) in the parasite’s genome, both of which are expressed in the intraerythrocytic stage of the parasite. Many antiplasmodial pantothenate analogues, including pantothenol (PanOH), CJ-15,801, N-substituted pantothenamides (PanAms) and PanAm derivatives have been shown to inhibit P. falciparum growth by targeting its CoA biosynthesis and/or utilisation, although their exact mechanism of action in the parasite remains poorly characterised. In this study, a step-wise dose-escalating drug pressure regime with either PanOH or CJ-15,801 was used to generate resistant parasite lines. These parasite lines are cross-resistant to both PanOH and CJ-15,801, but exhibit different sensitivity profiles to the PanAm derivatives N5-trz-C1-Pan and N-PE-αMe-PanAm, consistent with these two groups of pantothenate analogues having different mechanisms of action. Whole-genome sequencing revealed that these parasites harbour mutations in Pfpank1. Some of these mutations significantly alter the activity of PfPanK, the parasite’s requirement for pantothenate and consequently their fitness compared to the Parent line. These results are consistent with PfPanK1 being the active PanK during this stage of the parasite’s lifecycle. When analysed in conjunction with what has been reported for other organisms, the results of functional enzymatic assays performed in this study revealed important information about the modes of action of these pantothenate analogues. PanOH and CJ-15,801 are predicted to inhibit PfPPCS (the second enzyme of CoA biosynthesis). Conversely, N5-trz-C1-Pan and N-PE-αMe-PanAm are hypothesised to be metabolised into CoA analogues, which subsequently inhibit downstream CoA-utilising enzymes. In order to characterise the activity, conformation and potential interacting partners of PfPanK1 and PfPanK2, green fluorescent protein (GFP)-fused copies of these proteins were expressed in P. falciparum parasites to enable their purification. Results of western blot and mass spectrometry analyses of immunoprecipitated PfPanK are consistent with the native protein being a complex that is comprised of PfPanK1, PfPanK2 and the adapter protein Pf14-3-3I. This marks the first description of a heterodimeric PanK in nature. In silico analysis of the amino acid sequence of the two PfPanKs and interrogation of existing phosphoproteomic studies suggest that Pf14-3-3I binds to PfPanK2. Taken together, the research presented in this study has extended our understanding of the P. falciparum PanKs and, therefore, provided further insight into the CoA biosynthesis pathway as an antimalarial drug target. Furthermore, the information generated in this study about the mechanisms of action of the pantothenate analogues will hopefully expedite the discovery of new antimalarials.

Malaria is a life-threatening vector borne infectious disease caused by protozoan parasites of the genus Plasmodium. More than 100 species of Plasmodium can infect numerous animal species such as reptiles, birds and various mammals. However, human malaria is caused by four Plasmodium species -Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae, and occasionally by the simian malaria parasite, Plasmodium knowlesi. Of these, P. falciparum and P. vivax are the major causative agents and P. falciparum is the most virulent. About 300-500 million malaria infections occur every year leading to over 1-2 million deaths, of which 75% occur in African children of less than 5 years infected with P. falciparum. In spite of major global efforts to eliminate this disease over the past few decades, it continues to persist as a major affliction of human-kind imposing serious health and economic burden, especially to the poor countries. In India, the present scenario is about 2 million malaria positive cases every year, with almost 50% being caused by P. falciparum. Although remarkable attempts have been made over the years to develop vaccines against sexual and asexual stages of malaria parasite, an effective vaccine is still not in sight and remains as a distant goal. Hence, highly potent, less toxic and affordable antimalarial drugs remain as a first line therapy for malaria. Unfortunately, these parasites have been evolving against every known antimalarial drug and many of these drugs have lost their potency due to rapid emergence and spread of drug resistant strains. With development of resistance against frontline antimalarials such as chloroquine and antifolates, artemisinin and its derivatives seem to be the only effective antimalarials. However, recent reports on the possible emergence of artemisinin resistant strains, have led to the implementation of artemisinin-based combination therapies as a strategy to prevent drug resistance. Also, this continuous emergence of drug resistance has necessitated the development of new antimalarial drugs to combat this disease. While, Anopheles mosquitoes transmit parasites that infect humans, monkeys and rodents, Culex and Aedes mosquitoes predominate in the natural transmission to birds, and vectors of reptilian parasites are largely unknown. Of the approximately 400 species of Anopheles throughout the world, about 60 are malaria vectors under natural conditions, and 30 of which are of major importance. Ironically, the strategies implemented for controlling Anopheles, have also been hampered by insecticide resistance and other practical difficulties that exist in the scope of their applicability. In the past few years several milestones have been achieved in parasite genome, transcriptome and proteome studies, which could be exploited for the development of new drugs and drug targets. One such promising target includes the metabolic pathways of the malaria parasite which differ significantly from its human host. This thesis entitled “Unique Features of the Heme-Biosynthetic Pathway in Human Malaria Parasite, Plasmodium falciparum” unravels the unique biochemical features of heme-biosynthetic enzymes of P. falciparum, which have the potential for being drug targets. This pathway was first identified in this laboratory over 15 years ago. In the present study, five of the 7 enzymes of this pathway have been cloned, expressed, properties studied and sites of localization identified. With the knowledge on the first two enzymes coming from earlier studies, it is now possible to depict the unique hybrid pathway for heme biosynthesis in P. falciparum with full experimental validation.