Molecular Mechanics Study of Protein Folding and Protein-Ligand Binding

In this dissertation, molecular dynamics (MD) simulations were applied to study the effect of single point mutations on protein folding free energy and the protein-ligand binding in the bifunctional protein dihydrofolate reductase-thymidylate synthase (TS-DHFR) in plasmodium falciparum (pf). The main goal of current computational studies is to have a deeper understanding of factors related to protein folding stability and protein-ligand binding. Chapter two aims to seek solutions for improving the accuracy of predicting changes of folding free energy upon single point mutations in proteins. While the importance of conformational sampling was adequately addressed, the diverse dielectric properties of proteins were also taken into consideration in this study. Through developing a three-dielectric-constant model and broadening conformational sampling, a method for predicting the effect of point mutations on protein folding free energy is described, and factors of affecting the prediction accuracy are addressed in this chapter. The following two chapters focus on the binding process and domain-domain interactions in the bifunctional protein pfDHFR-TS. This protein usually plays as the target of antimalarial drugs, but the drug resistance in this protein has caused lots of problems. In chapter three, the mechanism of the development of drug resistance was investigated. This study indicated that the accumulation of mutations in pfDHFR caused obvious changes of conformation and interactions among residues in the binding pocket, which further weakened the binding affinity between pfDHFR and the inhibitor drug. Furthermore, the high rigidity and significantly weakened communications among key residues in the protein binding pocket were exhibited in the pfDHFR quadruple mutant. The rigid binding site was associated with the failure of conformational reorganization upon the binding of pyrimethamine in the quadruple mutant. Chapter four investigated the effect of the N-terminus in pfDHFR-TS on enzyme activity and domain-domain communications. This is the first computational study that focuses on the full-length pfDHFR-TS dimer. This study provided computational evidence to support that remote mutations could disturb the interactions and conformations of the binding site through disrupting dynamic motions in pfDHFR-TS

Drug resistant malaria in Papua New Guinea and molecular monitoring of parasite resistance

Malaria is a serious global health problem and in the absence of an effective vaccine, access to safe and effective treatment still remains the mainstay in the control of the disease. However, the efficacy of this control strategy is hampered by the emergence and spread of drug resistant malaria which may lead to excess of mortality. One of the greatest challenges for health authorities of malaria endemic countries is thus to decide on when and how antimalarial drug policy should be changed, so that most of the patients will fully recover from the disease and will be cleared from parasites. The current ‘gold standard’ for the assessment of antimalarial resistance is the estimation of in vivo drug efficacy, whereas in vitro drug sensitivity tests and the analysis of molecular resistance markers in the parasite serve as complementary tools. In the present study, we assessed the relevance of a new appraisal approach for malaria resistance: community-based cross-sectional surveys versus clinical malaria studies, and the usefulness of a new molecular technology for the identification of molecular markers in different parasite genes. The frequencies of single nucleotide polymorphisms (SNPs) in given resistance marker genes, as well as genotype patterns were analyzed in clinical samples and their role in predicting in vivo treatment response was investigated. Furthermore, community drug resistance profiles were correlated with the incidence risk of clinical treatment failure in order to evaluate the relevance and usefulness of such a novel approach in the management of drug use. In Papua New Guinea (PNG), the 4-aminoquinoline drugs amodiaquine (AQ) and chloroquine (CQ) have been first-line treatment against uncomplicated malaria until the late 1990s. At the same time, resistance of Plasmodium falciparum and P. vivax to these drugs had reached unacceptably high levels and health authorities were prompted to revise antimalarial treatment policy in 1997. First efficacy trials with the combination of AQ or CQ plus SP conducted between 1998 and 1999 showed good efficacy against falciparum and vivax malaria and the PNG Department of Health chose these combination regimens to replace the monotherapy with AQ or CQ as the standard first-line treatment against uncomplicated malaria in 2000. The in vivo studies we conducted between 2003 and 2005 were the first ones to assess the therapeutic efficacy of the newly introduced combination regimen against P. falciparum and P. vivax malaria using the revised WHO standard protocol. In our studies conducted in three different areas over the period of three consecutive years, we observed PCR-corrected treatment failure rates up to 28% for P. falciparum and 12% for P. vivax malaria. Regarding former drug history in PNG (i.e., long lasting 4-aminoquinoline use and sporadic use of SP as mass chemprophylaxis or partner drug with quinine for second-line treatment), we found a genetic background in the parasite population that is associated with high CQ as well as moderate pyrimethamine resistance. We also observed the emergence of mutations concordant with a sulphadoxine resistant phenotype, indicating that the efficacy of the sulpha component is already compromised. Further results that identified key pfdhps mutations to be most relevant in predicting treatment failure with the current first-line regimen corroborated our findings that AQ and CQ as inefficacious partner drugs of SP in the new standard treatment were not able to curb both, the progression of pyrimethamine resistance as well as the emergence of sulphadoxine resistance in PNG. We have shown that our community-based molecular monitoring approach was feasible in PNG and that molecular monitoring of parasite resistance can indeed be a valuable supplementary tool in malaria resistance surveillance. However, our data also clearly highlighted several drawbacks of the presently applied methods for the assessment of resistance, the most important being the lack of standardised methods that are applicable in different epidemiological settings. In addition, our data indicate that currently suggested public health models for the molecular monitoring of parasite resistance are not suitable for universal application in settings which are different with regard to several factors such as malaria endemicity, transmission intensity and drug use patterns. To summarize, decreasing in vivo efficacy of the current first-line regimen in PNG and the molecular drug resistance profile of the parasite population consistent with a CQ and SP resistant phenotype strongly indicate that a policy change to artemisinin-based combination therapy (ACT) has to be considered in the near future. We have shown that a careful baseline evaluation of the molecular resistance background is needed for the identification of the most relevant molecular markers for longitudinal monitoring in a given area. The novel DNA microarray-based method which allows the parallel analysis of multiple drug resistanceassociated SNPs has been proven to be a valuable tool to assess the usefulness of each known molecular marker in a particular region with specific drug use. Moreover, the new technology enabled the assessment of molecular markers on an epidemiological scale and hence opened the avenue for the investigation of a more comprehensive community-based monitoring programme. To conclude, the novel technical tool for the assessment of molecular markers of parasite resistance presented in the current study is cheap, easy to use, and applicable in laboratories with limited infrastructure. Moreover, the technology is highly versatile and allows rapid adaptation to specific monitoring needs, the most important at the moment being the close monitoring of resistance to the highly effective artemisinin derivates and potential partner drugs in ACTs. Though molecular markers have been proven to be useful as an early warning system, their usefulness in predicting treatment response and the progression of resistance is still limited. Hence, currently suggested public health models based on molecular data will have to include additional parameters for important determinants of parasite resistance and to be evaluated in varying epidemiological settings before molecular methods may eventually replace in vivo efficacy studies for the surveillance of resistance

Mutagénèse semi-aléatoire au site actif de la DHFR humaine : création et caractérisation de variantes hautement résistantes au MTX

La dihydrofolate réductase humaine (DHFRh) est une enzyme essentielle à la prolifération cellulaire. Elle réduit le dihydrofolate en tétrahydrofolate, un co-facteur impliqué dans la biosynthèse des purines et du thymidylate. La DHFRh est une cible de choix pour des agents de chimiothérapie comme le méthotrexate (MTX), inhibant spécifiquement l’enzyme ce qui mène à un arrêt de la prolifération et ultimement à la mort cellulaire. Le MTX est utilisé pour le traitement de plusieurs maladies prolifératives, incluant le cancer. La grande utilisation du MTX dans le milieu clinique a mené au développement de mécanismes de résistance, qui réduisent l’efficacité de traitement. La présente étude se penche sur l’un des mécanismes de résistance, soit des mutations dans la DHFRh qui réduisent son affinité pour le MTX, dans le but de mieux comprendre les éléments moléculaires requis pour la reconnaissance de l’inhibiteur au site actif de l’enzyme. En parallèle, nous visons à identifier des variantes plus résistantes au MTX pour leur utilisation en tant que marqueurs de sélection en culture cellulaire pour des systèmes particuliers, tel que la culture de cellules hématopoïétiques souches (CHS), qui offrent des possibilités intéressantes dans le domaine de la thérapie cellulaire. Pour étudier le rôle des différentes régions du site actif, et pour vérifier la présence d’une corrélation entre des mutations à ces régions et une augmentation de la résistance au MTX, une stratégie combinatoire a été dévelopée pour la création de plusieurs banques de variantes à des résidus du site actif à proximité du MTX lié. Les banques ont été sélectionnées in vivo dans un système bactérien en utilisant des milieux de croissance contenant des hautes concentrations de MTX. La banque DHFRh 31/34/35 généra un nombre considérable de variantes combinatoires de la DHFRh hautement résistantes au MTX. Les variantes les plus intéressantes ont été testées pour leur potentiel en tant que marqueur de sélection dans plusieurs lignées cellulaires, dont les cellules hématopoïétiques transduites. Une protection complète contre les effets cytotoxiques du MTX a été observée chez ces cellules suite à leur infection avec les variantes combinatoires. Pour mieux comprendre les causes moléculaires reliées à la résistance au MTX, des études de structure tridimensionnelle de variantes liées au MTX ont été entreprises. La résolution de la structure de la double variante F31R/Q35E lié au MTX a révélé que le phénotype de résistance était attribuable à d’importantes différences entre le site actif de la double variante et de l’enzyme native, possiblement dû à un phénomème dynamique. Une compréhension plus générale de la reconnaissance et la résistance aux antifolates a été réalisée en comparant des séquences et des structures de variantes de la DHFR résistants aux antifolates et provenant de différentes espèces. En somme, ces travaux apportent de nouveaux éléments pour la comprehension des intéractions importantes entre une enzyme et un ligand, pouvant aider au développement de nouveaux antifolates plus efficaces pour le traitement de diverses maladies. De plus, ces travaux ont généré de nouveaux gènes de résistance pouvant être utilisés en tant que marqueurs de sélection en biologie cellulaire.Human dihydrofolate reductase (hDHFR) is an enzyme that is essential to cell proliferation. It reduces dihydrofolate to tetrahydrofolate, an important cofactor involved in purine and thymidylate biosynthesis. hDHFR is a choice target for chemotherapeutic drugs like methotrexate (MTX), which specifically inhibits the enzyme, stopping cell proliferation and leading to cellular death. MTX is used for the treatment of many proliferative diseases, including cancers. Widespread use of MTX has lead to the development of resistance mechanisms appear which impair treatment efficiency. The present work focuses on a mechanism of resistance, namely mutations in hDHFR that reduce its affinity for MTX, to better understand the underlying mechanisms of inhibitor recognition at the active site of the enzyme. In parallel, we aim at identifying the most MTX-resistant variants to offer novel selectable markers for particular cell culture systems, such as hematopoietic cell culture, which offer important perspectives for cellular therapy. To study the role of different regions of the hDHFR active site, and to verify if a correlation exists between mutations in these regions and increased resistance to MTX, a combinatorial strategy was developed enabling the creation of several hDHFR variant libraries at active site residues located in proximity to bound MTX. The libraries were selected in vivo in a bacterial system using culture media containing high concentration of the inhibitor. One library in particular, hDHFR 31/34/35, yielded a considerable number of highly MTX-resistant combinatorial hDHFR variants. The most interesting candidates were tested for their potential as selectable markers in various cell lines, including transduced hematopoietic cells. Complete protection from MTX-cytotoxicity was obtained for these cells following infection with the combinatorial variants. To better understand the molecular causes of MTX resistance, resolution of the crystal structures of variant proteins in presence of MTX was attempted. Resolution of the F31R/Q35E double variant revealed that the resistance phenotype was related to important differences in the active site relative to WT, possibly attributable to a dynamic motion effect. A more general comprehension of antifolate recognition and resistance was achieved by sequence and structural comparison of antifolate-resistant DHFR variants from different species. Overall, our work contributes to the better understanding of enzyme-inhibitor interactions, which could provide new insights into the development of more efficient clinical therapies. In addition, this work has yielded novel drug-resistance genes useful as selectable markers for cellular biology

Development of Potent and Selective Inhibitors of Mycobacterium Tuberculosis, Plasmodium Falciparum and Staphylococcus Aureus Dihydrofolate Reductase

The goal of this study was to develop drugs that exclusively affect pathogenic dihydrofolate reductase (DHFR) without causing harm to the human counterpart. To achieve that goal, a well-known dihydrofolate reductase (DHFR) inhibitors, trimethoprim (TMP), methotrexate (MTX) and trimetrexate (TMQ), were modified, tested, and crystallized on Mycobacterium tuberculosis (Mtb) dihydrofolate reductase (DHFR), wild type and quadruple mutant Plasmodium falciparum (Pf) DHFR-thymidylate synthase (TS), Staphylococcus aureus DHFR, and human DHFR. We focused on the drug design to utilize the structural differences between the pathogenic DHFRs and the human DHFR; specifically, we focused on a pocket near the substrate binding site where Asp27 and Gln28 of Mtb DHFR, and Asp54 and Met55 of Pf DHFR-TS are located. The same site is closely packed in human DHFR. From the initial screening and designing process, C-8 benzyl-2,4-diaminoquinazoline TMQ analogs were found to have outstanding selectivity against Mtb and Pf DHFR. Co-crystal structures of C-8 benzyl TMQ analogs with Mtb and Pf DHFR showed that the flexibility of Gln28 in Mtb DHFR, and Met55 in Pf DHFR contributes to extra space and interaction with C-8 benzyl moiety. This flexibility, which is not available in the human DHFR, enables the TMQ analogs to bind exclusively to the pathogenic DHFRs. Our novel C-8 benzyl-2,4-diaminoquinazoline TMQ analogs exhibited great potency and selectivity toward pathogenic DHFRs. In addition, these C-8 benzyl-2,4-diaminoquinazoline TMQ analogs were potent on Staphylococcus aureus DHFR as well and we hypothesize based on our findings that our C-8 benzyl-2,4-diaminoquinazoline TMQ analogs have potential for selective, broad spectrum antimicrobials against whose DHFR share the common structural feature with Mtb or Pf DHFR, an acid residue and a flexible residue next to it

Advances in Malaria Pharmacology and the online Guide to MALARIA PHARMACOLOGY: IUPHAR Review X

Antimalarial drug discovery has until recently been driven by high-throughput phenotypic cellular screening, allowing millions of compounds to be assayed and delivering clinical drug candidates. In this review, we will focus on target-based approaches, describing recent advances in our understanding of druggable targets in the malaria parasite. Targeting multiple stages of the Plasmodium lifecycle, rather than just the clinically symptomatic asexual blood stage, has become a requirement for new antimalarial medicines, and we link pharmacological data clearly to the parasite stages to which it applies. Finally, we highlight the IUPHAR/MMV Guide to MALARIA PHARMACOLOGY, a web resource developed for the malaria research community that provides open and optimized access to published data on malaria pharmacology

Back to the Future: Lessons Learned in Modern Target-based and Whole-Cell Lead Optimization of Antimalarials

Antimalarial drug discovery has historically benefited from the whole-cell (phenotypic) screening approach to identify lead molecules in the search for new drugs. However over the past two decades there has been a shift in the pharmaceutical industry to move away from whole-cell screening to target-based approaches. As part of a Wellcome Trust and Medicines for Malaria Venture (MMV) funded consortium to discover new blood-stage antimalarials, we used both approaches to identify new antimalarial chemotypes, two of which have progressed beyond the lead optimization phase and display excellent in vivo efficacy in mice. These two advanced series were identified through a cell-based optimization devoid of target information and in this review we summarize the advantages of this approach versus a target-based optimization. Although the each lead optimization required slightly different medicinal chemistry strategies, we observed some common issues across the different the scaffolds which could be applied to other cell based lead optimization programs