Computer aided approaches against Human African Trypanosomiasis

The thesis presented here is divided into two parts under a common theme that is the use of computer based tools, genomics, and in vitro experiments to develop innovative ways of tackling Human African Trypanosomiasis (HAT). Part I of this thesis focused on the human host genetic determinants while Part II focused on the discovery of novel chemotherapeutics against the parasite. Part I is further sub-divided into two parts: The first involves a Candidate Gene Association Study (CGAS) on an African population to identify genetic determinants associated with disease and/or susceptibility to HAT. The second involves studying the effects of missense Single Nucleotide Variants (SNVs) on protein structure, dynamics, and function using Macrophage Migration Inhibitory Factor (MIF) as a case study. Part II is also sub-divided into two parts: The first involves a computer based rational drug discovery of potential inhibitors against the Trypanosoma the folate pathway; particularly by targeting Trypanosoma brucei Pteridine Reductase (TbPTR1) which is an enzyme used by trypanosomes to overcome T. brucei Dihydrofolate Reductase (TbDHFR) inhibition. Lastly the derivation of CHARMM force-field parameters that can be used to accurately model the geometry and dynamics of the T. brucei Phosphodiesterase B1 enzyme (TbrPDEB1) bimetallic active site center. The derived parameters were then used in MD simulations to characterise protein-ligand residue interactions that are important in TbrPDEB1 inhibition with the goal of targeting the cyclic Adenosine Monophosphate (cAMP) signalling pathway. In the CGAS we were unable to detect any genetic associations in the Ugandan cohort analysed that passed correction for multiple testing in spite of the study being sufficiently powered. Additionally, our study found no association of the Apo lipoprotein 1 (APOL1) G2 allele association with protection against acute HAT that has been previously reported. Future investigations for example, Genome Wide Association Studies using larger samples sizes (>3000 cases and controls) are required. Macrophage migration inhibitory factor (MIF) is a cytokine that is important in both innate and adaptive immunity that has been shown to play a role in T. brucei pathogenicity using murine models. A total of 27 missense SNVs were modelled using homology modelling to create MIF protein mutants that were investigated using in silico effect prediction tools, molecular dynamics (MD), Principal Component Analysis (PCA), and Dynamic Residue Network (DRN) analysis. Our results demonstrate that mutations P2Q, I5M, P16Q, L23F, T24S, T31I, Y37H, H41P, M48V, P44L, G52C, S54R, I65M, I68T, S75F, N106S, and T113S caused significant conformational changes. Further, DRN analysis showed that residues P2, T31, Y37, G52, I65, I68, S75, N106, and T113S are part of a similar local residue interaction network with functional significance. These results show how polymorphisms such as missense SNVs can affect protein conformation, dynamics, and function. Trypanosomes are auxotrophic for folates and pterins but require them for survival. They scavenge them from their hosts. PTR1 is a multifunctional enzyme that is unique to trypanosomatids that reduces both pterins and folates. In the presence of DHFR inhibitors, PTR1 is over-expressed thus providing an escape from the effects of DHFR inhibition. Both TbPTR1 and TbDHFR are pharmacologically and genetically validated drug targets. In this study 5742 compounds were screened using molecular docking, and 13 promising binding modes were further analysed using MD simulations. The trajectories were analysed using RMSD, Rg, RMSF, PCA, Essential Dynamics Analysis (EDA), Molecular Mechanics Poisson–Boltzmann surface area (MM-PBSA) binding free energy calculations, and DRN analysis. The computational screening approach allowed us to identify five of the compounds, named RUBi004, RUBi007, RUBi014, RUBi016 and RUBi018 that exhibited antitrypanosomal growth activities against trypanosomes in culture with IC50 values of 12.5 ± 4.8 μM, 32.4 ± 4.2 μM, 5.9 ± 1.4 μM, 28.2 ± 3.3 μM, and 9.7 ± 2.1 μM, respectively. Further when used in combination with WR99210 a known TbDHFR inhibitor RUBi004, RUBi007, RUBi014 and RUBi018 showed antagonism while RUBi016 showed an additive effect. These results indicate that the four compounds might be competing with TbDHFR while RUBi016 might be more specific for TbPTR1. These compounds provide scaffolds that can be further optimised to improve their potency and specificity. Lastly, using a systematic approach we derived CHARMM force-field parameters to accurately describe the TbrPDEB1 bi-metal catalytic center. For dynamics, we employed mixed bonded and non-bonded approach. We optimised the structure using a two-layer QM/MM ONIOM (B3LYP/6-31(g): UFF). The TbrPDEB1 bi-metallic center bonds, angles, and dihedrals were parameterized by fitting the energy profiles from Potential Energy Surface (PES) scans to the CHARMM potential energy function. The parameters were validated by means of MD simulations and analysed using RMSD, Rg, RMSF, hydrogen bonding, bond/angle/dihedral evaluations, EDA, PCA, and DRN analysis. The force-field parameters were able to accurately reproduce the geometry and dynamics of the TbrPDEB1 bi-metal catalytic center during MD simulations. Molecular docking was used to identify 6 potential hits, that inhibited trypanosome growth in vitro. The derived force-field parameters were used to simulate the 6 protein-ligand complexes with the aim of elucidating crucial protein-ligand residue interactions. Using the most potent ligand RUBi022 that had an IC50 of 14.96 μM we were able to identify key residue interactions that can be of use in in silico prediction of potential TbrPDEB1 inhibitors. Overall we demonstrate how bioinformatics tools can complement current disease eradication strategies. Future work will focus on identifying variants identified in Genome Wide Association Studies and partnering with wet labs to carry out further enzyme-ligand activity relationship studies, structure determination or characterisation of appropriate protein-ligand complexes by crystallography, and site specific mutation studie

Theoretical Model of EphA2-Ephrin A1 Inhibition

This work aims at the theoretical description of EphA2-ephrin A1 inhibition by small molecules. Recently proposed ab initio-based scoring models, comprising long-range components of interaction energy, is tested on lithocholic acid class inhibitors of this protein⁻protein interaction (PPI) against common empirical descriptors. We show that, although limited to compounds with similar solvation energy, the ab initio model is able to rank the set of selected inhibitors more effectively than empirical scoring functions, aiding the design of novel compounds

Leishmaniasis

Leishmaniasis is a major global health challenge, affecting approximately 12 million of the poorest people in 100 countries. It is a deforming and fatal disease in the visceral form. Therapies for leishmaniasis are numerically restricted, basically consisting of the administration of miltefosine, pentavalent antimonials, amphotericin B, or pentamidine. This is an important vulnerability against therapy efficiency that must be overcome by the scientific community. This book discusses important aspects of the disease, such as treatment, epidemiology, and molecular and cell biology. The information contained herein is important for young researchers as they seek to develop safe and effective treatments for this neglected tropical disease

Design and synthesis of membrane-bound pyrophosphatase inhibitors against pathogenic protozoan parasites

Pathogenic protozoan parasites cause devastating diseases, such as malaria (Plasmodium spp.), toxoplasmosis (Toxoplasma spp.), leishmaniasis (Leishmania spp.) and trypanosomiasis (Trypanosoma spp.), which have an enormous health, social and economic impact, particularly in tropical regions of the world. For example, nearly half of the population of the world is at risk of malaria that is responsible for more than 600,000 deaths annually. Interestingly, all of these parasites have in common a homodimeric integral protein, membrane-bound pyrophosphatase (mPPase), consisting of 15 to 17 helices with a molecular weight of 70 to 81 kDa. These mPPase enzymes hydrolyze pyrophosphate, a by-product of many biological processes. Besides taking care of excess pyrophosphate, the energy released through hydrolysis of phosphoanhydride bonds is coupled with the pumping of protons or sodium ions thus creating an ion gradient across the membrane. Consequently, mPPases play an important role in the survival of many organisms under diverse stress situations due to osmotic stress or other energy limitations. Inhibiting the function of mPPases bears promise from a drug discovery perspective, as remarkably these enzymes do not exist in animals or humans. Hitherto, only crystal structures of the hyperthermophilic bacterium Thermotoga maritima mPPase (TmPPase) and mung bean Vigna radiata (L.) R. Wilczek mPPase have been solved and are amenable for structure-based drug design. Still, most known inhibitors, such as pyrophosphate and bisphosphonate derivatives, are not optimal as drugs due to features including poor stability and extensive hydrophilicity. The focus of this study was to explore membrane-bound pyrophosphatase as a potential drug target for pathogenic protozoan parasites, mainly Plasmodium falciparum. Since most of the currently known inhibitors are phosphorus-based, thus limiting their therapeutic usage, the aim of this thesis was to develop new organic mPPase inhibitors with potential as an alternative approach in the treatment of malaria and other related parasitic diseases. By developing nonphosphorus inhibitors targeting mPPase, the intention was to block the essential ion pump of these parasites. The compounds obtained were first tested in a 96-well plate in vitro screening assay using thermostable TmPPase as a model enzyme, resulting in the construction of a small library of drug-like compounds (<500 Da) containing different scaffolds with low micromolar activities. Selected top hits were studied further and evaluated against purified mPPase from P. falciparum (PfPPase-VP1) expressed in baculovirus-infected insect cells. In addition, their ability to inhibit the growth of P. falciparum in a survival assay in erythrocytes was studied. In publication I, the discovery and synthesis of the first nonphosphorus, non-substrate analog inhibitor of Thermotoga maritima mPPase (TmPPase), with a 6-(aminomethyl)benzo[d]thiazol-2-amine core, as well as preliminary structure-activity relationships (SARs) and a X-ray crystal structure of this allosteric inhibitor bound to TmPPase are reported. Unfortunately, the allosteric binding region was not preserved in parasitic mPPase thus indicating that there are alternative ways to inhibit mPPase enzymes. In the subsequent study, publication II, the chemical exploration of phosphate isosteres, which finally led us to 2-bromophenyl 5-arylisoxazole-3-carboxylates, is presented. As some antiparasitic activity was maintained, it is assumed that these inhibitors bind orthosterically in the preserved region of the enzyme even though it has not yet been confirmed by sufficient crystallographic data. Publication III further explores structural analogs of the best isoxazole inhibitor (publication II), which resulted in discovering a new bicyclic scaffold, with a 4,5-dihydropyrrolo[3,4-c]pyrazol-6(1H)-one core, and its related pyrazolo[1,5-a]pyrimidine analogs. These compounds seem promising also against PfPPase and, for that reason further studies thereof are ongoing. Additionally, publication IV describes a cross-project in vitro screening that got us to study azulenes and benzazulenes as potential TmPPase inhibitors. These low micromolar TmPPase inhibitors retained similar activity also in a P. falciparum survival assay in erythrocytes. Altogether, these studies offer a different approach for further development of novel drugs against malaria and other diseases caused by pathogenic protozoan parasites.Sjukdomsframkallande urdjursparasiter orsakar förödande sjukdomar, såsom malaria (Plasmodium spp.), toxoplasmos (Toxoplasma spp.), leishmaniasis (Leishmania spp.) och trypanosomiasis (Trypanosoma spp.). Dessa sjukdomar leder till enorma hälsomässiga, sociala och ekonomiska konsekvenser, särskilt i tropiska områden i världen. Till exempel malaria leder till mer än 600 000 dödsfall årligen och nästan hälften av världens befolkning befinner sig i riskzonen för att insjukna. Intressant nog har alla dessa parasiter gemensamt ett homodimert integralt protein, membranbundet pyrofosfatas (eng. membrane-bound pyrophosphatase, mPPase), bestående av 15 till 17 helixar med en molekylvikt på 70 till 81 kDa. Dessa mPPase-enzymer hydrolyserar pyrofosfat, en biprodukt i många biologiska processer. Förutom att ta hand om överskottet av pyrofosfat, kopplas energin som frigörs vid hydrolys av fosfoanhydridbindningar till pumpning av protoner eller natriumjoner, vilket skapar en jongradient över membranet. mPPaser har således en viktig roll för överlevnaden av många organismer under diverse stressituationer på grund av osmotisk stress eller andra energibegränsningar. Att hämma funktionen av mPPaser kunde vara ett lovande alternativt för attt upptäckta nya läkemedel, eftersom dessa enzymer inte finns i djur eller människor. Hittills har endast kristallstrukturer av den hypertermofila bakterien Thermotoga maritima mPPase (TmPPase) och mungböna Vigna radiata (L.) R. Wilczek mPPase lösts och är tillgängliga för strukturbaserad läkemedelsdesign. De flesta kända hämmare, såsom pyrofosfat- och bisfosfonatderivat, är inte optimala som läkemedel på grund av egenskaper inklusive dålig stabilitet och omfattande hydrofilicitet. Fokus för denna studie var att utforska membranbundet pyrofosfatas som ett potentiellt läkemedelsmål för sjukdomsframkallande urdjursparasiter, främst Plasmodium falciparum. De flesta av de för närvarande kända hämmarna är fosforbaserade, vilket begränsar deras terapeutiska användning. Syftet med denna avhandling var således att utveckla nya organiska mPPase-hämmare med utvecklingsmöjligheter som ett alternativt tillvägagångssätt vid behandling av malaria och andra relaterade parasitsjukdomar. Avsikten var att blockera den essentiella jonpumpen hos dessa parasiter genom att utveckla fosforfria hämmare gentemot mPPase. De erhållna föreningarnas biologiska aktivitet utvärderades först in vitro med termostabilt TmPPase som modellenzym i 96-brunnsplattor, vilket resulterade i uppbyggnaden av ett litet bibliotek av läkemedelsliknande föreningar (<500 Da) bestående av olika grundstommar (eng. scaffolds) med låga mikromolära aktiviteter. De mest lovande föreningarna studerades vidare och testades mot renat mPPase från P. falciparum (PfPPase-VP1) uttryckt i baculovirusinfekterade insektsceller. Dessutom studerades deras förmåga att hämma tillväxten av P. falciparum i en överlevnadsanalys i erytrocyter. I publikation I rapporteras upptäckten och syntesen av den första fosforfria TmPPase-hämmaren, som har en 6-(aminometyl)benso[d]tiazol-2-amin -stomme och således inte är en substratanalog, samt preliminära struktur-aktivitetsförhållanden (SAR) och en röntgenkristallstruktur för denna allosteriska hämmare bunden till TmPPase. Tyvärr bevarades inte den allosteriska bindningsregionen i parasitiskt mPPase vilket indikerar att det finns alternativa sätt att hämma mPPase-enzymer. I den efterföljande studien, publikation II, presenteras diverse fosfatisosterer, som slutligen ledde oss till 2-bromfenyl-5-arylisoxazol-3-karboxylater. Eftersom viss antiparasitisk aktivitet bibehölls, antas det att dessa hämmare binder ortosteriskt i den konserverade regionen av enzymet även om detta ännu inte har bekräftats av tillräcklig kristallografisk data. För publikation III studerades ytterligare strukturella analoger av den bästa isoxazolhämmaren (publikation II), vilket resulterade i upptäckten av en ny bicyklisk grundstomme, med en 4,5-dihydropyrrolo[3,4-c]pyrazol-6(1H)-on -stomme, och dess relaterade pyrazolo[1,5-a]pyrimidinanaloger. Dessa föreningar verkar lovande även mot PfPPase och följaktligen pågår ytterligare studier av dessa. I publikation IV beskrivs en in vitro screening av föreningar från anknutna projekt som fick oss att studera azulener och bensazulener som potentiella TmPPase-hämmare. Dessa låga mikromolära TmPPase-hämmare bibehöll liknande aktivitet även i en P. falciparum-överlevnadsanalys i erytrocyter. Sammantaget erbjuder dessa studier ett annat tillvägagångssätt för vidareutveckling av nya läkemedel mot malaria och andra sjukdomar orsakade av patogena protozoparasiter

Kinetoplastid Genomics and Beyond

This book includes a collection of eight original research articles and three reviews covering a wide range of topics in the field of kinetoplastids. In addition, readers can find a compendium of molecular biology procedures and bioinformatics tools

Découverte d'inhibiteurs de la dihydrofolate réductase R67 impliquée dans la résistance au triméthoprime

Le triméthoprime (TMP) est un antibiotique communément utilisé depuis les années 60. Le TMP est un inhibiteur de la dihydrofolate réductase (DHFR) bactérienne chromosomale. Cette enzyme est responsable de la réduction du dihydrofolate (DHF) en tétrahydrofolate (THF) chez les bactéries, qui lui, est essentiel à la synthèse des purines et ainsi, à la prolifération cellulaire. La résistance bactérienne au TMP est documentée depuis plus de 30 ans. Une des causes de cette résistance provient du fait que certaines souches bactériennes expriment une DHFR plasmidique, la DHFR R67. La DHFR R67 n'est pas affectée par le TMP, et peut ainsi remplacer la DHFR chromosomale lorsque celle-ci est inhibée par le TMP. À ce jour, aucun inhibiteur spécifique de la DHFR R67 est connu. En découvrant des inhibiteurs contre la DHFR R67, il serait possible de lever la résistance au TMP que la DHFR R67 confère aux bactéries. Afin de découvrir des inhibiteurs de DHFR R67, les approches de design à base de fragments et de criblage virtuel ont été choisies. L'approche de design à base de fragments a permis d'identifier sept composés simples et de faible poids moléculaire (fragments) inhibant faiblement la DHFR R67. À partir de ces fragments, des composés plus complexes et symétriques, inhibant la DHFR R67 dans l'ordre du micromolaire, ont été élaborés. Des études cinétiques ont montré que ces inhibiteurs sont compétitifs et qu'au moins deux molécules se lient simultanément dans le site actif de la DHFR R67. L'étude d'analogues des inhibiteurs micromolaires de la DHFR R67 a permis de déterminer que la présence de groupements carboxylate, benzimidazole et que la longueur des molécules influencent la puissance des inhibiteurs. Une étude par arrimage moléculaire, appuyée par les résultats in vitro, a permis d'élaborer un modèle qui suggère que les résidus Lys32, Gln67 et Ile68 seraient impliqués dans la liaison avec les inhibiteurs. Le criblage virtuel de la librairie de 80 000 composés de Maybridge avec le logiciel Moldock, et les essais d'inhibition in vitro des meilleurs candidats, a permis d'identifier quatre inhibiteurs micromolaires appartenant à des familles distinctes des composés précédemment identifiés. Un second criblage virtuel, d'une banque de 6 millions de composés, a permis d'identifier trois inhibiteurs micromolaires toujours distincts. Ces résultats offrent la base à partir de laquelle il sera possible de développer iv des composés plus efficaces et possédant des propriétés phamacologiquement acceptables dans le but de développer un antibiotique pouvant lever la résistance au TMP conféré par la DHFR R67.Trimethoprim (TMP) is a common antibiotic which is used since the 60's. TMP is an inhibitor of the bacterial chromosomal dihydrofolate reductase (DHFR). This enzyme catalyses the reduction of the dihydrofolate (DHF) to tetrahydrofolate (THF) which is essential to the biosynthesis of purines thus to cellular proliferation. Bacterial TMP resistance is documented since about 30 years. One of the cause of this resistance comes from the fact that certain bacteria express a plasmidic DHFR, the R67 DHFR, which confers TMP resistance. The R67 DHFR is not inhibited by TMP and can replace the chromosomal DHFR when the latter is inhibited by TMP. The discovery of R67 DHFR inhibitors would allow to break the trimethoprim resistance granted by R67 DHFR. In order to discover R67 DHFR inhibitors, fragment based design and virtual screening approaches were selected. By fragment based design, seven simple compounds with a low molecular mass which inhibited weakly R67 DHFR (fragments) were identified. From these fragments, more complex and symmetrical compounds inhibiting R67 DHFR in the micromolar range were identified. Kinetic studies showed these inhibitors were competitive and at least two molecules bind simultaneously to the active site of the R67 DHFR. Test of the micromolar inhibitors analog showed that the presence of carboxylate, benzimidazole and the length of the molecule all have an effect on the potency of the inhibitors. Molecular docking of the inhibitors, supported by in vitro data, were used to develop a model which suggest that residue like Lys32, Gln67 and Ile68 would be involved in the binding of the inhibitors to the R67 DHFR. Virtual screening of the 80 000 compound Maybridge library with Moldock software, followed by in vitro test of the best candidate, identified four micromolar inhibitors which are chemically distinct from the inhibitor beforehand identified. A second virtual screening of a 6 million compounds bank identified three micromolar inhibitors which are also distinct from the inhibitor beforehand identified. vi These results offer a basis which will allow further development of more potent inhibitors with more acceptable pharmacologic properties in order to develop an antibiotic which would break the TMP resistance granted by the R67 DHFR

Application of computer-aided drug design for identification of P. falciparum inhibitors

Malaria is a millennia-old disease with the first recorded cases dating back to 2700 BC found in Chinese medical records, and later in other civilizations. It has claimed human lives to such an extent that there are a notable associated socio-economic consequences. Currently, according to the World Health Organization (WHO), Africa holds the highest disease burden with 94% of deaths and 82% of cases with P. falciparum having ~100% prevalence. Chemotherapy, such as artemisinin combination therapy, has been and continues to be the work horse in the fight against the disease, together with seasonal malaria chemoprevention and the use of insecticides. Natural products such as quinine and artemisinin are particularly important in terms of their antimalarial activity. The emphasis in current chemotherapy research is the need for time and cost-effective workflows focussed on new mechanisms of action (MoAs) covering the target candidate profiles (TCPs). Despite a decline in cases over the past decades with, countries increasingly becoming certified malaria free, a stalling trend has been observed in the past five years resulting in missing the 2020 Global Technical Strategy (GTS) milestones. With no effective vaccine, a reduction in funding, slower drug approval than resistance emergence from resistant and invasive vectors, and threats in diagnosis with the pfhrp2/3 gene deletion, malaria remains a major health concern. Motivated by these reasons, the primary aim of this work was a contribution to the antimalarial pipeline through in silico approaches focusing on P. falciparum. We first intended an exploration of malarial targets through a proteome scale screening on 36 targets using multiple metrics to account for the multi-objective nature of drug discovery. The continuous growth of structural data offers the ideal scenario for mining new MoAs covering antimalarials TCPs. This was combined with a repurposing strategy using a set of orally available FDA approved drugs. Further, use was made of time- and cost-effective strategies combining QVina-W efficiency metrics that integrate molecular properties, GRIM rescoring for molecular interactions and a hydrogen mass repartitioning (HMR) molecular dynamics (MD) scheme for accelerated development of antimalarials in the context of resistance. This pipeline further integrates a complex ranking for better drug-target selectivity, and normalization strategies to overcome docking scoring function bias. The different metrics, ranking, normalization strategies and their combinations were first assessed using their mean ranking error (MRE). A version combining all metrics was used to select 36 unique protein-ligand complexes, assessed in MD, with the final retention of 25. From the 16 in vitro tested hits of the 25, fingolimod, abiraterone, prazosin, and terazosin showed antiplasmodial activity with IC50 2.21, 3.37, 16.67 and 34.72 μM respectively and of these, only fingolimod was found to be not safe with respect to human cell viability. These compounds were predicted active on different molecular targets, abiraterone was predicted to interact with a putative liver-stage essential target, hence promising as a transmission-blocking agent. The pipeline had a promising 25% hit rate considering the proteome-scale and use of cost-effective approaches. Secondly, we focused on Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) using a more extensive screening pipeline to overcome some of the current in silico screening limitations. Starting from the ZINC lead-like library of ~3M, hierarchical ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS) approaches with molecular docking and re-scoring using eleven scoring functions (SFs) were used. Later ranking with an exponential consensus strategy was included. Selected hits were further assessed through Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA), advanced MD sampling in a ligand pulling simulations and (Weighted Histogram Analysis Method) WHAM analysis for umbrella sampling (US) to derive binding free energies. Four leads had better predicted affinities in US than LC5, a 280 nM potent PfDXR inhibitor with ZINC000050633276 showing a promising binding of -20.43 kcal/mol. As shown with fosmidomycin, DXR inhibition offers fast acting compounds fulfilling antimalarials TCP1. Yet, fosmidomycin has a high polarity causing its short half-life and hampering its clinical use. These leads scaffolds are different from fosmidomycin and hence may offer better pharmacokinetic and pharmacodynamic properties and may also be promising for lead optimization. A combined analysis of residues’ contributions to the free energy of binding in MM-PBSA and to steered molecular dynamics (SMD) Fmax indicated GLU233, CYS268, SER270, TRP296, and HIS341 as exploitable for compound optimization. Finally, we updated the SANCDB library with new NPs and their commercially available analogs as a solution to NP availability. The library is extended to 1005 compounds from its initial 600 compounds and the database is integrated to Mcule and Molport APIs for analogs automatic update. The new set may contribute to virtual screening and to antimalarials as the most effective ones have NP origin.Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 202