Identification of Novel Potential Inhibitors of Pteridine Reductase 1
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molecules Article Identification of Novel Potential Inhibitors of Pteridine Reductase 1 in Trypanosoma brucei via Computational Structure-Based Approaches and in Vitro Inhibition Assays Magambo Phillip Kimuda 1,2,† , Dustin Laming 3,4 , Heinrich C. Hoppe 3,4 and Özlem Tastan Bishop 1,* 1 Research Unit in Bioinformatics (RUBi), Department of Biochemistry and Microbiology, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa; [email protected] 2 College of Veterinary Medicine, Animal Resources and Biosecurity (COVAB), Makerere University, P.O. Box 7062, Kampala 00256, Uganda 3 Department of Biochemistry and Microbiology, Rhodes University, Grahamstown 6140, South Africa; [email protected] (D.L.); [email protected] (H.C.H.) 4 Centre for Chemico- and Biomedicinal Research, Rhodes University, Grahamstown 6140, South Africa * Correspondence: [email protected]; Tel.: +27-46-603-8072 † Member of the Trypanogen Consortium, www.trypanogen.net. Academic Editor: Tiziano Tuccinardi Received: 12 November 2018; Accepted: 24 December 2018; Published: 1 January 2019 Abstract: Pteridine reductase 1 (PTR1) is a trypanosomatid multifunctional enzyme that provides a mechanism for escape of dihydrofolate reductase (DHFR) inhibition. This is because PTR1 can reduce pterins and folates. Trypanosomes require folates and pterins for survival and are unable to synthesize them de novo. Currently there are no anti-folate based Human African Trypanosomiasis (HAT) chemotherapeutics in use. Thus, successful dual inhibition of Trypanosoma brucei dihydrofolate reductase (TbDHFR) and Trypanosoma brucei pteridine reductase 1 (TbPTR1) has implications in the exploitation of anti-folates. We carried out molecular docking of a ligand library of 5742 compounds against TbPTR1 and identified 18 compounds showing promising binding modes. The protein-ligand complexes were subjected to molecular dynamics to characterize their molecular interactions and energetics, followed by in vitro testing. In this study, we identified five compounds which showed low micromolar Trypanosome growth inhibition in in vitro experiments that might be acting by inhibition of TbPTR1. Compounds RUBi004, RUBi007, RUBi014, and RUBi018 displayed moderate to strong antagonism (mutual reduction in potency) when used in combination with the known TbDHFR inhibitor, WR99210. This gave an indication that the compounds might inhibit both TbPTR1 and TbDHFR. RUBi016 showed an additive effect in the isobologram assay. Overall, our results provide a basis for scaffold optimization for further studies in the development of HAT anti-folates. Keywords: Human African Trypanosomiasis; pteridine reductase 1; PTR1; DHFR; anti-folates; anti-trypanosomal agents; molecular dynamics; dynamic residue network analysis; binding free energy; isobologram assay 1. Introduction African trypanosomes are flagellated hemo-parasites, transmitted by Tsetse flies, and cause zoonotic infection in mammalian hosts [1]. In animals the disease is known as Nagana while in humans it is known as Human African Trypanosomiasis (HAT) [2,3]. Acute HAT is caused by Trypanosoma brucei rhodesiense (Tbr) while chronic HAT is caused by Trypanosoma brucei gambiense (Tbg). Molecules 2019, 24, 142; doi:10.3390/molecules24010142 www.mdpi.com/journal/molecules Molecules 2018, 23, x FOR PEER REVIEW 2 of 26 Molecules 2019, 24, 142 2 of 25 humans it is known as Human African Trypanosomiasis (HAT) [2,3]. Acute HAT is caused by Trypanosoma brucei rhodesiense (Tbr) while chronic HAT is caused by Trypanosoma brucei gambiense This(Tbg neglected). This neglected tropical disease tropical (NTD) disease remains (NTD) of considerableremains of considerable public health public and animal health production and animal concernproduction [4,5]. concern [4,5]. TrypanosomesTrypanosomes are are unable unable to synthesize to synthesize folates folates and pterins and pterins de novo de [ 6novo]. Reduced [6]. Reduced folate and folate pterin and cofactorspterin cofactors are essential are essential for parasite for parasite survival survival where they where are they critical are incritical pathways in pathways such as such protein as protein and nucleicand nucleic acid biosynthesis acid biosynthesis [7]. In order [7]. In to order survive, to surv trypanosomesive, trypanosomes scavenge scavenge extracellular extracellular folate and folate pterin and precursorspterin precursors from their from hosts their [8,9]. hosts Hence, [8,9]. the pathwayHence, the is an pathway interesting is an drug interesting target. Drugs drug targeting target. Drugs the folatetargeting pathway the havefolate been pathway used inhave the been treatment used ofin several the treatment infections, of several most notably infections, in the most treatment notably of in bacterialthe treatment and malarial of bacterial infections and [ 10malarial]. However, infections their use[10]. in However, the treatment their and use management in the treatment of HAT and hasmanagement not been successful of HAT tohas date. not been successful to date. TheThe key key enzymes enzymes involved involved in trypanosome in trypanosome folate metabolismfolate metabolism are dihydrofolate are dihydrofolate reductase (DHFR)reductase and(DHFR) pteridine and reductase pteridine1 reductase (PTR1) (Figure 1 (PTR1)1)[ 11(Figure–13]. DHFR1) [11–13]. (EC 1.5.1.3)DHFR is(EC an 1.5.1.3) NADPH-dependent is an NADPH- enzymedependent that catalyzesenzyme reductionthat catalyzes of folate redu to dihydrofolatection of folate (H 2F),to anddihydrofolate H2F to tetrahydrofolate (H2F), and (HH24FF) to (Figuretetrahydrofolate1)[ 11,12]. Folate (H4F) (Figure is essentially 1) [11,12]. a pteridine Folate is that essentiall has beeny a conjugatedpteridine that to phas-aminobenzoic been conjugated acid to (pABA)p-aminobenzoic that is glutamylated acid (pABA) (Figure that1 )[is 14glutamylated]. DHFR is a (Figure validated 1) and[14]. primary DHFR is target a validated of most and anti-folate primary drugstarget [12 of]. most However, anti-folate the use drugs of traditional [12]. However, anti-folates the use against of traditional DHFR in anti-folates trypanosomatids against suchDHFR as in Trypanosomatrypanosomatidsand Leishmania such as Trypanosomahas been largely and unsuccessfulLeishmania has [12 been,13,15 largely,16]. unsuccessful [12,13,15,16]. FigureFigure 1. The1. roleThe ofrole dihydrofolate of dihydrofolate reductase reductase (DHFR) and(DHF pteridineR) and reductase pteridine 1 (PTR1)reductase in trypanosome 1 (PTR1) in folatetrypanosome and pterin folate metabolism. and pterin Trypanosomes, metabolism. which Trypanosomes, are auxotrophic which for are folates auxotrophic and pterins, for folates salvage and thempterins, from salvage the host. them The structuresfrom the host. of folate The andstructures biopterin of folate are shown and biopterin (A). Folates are andshown pterins (A). areFolates taken and uppterins by transporters are taken (the up folate-biopterinby transporters transporter(the folate-b superfamilyiopterin transporter includes superfamily biopterin transporter includes 1biopterin (BT1) andtransporter folate transporter 1 (BT1) 1and (FT1)) folate after transporter which they 1 are(FT1)) reduced after towhich their they functional are reduced cofactors to their (B). functional cofactors (B). PTR1 (EC 1.5.1.33), which is a short-chain dehydrogenase reductase family member and an NADPH-dependentPTR1 (EC 1.5.1.33), enzyme, which is is unique a short-chain to trypanosomatids dehydrogenase [8]. It reductase is important family in the member reduction and of an biopterinNADPH-dependent to dihydrobiopterin enzyme, (H is2B), unique and of to H trypanosomat2B to tetrahydrobiopterinids [8]. It is important (H4B) (Figure in the1). PTR1reduction also of reducesbiopterin folate to to dihydrobiopterin H2F, and H2F to H(H4F2B), (Figure and 1of)[ H8].2B In to trypanosomatids, tetrahydrobiopterin PTR1, (H which4B) (Figure is less susceptible1). PTR1 also to traditionalreduces folate anti-folate to H2 inhibition,F, and H2F contributes to H4F (Figure about 10%1) [8]. to total In trypanosomatids, folate metabolism [PTR1,13]. It iswhich important is less tosusceptible note that studies to traditional have shown anti-folate that under inhibition, DHFR cont inhibitionributes PTR1 about is 10% over-expressed, to total folate thus metabolism promoting [13]. anti-folateIt is important resistance to note in Leishmania that studies major haveand shownTrypanosoma that under cruzi DHFR[8,13,15 inhibition,16]. This PTR1 has been is over-expressed, proposed as thethus key promoting mechanism anti-folate by which trypanosomatids resistance in Leishmania are able major to resist and anti-folates Trypanosoma targeting cruzi [8,13,15,16]. DHFR [8,13, 15This,16 has]. Genebeen knock proposed down as and the knock key outmechanism studies in byT. which brucei havetrypanosomatids shown that PTR1 are able is essential to resist for anti-folates parasite survival.targeting As DHFR such, its [8,13,15,16]. inhibition aloneGene mightknock be down sufficient and knock to negatively out studies impact in parasiteT. brucei survival have shown [17,18 that]. PTR1There is essential are several for studies parasite that survival. have reported As such, successful its inhibition combination alone might of PTR1 be andsufficient DHFR to inhibitors negatively inimpact order to parasite achieve survival synergistic [17,18]. inhibition of the trypanosomatid folate pathway in T. cruzi, L. major and T. brucei [18–22]. However, the identification