High-resolution crystal structure of Trypanosoma brucei pteridine reductase 1 in complex with an innovative tricyclic-based inhibitor

Abstract

The protozoan parasite Trypanosoma brucei is the etiological agent of human African trypanosomiasis (HAT). HAT, together with other neglected tropical diseases, causes serious health and economic issues, especially in tropical and subtropical areas. The classical antifolates targeting dihydrofolate reductase (DHFR) are ineffective towards trypanosomatid parasites owing to a metabolic bypass by the expression of pteridine reductase 1 (PTR1). The combined inhibition of PTR1 and DHFR activities in Trypanosoma parasites represents a promising strategy for the development of new effective treatments for HAT. To date, only monocyclic and bicyclic aromatic systems have been proposed as inhibitors of T. brucei PTR1 (TbPTR1); nevertheless, the size of the catalytic cavity allows the accommodation of expanded molecular cores. Here, an innovative tricyclic-based compound has been explored as a TbPTR1-targeting molecule and its potential application for the development of a new class of PTR1 inhibitors has been evaluated. 2,4-Diaminopyrimido[4,5-b]indol-6-ol (1) was designed and synthesized, and was found to be effective in blocking TbPTR1 activity, with a K i in the low-micromolar range. The binding mode of 1 was clarified through the structural characterization of its ternary complex with TbPTR1 and the cofactor NADP(H), which was determined to 1.30 Å resolution. The compound adopts a substrate-like orientation inside the cavity that maximizes the binding contributions of hydrophobic and hydrogen-bond interactions. The binding mode of 1 was compared with those of previously reported bicyclic inhibitors, providing new insights for the design of innovative tricyclic-based molecules targeting TbPTR1.

Evidence of Pyrimethamine and Cycloguanil Analogues as Dual Inhibitors of Trypanosoma brucei Pteridine Reductase and Dihydrofolate Reductase

“…The structure of Tb PTR1 in complex with the cofactor NADP(H) and PYR was obtained with a 1.34 Å resolution, providing a detailed view of the inhibitor interactions within the enzyme catalytic cavity ( Table S1, Supplementary Materials ). The crystal asymmetric unit includes a whole functional Tb PTR1 tetramer, whose structure is highly conserved compared with those previously described (subunits were completely traced apart for two surface-exposed loops including residues 103–113 and 143–151, usually poorly organized in Tb PTR1 structures) [ 15 , 16 , 17 , 21 , 23 ]. The active site is an L-shaped depression mainly built within a single protein chain having one end blocked by the C-terminus of a partner subunit.…”

“…The crystals of the complex belonged to the monoclinic space group P2 1 with a functional tetramer in the asymmetric unit. The structure was solved by the molecular replacement technique as implemented in MOLREP [ 56 ] and a functional enzyme tetramer (PDB code 6TBX [ 21 ], excluding non-protein atoms and water molecules) was used as searching model. The model was refined using REFMAC5 [ 57 ] from the CCP4 suite.…”

“…Targeting the enzymes of the folate metabolism was proven as a successful strategy against bacterial infections [ 13 ] and malaria [ 14 ], and, more recently, it has been exploited also for the development of novel antiparasitic treatments [ 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 ]. Trypanosomes are unable to synthesize folates and pterins required for critical cellular metabolic pathways, such as the biosynthesis of nucleic acids and proteins [ 25 ].…”

“…The pteridine and pyrimidine core structures have largely been explored with the intent to discover new anti-trypanosomatidic drugs, also taking advantage of the information derived from the available crystal structures, which allowed the further decoration of their scaffolds [ 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 ]. Pursuing our interest in the search of more effective agents against the T. brucei parasite, we deemed worthwhile the study of the anti-trypanosomatidic activity of two known drugs, pyrimethamine (PYR) and MTX ( Figure S1 , Supplementary Materials ).…”

Repurposing the Trypanosomatidic GSK Kinetobox for the Inhibition of Parasitic Pteridine and Dihydrofolate Reductases

“…Under these conditions, PTR1 expression levels highly increase, and this can guarantee the production of 10% of tetrahydrofolate required by the cell to sustain the parasite survival [18]. An effective treatment of trypanosomatid infections could be achieved through the simultaneous inhibition of DHFR-TS and PTR1 by a single drug or a combination of compounds that are specific and selective inhibitors of each target [19].…”