Adenosine analogues as inhibitors of Trypanosoma brucei phosphoglycerate kinase: Elucidation of a novel binding mode for a 2-amino-N-6-substituted adenosine

As part of a project aimed at structure-based design of adenosine analogues as drugs against African trypanosomiasis, N-6-, 2-amino-N-6-, and N-2-subs tituted adenosine analogues were synthesized and tested to establish struct ure-activity relationships for inhibiting Trypanosoma brucei glycosomal pho sphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH ), and glycerol-3-phosphate dehydrogenase (GPDH). Evaluation of X-ray struc tures of parasite PGK, GAPDH, and GPDH complexed with their adenosyl-bearin g substrates led us to generate a series of adenosine analogues which would target all three enzymes simultaneously. There was a modest preference by PGK for NG-substituted analogues bearing the 2-amino group. The best compou nd in this series, 2-amino-N-6-[2 "-(p-hydroxyphenyl)ethyl]adenosine (46b), displayed a 23-fold improvement over adenosine with an IC50 of 130 muM. 2- [[2 "-(p-Hydroxyphenyl)ethyl]amino]adenosine (46c) was a weak inhibitor of T. brucei PGK with an IC50 of 500 muM. To explore the potential of an addit ive effect that having the N-6 and N-2 substitutions in one molecule might provide, the best ligands from the two series were incorporated into N-6,N- 2-disubstituted adenosine analogues to yield N-6-(2 " -phenylethyl)-2-[(2 " -phenylethyl)amino]adenosine (69) as a 30 muM inhibitor of T. brucei PGK w hich is 100-fold more potent than the adenosine template. In contrast, thes e series gave no compounds that inhibited parasitic GAPDH or GPDH more than 10-20% when tested at 1.0 mM. A 3.0 Angstrom X-ray structure of a T, bruce i PGK/46b complex revealed a binding mode in which the nucleoside analogue was flipped and the ribosyl moiety adopted a syn conformation as compared w ith the previously determined binding mode of ADP. Molecular docking experi ments using QXP and SAS program suites reproduced this "flipped and rotated " binding mode.