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)-substituted adenosine analogues were synthesized and tested to establish structure-activity relationships for inhibiting Trypanosoma brucei glycosomal phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glycerol-3-phosphate dehydrogenase (GPDH). Evaluation of X-ray structures of parasite PGK, GAPDH, and GPDH complexed with their adenosyl-bearing 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 N(6)-substituted analogues bearing the 2-amino group. The best compound in this series, 2-amino-N(6)- [2''(p-hydroxyphenyl)ethyl]adenosine (46b), displayed a 23-fold improvement over adenosine with an IC(50) of 130 microM. 2-[[2''-(p-Hydroxyphenyl)ethyl]amino]adenosine (46c) was a weak inhibitor of T. brucei PGK with an IC(50) of 500 microM. To explore the potential of an additive 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 microM inhibitor of T. brucei PGK which is 100-fold more potent than the adenosine template. In contrast, these series gave no compounds that inhibited parasitic GAPDH or GPDH more than 10-20% when tested at 1.0 mM. A 3.0 A X-ray structure of a T. brucei PGK/46b complex revealed a binding mode in which the nucleoside analogue was flipped and the ribosyl moiety adopted a syn conformation as compared with the previously determined binding mode of ADP. Molecular docking experiments using QXP and SAS program suites reproduced this "flipped and rotated" binding mode.