Table 1.
Data collection and refinement statistics.
Fig 1.
(a) DLAC screening tests reveal N6-methyl adenosine (D30) as a putative hit component against AlaDH. D28, C2-methyl adenosine; D29, cyclohexyl 2-[[5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl]acetate; D30, N6-methyl adenosine; D31, 4-[[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl]but-2-ynyl furan-2-carboxylate; D32, N-[4-(4-butanoylpiperazin-1-yl)-3-chlorophenyl]-5-nitrofuran-2-carboxamide; D33, 5-(1,3-benzodioxol-5-yl)-N-(5-chloro-2-hydroxyphenyl)-7-(trifluoromethyl)-1,5,6,7-tetrahydro pyrazolo[1,5-a]pyrimidine-2-carboxamide; D34, 5-(4-bromophenyl)-N-[(1,5-dimethylpyrazol-4-yl)methyl]-7-(trifluoromethyl)-1,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide; D35, 4-methyl-N-(5-pyridin-4-yl-1,3,4-oxadiazol-2-yl)benzamide; D36, benzyl (2S)-4-methyl-2-[[4-[(3-methylphenyl) carbamoyl]-1H-imidazole-5-carbonyl]amino]pentanoate. The gels are cropped from the full-length gels which are presented in S2a and S2c Fig in S1 File. ATP is used as a positive control based on our previous data [25], where NADH and ATP (but not NAD) bound strongly with this assay (b) Inhibition effect of N6-methyl adenosine is confirmed using enzyme kinetic studies.
Fig 2.
Crystallographic AlaDH structure.
(a) Crystal structure of AlaDH bound to N6-methyl adenosine. Dashed circle demarcates the hydrophobic binding groove. (b) Close-up view of the nucleoside/inhibitor binding site. The electron density map is a polder omit map [48] at 3σ. (c) Crystal structure of AlaDH bound to NAD (PDB code 2VHX, [34]). Atom colors are red (oxygen), blue (nitrogen), yellow (ligand carbons) and gray (protein carbons). Hydrogen bonds are shown as dashed lines with distances in angstroms.
Fig 3.
Characterization of synthesized compounds.
(a) Analysis of six synthesized adenosine analogue compounds (including the lead compound, N6-methyl adenosine) with AlaDH by DLAC and densitometry analyses. The gel is cropped from the full-length gel that is presented in S6 Fig in S1 File. (b) Lineweaver-Burk plots to calculate the Ki of five compounds based on their activity inhibition at indicated concentrations against AlaDH.
Table 2.
Km of NAD and inhibition constants (Ki) of adenosine analogue compounds for Mycobacterium tuberculosis L-alanine dehydrogenase.
Fig 4.
AlaDH crystal structure in complex with adenosine-based inhibitors.
(a) N6-isobutyl adenosine inhibitor binding site showing hydrophobic interactions and hydrogen bonds. Electron density is shown as polder omit map at 3σ [48]. (b) Crystallographic structure bound to N6-isobutyl adenosine and (c) N6-methyl adenosine. Dashed lines show closest interatomic distances between the N6-methyl or N6-isobutyl moieties to surrounding hydrophobic residues. It can be seen that each distance for the N6-methyl moiety is longer than the corresponding nearest distance to the N6-isobutyl moiety (6.6 vs. 4.4 Å for A222; 4.1 vs. 4.0 Å for L225, 3.9 vs. 3.6 Å for L249; 4.4 vs. 3.9 Å for I199).