The manner in which various residue side
The manner in which various residue side chains are oriented in the active site of HsHxKIV was a driving factor for why glucosamine analogues could not bind, as we previously proposed . These compounds would have difficulty managing access into the active site based on residue P153 in addition to side chains that could potentially cause steric hindrance (i.e. T168, N166, Q286, and K56). Our previous work considered the possibility of HPOP-GlcN as a potential inhibitor to bind in the active site of HsHxKIV, through a molecular modeling analysis. This assumed that the T7 Tag Peptide moiety of HPOP-GlcN would maintain the key binding interactions at the glucose binding site . We reasoned that HPOP-GlcN (and similar analogues) may not have the ability to bind favorably in HsHxKIV, because in the closed conformation a narrow channel (bordered by residues P153 and T168) that the HPOP-GlcN linker group would need to extend through, would certainly have unavoidable close contact interactions (e.g. the rigid side chain of P153, in particular). The other HsHxKIV residues in the vicinity, such as K56, N166, T168, and Q286 have flexible side chains and could possibly reposition, but overall, the active site appeared too congested for HPOP-GlcN. On the other hand, TcGlcK was observed to have a much wider channel (bordered by residues P94 and N105) and was shown to accommodate HPOP-GlcN in the active site, as evidenced from a TcGlcK – HPOP-GlcN complex from crystallography (see PDB entry 5BRF ). P94 (TcGlcK) was the conserved residue to P153 (HsHxKIV) and P94 (TcGlcK) was observed to be positioned in a different manner that could avoid the close contact of the HPOP-GlcN linker moiety. Moreover, enzyme – inhibitor Ki values determined for TcGlcK and HsHxKIV revealed a value of 186 for a TcGlcK selectivity ratio . In glucokinase from L. braziliensis, residue P106 (LbGlcK) is the conserved residue of both P153 (HsHxKIV) (Fig. 2) and P94 (TcGlcK) (Figure S4, SI). Although our LbGlcK structure is observed in the open conformation, P106 (LbGlcK) is located on the small domain, and the P106 side chain is therefore shifted away from the active site. We cannot accurately predict if P106 would deter these kinds of glucosamine analogues to bind. Interestingly however, it does not appear that the P106 side chain would interfere with a bound HPOP-GlcN inhibitor in the active site and this is based off of a superposition from the A-chains of LbGlcK and the TcGlcK – HPOP-GlcN complex. With active site comparisons between LbGlcK and TcGlcK appearing to be rather close from a structural superposition (Figure S3, SI), it appears that glucosamine analogues have the ability to bind in the active site of LbGlcK. In order to test this hypothesis, we screened LbGlcK against four glucosamine analogue compounds that were previously determined as strong competitive inhibitors of TcGlcK . These compounds were BENZ-GlcN, CBZ-GlcN, HPOP-GlcN, and DBT-GlcN. The primary screening involved having percent LbGlcK inhibition to be greater than 34.2% (a calculated cutoff percentage) as the concentration of compounds was set to 20 μM, in order to be considered as candidate hits. From the series tested, none of the compounds were candidate hits. HPOP-GlcN was the stronger LbGlcK inhibitor of the series, in which chlorophyll a inhibited LbGlcK by 26.9 ± 1.2%. Inhibition by the other compounds was weaker, such as BENZ-GlcN with 14.2 ± 3.9% inhibition, DBT-GlcN with 13.7 ± 3.0% inhibition, and CBZ-GlcN with 10.6 ± 3.1% inhibition. The cutoff percentage of 34.2% was a calculated value that takes into consideration the positive controls when analyzed by the fluorescence microplate reader, and also, the variability that occurs from the fluorescence readout. A candidate hit was defined as an activity measurement being three standard deviations away from the mean of the positive controls and additional information on the calculation is provided in the SI section. In various target-based high-throughput screening campaign studies [, , ], the progression cascades (screening compounds to confirmed hits) reveal that compounds not passing requirements in the primary screen step will not proceed into in vitro parasite biological assays. As such, the four weak glucosamine analogue inhibitors of LbGlcK were excluded from any anti-Leishmania in vitro parasite testing. Since HPOP-GlcN had a higher inhibitory strength, its mode of inhibition and inhibitory constant (Ki) were sought after. The Ki for HPOP-GlcN vs. LbGlcK was determined to be 56.9 ± 16.6 μM and the mode of inhibition was competitive (see Figure S5 for the Lineweaver-Burk plot). The Ki was observed at a much higher value when compared to TcGlcK/HPOP-GlcN (Ki = 1.3 ± 0.6 μM) , in which HPOP-GlcN exhibited a 43.8-fold weaker inhibition to LbGlcK. For selectivity vs. HsHxKIV (Ki = 242 ± 0.6 μM) , there was a 4.25 selectivity index that indicated HPOP-GlcN was not very selective towards LbGlcK. In terms of the observed competitive inhibition, HPOP-GlcN is binding in the active site of LbGlcK and this corroborates well for a glucosamine analogue inhibitor having the expectation to bind in the LbGlcK active site based on superposition structural data, as revealed in Figure S3.