Summary /

ML251 showed promising ADME profile (Table 1).

Table 1. ADME profile for qHTS hit 1 (CID-2431573) and 30 (ML251).

Summary /

While a number of analogs had been generated in the first two rounds of synthesis that possessed comparable activity to the original hit 1, failure to improve upon this potency led to the decision to utilize the previously solved ATP-bound crystal structure of Tb PFK as a basis for molecular modeling efforts to guide future rounds of analog development. Thermal melt data suggested that 1 engages in competition for binding with F6P, as stabilization of Tb PFK is observed in the presence of ATP and inhibitor, but not in the presence of F6P and inhibitor. This finding led to docking of 1 in the sugar binding site and conformational energy minimization delivered a binding orientation in which the 3,4- dichlorobenzyl functionality of 1 extends into a hydrophobic pocket generated by the Leu346, Ile347, and Ile349 residues of Tb PFK. Important hydrogen bonding interactions are also predicted in this rendering for the sulfonamide linker with the Arg383 residue. Lastly, the modeling predicts occupation of the 5-methyl-3-aminoisoxazole functionality within a highly hydrophilic region of the Tb PFK active site. It is within this site that the LYS226 residue is predicted to engage in a hydrogen bond with the nitrogen center of the isoxazole functionality of 1. This suggested mode of binding was further corroborated by mechanistic enzyme studies that were performed on ML251.

Figure 3. Modeling of CID-2431573 in F6P binding site of Tb PFK. A) Rendering of the tunnel-like active site of Tb PFK and docking of CID-2431573 (pink) in the F6P binding site (ATP is shown in gold); B) Alternative view showing hydrophobic pocket that is predicted to be occupied by the dichlorobenzyl functionality of CID-2431573 (pink); C) Interior of Tb PFK active site and predicted interaction with CID-2431573 (cyan).

Summary /

Inclusion of saturating concentrations of ATP or F6P in the ADP-Glo inhibition assay showed a marked right-shift in ML251 potency under high F6P conditions (Figure 13, Panel A); the IC50 of ML251 against Tb PFK shifted from 365 nM under standard conditions to 2.06 μM in the presence of saturating F6P, demonstrating a 5.6-fold shift in potency. Inclusion of saturating ATP, by contrast, demonstrated virtually no change in ML251 potency, with an IC50 of 326 nM. These observations were followed up with a mechanistic competition study in which individual substrates and ML251 were titrated in a coupled PFK assay system. ML251 demonstrated a minimal effect on the Km of ATP, yet appeared to affect Vmax (Figure 13, Panel B, transformed to Lineweaver-Burk plot for ease of visualization), suggesting that it is noncompetitive with respect to ATP. F6P, conversely, showed no change in Vmax in the presence of ML251, but did show a shift in Km, suggesting that the compound may be competitive with respect to F6P (Figure 13, Panel C). These observations agree with the results of both our biochemical assays and molecular modeling in suggesting that the lead chemical series preferentially binds in the F6P region of the Tb PFK active site.

Figure 4. Evaluation of substrate competition of ML251 with F6P and ATP in Tb PFK. A. Inhibitory activity of ML251 in ADP-Glo assay under standard (white circles), saturating ATP (light grey circles) and saturating F6P (dark grey circles) conditions; B. Lineweaver-Burk plot of competition relationship between ML251 and ATP; C. Lineweaver Burk plot of competition relationship between ML251 and F6P.