Since the flexible protein region in proximity of the S1 pocket is distant from the highly conserved catalytic site of zinc-dependent metalloprotease enzymes, this region may be targeted in the search for selective small molecule inhibitors of LF. Initially, we looked for compounds that are capable of binding to the S1 pocket and to its unexplored adjacent region. Our preliminary docking studies suggested that a sulfonamide biphenyl substructure was capable of binding to the open channel that bridges the S1 pocket and the adjacent protein region. A p-substituent on the second phenyl ring of the sulfonamide biphenyl group would extend into the neighboring protein region. Hence, we first analyzed three compounds (6–8, ) that were initially selected by virtual screening from over 200 compounds containing a sulfonamide biphenyl group in a commercially available library of small molecules (Chembridge). The measured LF inhibition for the three compounds, 6–8, is 10%, 34% and 84% at 100μM, respectively with compound 8 displaying an IC50 value of 12 μM, in subsequent dose response measurements. Based on the predicted binding pose for compound 8 (), the following considerations can be made: a) one of its two pyridine rings is located near the Zn2+ possibly involved in a cation-π interaction; b) one sulfonamide group, which forms hydrogen bonds with Ser655, Lys656 and Glu687, allows compound 8 to fit with its biphenyl group in the S1 pocket and through the adjacent channel; c) the second sulfonamide-pyridyl group on the other end is bound to the adjacent region outside the S1 pocket. Comparing the chemical structures and activities of compounds 6–8, it is reasonable to speculate that the observed increased inhibition of compound 8 versus the others can be attributable to ability of the compound to extend into the sub-pocket near the S1 pocket. Hence, based on these observations additional ten compounds 9–18 () were selected and tested for their ability to inhibit the LF catalytic activity in vitro. Two compounds emerged that exhibited increased potency with IC50 values of 3.8 μM (compound 9) and 3.2 μM (compound 18) ( and , ). Because these compounds lack the typical hydroxamate Zn2+ chelating group and possess substructures that we hypothesize to occupy a non-conserved region adjacent to the S1 pocket, we anticipate that such compounds would result more selective against LF compared to various human metalloproteases or other Zn2+ ion containing enzymes. In fact, when tested against the most related human metalloproteases MMP-2 and MMP-9, both compounds 9 and 18 did not elicit any significant inhibition when tested at up to 100 μM concentration. In contrast, the hydroxamate-based compound 4 () that has an IC50 value of 0.8μM in our LF assay, significantly inhibits MMP-2 (IC50 = 4.4μM) and MMP-9 (IC50 = 21μM) ().
Small molecule structures with percentage inhibition of lethal factor at 100 μM.
Figure 3 Comparison of the binding poses of lethal factor inhibitors in the X-ray structures of (A) inhibitor 1 (PDB-1PWP), (B) inhibitor 4 (PDB-1YQY), (C) inhibitor 5 (PDB-1PWQ), (D) inhibitor 8: as predicted binding pose in PDB-1PWQ. Chemical structures are (more ...)
Comparison of IC50 values for LF inhibitors against two human Metalloproteases, MMP-2 and MMP-9.
Dose response curves for the inhibition of Lethal factor by compounds 4, 9 and 18.
By superimposing the LF docked binding poses of compounds 8, 9 and 18, we generated a new pharmacophore model that summarizes our experimental observations (). The pharmcophore model contains the Zn2+ interacting moiety group (A) linked to a longer substructure (C) suitable to fit the S1 pocket and to protrude into the adjacent region; the connection between (A) and (C) is best obtained with a sulfonamide group (B) capable of providing the proper angle between (A) and (C). Finally, the substructure (D) occupies the newly identified binding region in proximity of the S1 pocket. The pharmacophore model previously reported was derived from planar and rigid LF inhibitors that only bind to the catalytic site8. This new model generates a number of testable hypotheses that could further delineate the structural determinants for potent and selective LF inhibitors. For example, it would be of interesting to test the effect of replacing the cation-π interactions provided by the moiety (A) with traditional metal-chelating groups. Also, the nature of the (D) moiety makes it amenable to further iterative optimizations. Hence, together with the identified compounds, the model may be useful to guide the design of more potent and selective inhibitors against Anthrax.
Figure 5 (a) Superimposition of predicted binding poses of compounds 8, 9 and 18. (b) Novel pharmacophore model for the lethal factor inhibitors. Red ellipse (A) is an electron rich group, presumably capable of interacting with the metal-ion; the orange ellipse (more ...)