Rationalizing the Binding Kinetics for the Inhibition of the Burkholderia pseudomallei FabI1 Enoyl-ACP Reductase

Abstract

There is growing awareness of the link between drug-target residence time and in vivo drug activity, and there are increasing efforts to determine the molecular factors that control the lifetime of a drug-target complex. Rational alterations in drug-target residence time require knowledge of both the ground and transition states on the inhibition reaction coordinate, and we have determined the structure-kinetic relationship for 22 ethyl or hexyl substituted diphenyl ethers that are slow-binding inhibitors of bpFabI1, the enoyl-ACP reductase FabI1 from Burkholderia pseudomallei. Analysis of enzyme inhibition using a 2D-kinetic map demonstrates that the ethyl and hexyl diphenyl ethers fall into two distinct clusters. Modifications to the ethyl diphenyl ether B ring result in changes to both on and off-rates, where residence times of up to ~700 min (~11 h) are achieved by either ground state stabilization (PT444) or transition state destabilization (slower on-rate) (PT404). By contrast, modifications to the hexyl diphenyl ether B ring result in residence times of 300 min (~5 h) through changes in only ground state stabilization (PT119). Structural analysis of 9 enzyme:inhibitor complexes reveal that the variation in structure-kinetic relationships can be rationalized by structural rearrangements of bpFabI1 and subtle changes to the orientation of the inhibitor in the binding pocket. Finally, we demonstrate that three compounds with residence times on bpFabI1 of between 118 min (~2 h) and 670 min (~11 h) have in vivo efficacy in an acute B. pseudomallei murine infection model using the virulent B. pseudomallei strain Bp400.

A triclosan‐resistance protein from the soil metagenome is a novel enoyl‐acyl carrier protein reductase: Structure‐guided functional analysis

“…There are many hydrophobic interactions between triclosan and ENR, but only one possible hydrogen bond is present between the 2‐OH group and tyrosine in the Tyr–Lys pair (i.e., Tyr156–Lys163 in Ec FabI and Tyr157–Lys164 in Sa FabI). In fact, this triclosan‐binding site also serves as other inhibitors [9,27,30–32] and coincides with that of a fatty acid substrate in the structure of FabI of M . tuberculosis (PDB id http://www.rcsb.org/pdb/search/structidSearch.do?structureId=1BVR) [28].…”

Ligand‐ and Structure‐Based Approaches of Escherichia coli FabI Inhibition by Triclosan Derivatives: From Chemical Similarity to Protein Dynamics Influence

“…Interestingly, studies with FabI1 Enoyl-ACP Reductase from Burkholderia pseudomallei have shown that αhelix 6 (the so-called substrate binding-loop, represented by the residues Thr194-Gly199, original numbering from BpFabI1), becomes ordered and closed upon ligand binding. [31] In our EcFabI studies, also a flexible loop (comprised by the residues 191 to 205) covers the active site, as mentioned previously, which contains the key residues related to the inhibitor interaction ( Figure 4A), and were kept ordered during most of the simulations with the exception of compound 24 and the neutral forms of compound 41 and TCL ( Figure 4C-H and Supporting information, Figure 4, showing the comparison between the ligand-free and bound systems' RMSD values).…”

“…Kinetic and structural studies with the compound 41 related the effect of 5-ethyl substituents with the increases in the residence time, which presumably is related to the stabilization of the ground state's protein conformation. [31] Additionally, three-dimensional structures of BpFabI1 co-crystallised with different TCL analogues showed that Phe203 rotates toward Leu207 displacing Ala197 (equivalent to the Ala196 of EcFabI), upon ligand binding, which creates a hydrophobic pocket that accommodates the 5-ethyl group. Complementarily, in EcFabI, the Ile200 and Phe203 amino-acids compose the beginning of the short loop (near the α-helix-8) and end, which surrounds the ethyl moiety in our simulations.…”

“…Alternatively, a special type of two‐step process involves rapid formation of EI, which is then followed by a slow induced‐fit in the structure of complex (EI*), which for FabI would be related to the SBL stabilization. FabI is suggested to have a special kind of two‐step mechanism that is kinetically indistinguishable from the one‐step mechanism, in which the free energy of EI* is much lower than that of EI and the initial formation of EI cannot be detected at low inhibitor concentrations.…”

“…The increase in residence time can be described in terms of either by the stabilisation of the EI/EI* or the disruption of the TS. It has been shown that hydrophobic 5‐substituents can enhance residence time and affinity of diphenyl‐ethers in both Sa FabI and Bp FabI1 by both of those mechanisms. We could suggest that this residence time and affinity gain rise from the displacement of high energy water molecules (Figure F, sites 3 and 12) by the hydrophobic moieties in those compounds.…”