Molecular Recognition at the Active Site of Factor Xa: Cation–π Interactions, Stacking on Planar Peptide Surfaces, and Replacement of Structural Water

Abstract

Factor Xa, a serine protease from the blood coagulation cascade, is an ideal enzyme for molecular recognition studies, as its active site is highly shape-persistent and features distinct, concave sub-pockets. We developed a family of non-peptidic, small-molecule inhibitors with a central tricyclic core orienting a neutral heterocyclic substituent into the S1 pocket and a quaternary ammonium ion into the aromatic box in the S4 pocket. The substituents were systematically varied to investigate cation-π interactions in the S4 pocket, optimal heterocyclic stacking on the flat peptide walls lining the S1 pocket, and potential water replacements in both the S1 and the S4 pockets. Structure-activity relationships were established to reveal and quantify contributions to the binding free enthalpy, resulting from single-atom replacements or positional changes in the ligands. A series of high-affinity ligands with inhibitory constants down to K(i)=2 nM were obtained and their proposed binding geometries confirmed by X-ray co-crystal structures of protein-ligand complexes.

The Surprising Importance of Peptide Bond Contacts in Drug–Protein Interactions

“…A subset of the results of the difference F‐SAPT analysis are presented in Figure . The ab initio SAPT0/jun‐cc‐pVDZ computations indicate that the enhancement in ΔΔ G bind is largely driven by ΔΔ E int —the latter quantity always favors the Cl variant, and the magnitudes track quite well with the experimental IC 50 or K i enhancements across the four ligands tested [e.g., an enhancement of 2.5 (calculated) versus 2.3 (experimental) kcal mol −1 for 3ENS] . Having established this, we move on to determining the origins of ΔΔ E int via F‐SAPT.…”

“…As seen in Panels A and B of Figure , the largest contributors are the backbone peptide bonds: for 3ENS, these provide the leading seven contributions by magnitude to ΔΔ E int . On further reflection, this finding is certainly plausible considering the effect of Cl versus Me on the dipole of the aromatic group, and therefore, the differential dipole‐dipole interactions between the ligand and CONH peptide bond moieties (the potential importance of protein amides lining the fXa S1 pocket to binding ligands with aromatic P1 groups is discussed, though not in the context of Cl substitutents, in ref …”

Cation–π Interactions Contribute to Substrate Recognition in γ‐Butyrobetaine Hydroxylase Catalysis

“…Similar trends have been observed in inhibition studies on the serine protease factor Xa, which contains an aromatic cage in its “S4” substrate residue binding pocket. That is, a ligand possessing a quaternary ammonium moiety inhibits factor Xa to a similar extent as one with an analogous phosphonium group, whereas the neutral carba analogue is substantially (60‐fold) less potent …”

Optimization of 3,5-Dimethylisoxazole Derivatives as Potent Bromodomain Ligands

“…It seemed possible that an increase in affinity would be achieved if the ligand was able to displace, or hydrogen-bond to, one of the ZA channel water molecules. 32−35 …”