Pushing the Limits of Detection of Weak Binding Using Fragment-Based Drug Discovery: Identification of New Cyclophilin Binders

Abstract

Fragment-based drug discovery is an increasingly popular method to identify novel small-molecule drug candidates. One of the limitations of the approach is the difficulty of accurately characterizing weak binding events. This work reports a combination of X-ray diffraction, surface plasmon resonance experiments and molecular dynamics simulations for the characterization of binders to different isoforms of the cyclophilin (Cyp) protein family. Although several Cyp inhibitors have been reported in the literature, it has proven challenging to achieve high binding selectivity for different isoforms of this protein family. The present studies have led to the identification of several structurally novel fragments that bind to diverse Cyp isoforms in distinct pockets with low millimolar dissociation constants. A detailed comparison of the merits and drawbacks of the experimental and computational techniques is presented, and emerging strategies for designing ligands with enhanced isoform specificity are described.

Best Practices for Alchemical Free Energy Calculations [Article v1.0]

“…This strategy, however, can fail when the dissociation rate of the ligand has the same or smaller order of magnitude than the length of the simulation. This is typical of weak binders such as fragments binding shallow pockets with µM-mM affinities [62,152]. In this case, using a flat-bottom or harmonic restraint between receptor and ligand in the bound state(s) can prevent dissociations [83,152].…”

“…This is typical of weak binders such as fragments binding shallow pockets with µM-mM affinities [62,152]. In this case, using a flat-bottom or harmonic restraint between receptor and ligand in the bound state(s) can prevent dissociations [83,152]. We stress that this is normally avoided as it generally introduces bias in the free energy estimate, which is why the restraint is usually activated only in the intermediate states in absolute calculations.…”

Binding Modes of Ligands Using Enhanced Sampling (BLUES): Rapid Decorrelation of Ligand Binding Modes via Nonequilibrium Candidate Monte Carlo

“…28–30 Here, our primary focus is on different “binding modes”: defined as different metastable conformations of a fragment-like ligand within a single relatively rigid protein cavity. Metastable binding modes are thus those which are slow to interconvert on a simulation timescale z .…”

A computationally designed binding mode flip leads to a novel class of potent tri-vector cyclophilin inhibitors

“…Thus, further evaluation of the present tri-vector Cyp inhibitors in a range of in vitro and in vivo disease models implicating Cyps seems to be warranted, particularly in instances where CsA toxicity and off-target effects have been a cause for concern. Alternatives to the aniline moiety in 15 to alleviate toxicity concerns have been suggested elsewhere 21,45. Immunosuppression via inhibition of calcineurin phosphatase activity is a well-known feature of CsA.…”