Purpose We previously identified an oxysterol, VP1-001 (also known as compound 29), that partially restores the transparency of lenses with cataracts. To understand the mechanism of VP1-001, we tested the ability of its enantiomer, ent -VP1-001, to bind and stabilize αB-crystallin (cryAB) in vitro and to produce a similar therapeutic effect in cryAB(R120G) mutant and aged wild-type mice with cataracts. VP1-001 and ent -VP1-001 have identical physicochemical properties. These experiments are designed to critically evaluate whether stereoselective binding to cryAB is required for activity. Methods We compared the binding of VP1-001 and ent -VP1-001 to cryAB using in silico docking, differential scanning fluorimetry (DSF), and microscale thermophoresis (MST). Compounds were delivered by six topical administrations to mouse eyes over 2 weeks, and the effects on cataracts and lens refractive measures in vivo were examined. Additionally, lens epithelial and fiber cell morphologies were assessed via transmission electron microscopy. Results Docking studies suggested greater binding of VP1-001 into a deep groove in the cryAB dimer compared with ent -VP1-001. Consistent with this prediction, DSF and MST experiments showed that VP1-001 bound cryAB, whereas ent -VP1-001 did not. Accordingly, topical treatment of lenses with ent -VP1-001 had no effect, whereas VP1-001 produced a statistically significant improvement in lens clarity and favorable changes in lens morphology. Conclusions The ability of VP1-001 to bind native cryAB dimers is important for its ability to reverse lens opacity in mouse models of cataracts.

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Transcription factors (TFs) are one of the most promising but underutilized classes of drug targets. The high degree of intrinsic disorder in both the structure and the interactions (i.e., “fuzziness”) of TFs is one of the most important challenges to be addressed in this context. Here, we discuss the impacts of fuzziness on transcription factor drug discovery, describing how disorder poses fundamental problems to the typical drug design, and screening approaches used for other classes of proteins such as receptors or enzymes. We then speculate on ways modern biophysical and chemical biology approaches could synergize to overcome many of these challenges by directly addressing the challenges imposed by TF disorder and fuzziness.