It is theoretically possible to avoid misfolding into non-covalent lasso entanglements using small molecule drugs

A novel class of protein misfolding characterized by either the formation of non-native noncovalent lasso entanglements in the misfolded structure or loss of native entanglements has been predicted to exist and found circumstantial support through biochemical assays and limited-proteolysis mass spectrometry data. Here, we examine whether it is possible to design small molecule compounds that can bind to specific folding intermediates and thereby avoid these misfolded states in computer simulations under idealized conditions (perfect drug-binding specificity, zero promiscuity, and a smooth energy landscape). Studying two proteins, type III chloramphenicol acetyltransferase (CAT-III) and D-alanyl-D-alanine ligase B (DDLB), that were previously suggested to form soluble misfolded states through a mechanism involving a failure-to-form of native entanglements, we explore two different drug design strategies using coarse-grained structure-based models. The first strategy, in which the native entanglement is stabilized by drug binding, failed to decrease misfolding because it formed an alternative entanglement at a nearby region. The second strategy, in which a small molecule was designed to bind to a non-native tertiary structure and thereby destabilize the native entanglement, succeeded in decreasing misfolding and increasing the native state population. This strategy worked because destabilizing the entanglement loop provided more time for the threading segment to position itself correctly to be wrapped by the loop to form the native entanglement. Further, we computationally identified several FDA-approved drugs with the potential to bind these intermediate states and rescue misfolding in these proteins. This study suggests it is possible for small molecule drugs to prevent protein misfolding of this type. A variety of diseases are caused by protein misfolding. Therefore, the recent evidence suggesting there is an entire unexplored class of protein misfolding that may be widespread opens the possibility they contribute to disease and may be therapeutic targets. Here, we bypass the question of what diseases such misfolding may contribute to and ask whether it is even possible to restore proper folding and function to proteins that misfold in this manner. We tried the most obvious strategy first: in the computer simulations we created a drug that binds to the folded, native entanglement. The rationale being that this would thermodynamically stabilize the native state relative to misfolded states, and thereby shift the population of molecules to the folded states. But thermodynamic reasoning neglects the folding pathways and kinetics connecting metastable states on the energy landscape, and this strategy had the unintended consequence of slowing down native state formation by stabilizing the entanglement loop without the threading segment piercing it, resulting in the loss of the native entanglement and formation of a non-native entanglement. Building on this observation, we took a different route, and designed a drug that would delay formation of the entanglement loop allowing more time for the threading segment to position itself and allow proper folding of the native entanglement. While this study was carried out using coarse-grained structure-based models, the results indicate it is possible in principle for drugs to be designed to avoid such misfolding and suggest our second design strategy is more likely to work in future experiments.