Rational Polypharmacology: Systematically Identifying and Engaging Multiple Drug Targets To Promote Axon Growth

Abstract

Mammalian Central Nervous System (CNS) neurons regrow their axons poorly following injury, resulting in irreversible functional losses. Identifying therapeutics that encourage CNS axon repair has been difficult, in part because multiple etiologies underlie this regenerative failure. This suggests a particular need for drugs that engage multiple molecular targets. Although multi-target drugs are generally more effective than highly selective alternatives, we lack systematic methods for discovering such drugs. Target-based screening is an efficient technique for identifying potent modulators of individual targets. In contrast, phenotypic screening can identify drugs with multiple targets; however, these targets remain unknown. To address this gap, we combined the two drug discovery approaches using machine learning and information theory. We screened compounds in a phenotypic assay with primary CNS neurons and also in a panel of kinase enzyme assays. We used learning algorithms to relate the compounds’ kinase inhibition profiles to their influence on neurite outgrowth. This allowed us to identify kinases that may serve as targets for promoting neurite outgrowth, as well as others whose targeting should be avoided. We found that compounds that inhibit multiple targets (polypharmacology) promote robust neurite outgrowth in vitro. One compound with exemplary polypharmacology, was found to promote axon growth in a rodent spinal cord injury model. A more general applicability of our approach is suggested by its ability to deconvolve known targets for a breast cancer cell line, as well as targets recently shown to mediate drug resistance.

The mTOR Substrate S6 Kinase 1 (S6K1) Is a Negative Regulator of Axon Regeneration and a Potential Drug Target for Central Nervous System Injury

“…The neurite outgrowth assay was performed as previously described (Al-Ali et al, 2013b), with minor modifications. Briefly, compounds were tested with rat embryonic (E18) hippocampal neurons cultured at low density for 48 h. Neurons were plated at 1500 -1800 cells/well in 96-well plates coated overnight with poly-D-lysine.…”

“…The following day, siRNA solutions were prepared in 96-well plates at 2 M in 100 l of media and allowed to equilibrate for 1 h in a CO 2 incubator. Immediately before treatment, 100 l of media was removed from the cells and replaced with 50 l of siRNA solution (duplicate wells for each treatment) to bring the final siRNA reagent concentration to 1 M (per vendor recommendation) in a total volume of 100 l. Cells were incubated for 96 h, then fixed and analyzed for neurite outgrowth as previously described for the neurite outgrowth assay (Al-Ali et al, 2013b).…”

“…A set of small-molecule kinase inhibitors was selected from the hits in our phenotypic screen to represent a variety of chemical scaffolds, distinct kinase inhibition profiles, and potentially diverse mechanisms for promoting neurite outgrowth (Al-Ali et al, 2013b. In the present study, we investigated the effect of these representative compounds on S6 phosphorylation, in primary hippocampal neurons.…”

“…We previously screened a large number of kinase inhibitors in a neurite outgrowth assay using primary hippocampal neurons, and identified "hit" compounds that promote neurite outgrowth (Al-Ali et al, 2013b). This phenotypic assay is reliable (ZЈ factor Ͼ 0.7) and has identified both chemical and genetic perturbagens that promote axon growth from a variety of CNS neurons (Al-Ali et al, 2013a, 2017.…”

“…This phenotypic assay is reliable (ZЈ factor Ͼ 0.7) and has identified both chemical and genetic perturbagens that promote axon growth from a variety of CNS neurons (Al-Ali et al, 2013a, 2017. In a follow-up study, we used machine learning to relate data from the phenotypic screen of kinase inhibitors to kinase profiling data, which allowed us to identify (and verify) "target" kinases whose inhibition induces neurite outgrowth (Al-Ali et al, 2015).…”