Table 1.

Effects of chemical perturbations

Compound	Target and Mode of Action	Primary Cellular Effects	Connection to α-Synuclein	Hypothesized Effects on α-Synuclein Fitness Landscape
Dopamine	Covalent modification of α-synuclein41	Neurotransmitter	α-Synuclein deficiency alters dopamine signaling;12 dopaminergic neurons degenerate selectively in PD;29 dopamine promotes oligomer formation, inhibits fibrillization41	Residues that interact with dopamine will show disruptive mutations selectively in the presence of dopamine; sequence signatures of oligomers (e.g., positions sensitive to steric hindrance) will emerge
Menadione	Quinone reduction by molecular oxygen generates superoxide radicals	Induces oxidative stress	Various α-synuclein species induce oxidative stress;42 oxidative modification of α-synuclein is observed in patients43 and inhibits fibrillization;44 menadione exacerbates α-synuclein toxicity in yeast45	Residues whose oxidation contributes to α-synuclein toxicity will become more sensitive to mutation; compromised capacity to resolve oxidative stress might reveal conformations that drive toxicity by increasing oxidative stress
Melatonin	Scavenges free radicals	Relieves oxidative stress	Various α-synuclein species induce oxidative stress;42 oxidative modification of α-synuclein is observed in patients43 and inhibits fibrillization;44 melatonin attenuates α-synuclein toxicity in yeast challenged by menadione45	Residues that become modified by oxidative stress will be less sensitive to mutation; enhanced resilience to oxidative stress might reveal conformations that drive toxicity through other mechanisms
Miconazole	Inhibits 14α-sterol demethylase, reducing ergosterol production46	Decreases membrane sterol content, affecting membrane fluidity and integrity46	Sterol content modulates the membrane-binding affinity of α-synuclein47	Membrane-contacting residues will be selectively affected; cells might maintain resistance only to variants with the weakest membrane affinity
Brefeldin A	Inhibits COP-I coat assembly	Disrupts Golgi-ER and other membrane transport processes; induces the unfolded protein response	ER-Golgi trafficking genes modify α-synuclein toxicity;20 α-synuclein inclusions involve clustered ER/Golgi vesicles18,19	Because cells will be more sensitive to agents that interfere with trafficking, cells should be more sensitive to all variants except those with the weakest membrane affinity, which are less likely to affect trafficking; sequence signatures of alternatively localized α-synuclein should emerge
MG-132	Covalent inhibitor of the 20S proteasome	Reduces degradation of ubiquitin-modified proteins	Lewy bodies are enriched in ubiquitinated proteins, including α-synuclein;48 ubiquitination is a common cellular strategy for mitigating misfolded proteins; α-synuclein impairs proteasome function49	Residues whose ubiquitination modifies α-synuclein toxicity will be less sensitive to mutation; species that are more efficiently ubiquitinated and cleared will become apparent
Tunicamycin	Inhibits glycosyl-transferases involved in glycoprotein synthesis50	Creates a burden of misfolded ER proteins; induces the unfolded protein response50	α-Synuclein accumulation induces the unfolded protein response;21 α-synuclein interferes with glycoprotein maturation;21 tunicamycin induces α-synuclein oligomerization in cellular models36	Cells will become less resistant to all variants except those with the weakest membrane affinity, which are less likely to affect glycoprotein maturation; additional sequence determinants of oligomerization might appear; sequence determinants of species resolved by the unfolded protein response will appear
Geldanamycin	ADP/ATP-competitive inhibitor of Hsp9051	Reduces proteostatic capacity51	Hsp90 reduces α-synuclein aggregation and toxicity by modifying oligomers52	Sequence signatures of conformations resolved by Hsp90 will appear; residues that directly interact with Hsp90 will be less sensitive to mutation
Rapamycin	Inhibitor of mTORC1 formation53	Inhibits translation; induces autophagy53	α-Synuclein aggregates are cleared by autophagy;37 α-synuclein aggregation impairs autophagy54	Variants with sub-saturating toxicity will increase in fitness due to reduced translation; residues that mediate trafficking to autophagosomes will be more sensitive to mutation; sequence signatures of toxic species not resolved by autophagy will be revealed
Spermidine	Inhibits histone acetyltransferases, altering the epigenetic state of histone H3 and inducing transcription of autophagy-related genes55	Induces autophagy; suppresses oxidative stress55	α-Synuclein aggregates are cleared by autophagy;37 synuclein aggregation impairs autophagy54	Residues that mediate trafficking to autophagosomes will be more sensitive to mutation; sequence signatures of toxic species not resolved by autophagy will be revealed