Pervasive aggregation and depletion of host and viral proteins in response to cysteine-reactive electrophilic compounds

Abstract

Protein homeostasis is tightly regulated, with damaged or misfolded proteins quickly eliminated by the proteasome and autophagosome pathways. By co-opting these processes, targeted protein degradation technologies enable pharmacological manipulation of protein abundance. Recently, cysteine-reactive molecules have been added to the degrader toolbox, which offer the benefit of unlocking the therapeutic potential of ‘undruggable’ protein targets. The proteome-wide impact of these molecules remains to be fully understood and given the general reactivity of many classes of cysteine-reactive electrophiles, on- and off-target effects are likely. Using chemical proteomics, we identified a cysteine-reactive small molecule degrader of the SARS-CoV-2 non- structural protein 14 (nsp14), which effects degradation through direct modification of cysteines in both nsp14 and in host chaperones together with activation of global cell stress response pathways. We find that cysteine-reactive electrophiles increase global protein ubiquitylation, trigger proteasome activation, and result in widespread aggregation and depletion of host proteins, including components of the nuclear pore complex. Formation of stress granules was also found to be a remarkably ubiquitous cellular response to nearly all cysteine-reactive compounds and degraders. Collectively, our study sheds light on complexities of covalent target protein degradation and highlights untapped opportunities in manipulating and characterizing proteostasis processes via deciphering the cysteine-centric regulation of stress response pathways.

Reversible Covalent Inhibition─Desired Covalent Adduct Formation by Mass Action

“…Performing competition reactions of the protein target and glutathione with RCIs in addition to monitoring for glutathione depletion in cells , can provide helpful information on the effect of RCIs on electrophilic stress in cells. Furthermore, covalent inhibitors may induce misfolding of proteins which could lead to a global protein misfolding profiles and responses. , …”

“… There should be a minimal effect on cellular stress responses. Covalent inhibition of an enzyme may lead to misfolding of the protein. , If this is nonspecific, this may lead to global protein misfolding stress responses. Examinations of protein profiles can be performed as previously described, and comparisons with efforts to probe reactive residues of the proteome − ,, to ensure limited off-target activity.…”

“…Furthermore, covalent inhibitors may induce misfolding of proteins which could lead to a global protein misfolding profiles and responses. 57 , 58 …”

Proteomic Ligandability Maps of Spirocycle Acrylamide Stereoprobes Identify Covalent ERCC3 Degraders

“…20−23 While fragment electrophiles provide arguably the broadest initial portrait of covalent binding sites throughout the proteome, the hit compounds emerging from fragment-based screens are often weak in potency (high-μM) and promiscuous in protein interactions and therefore challenging to use in cell biology studies without further optimization. 24 We have found alternatively that electrophilic stereoprobes frequently engage cysteine residues with site specificity (only a single cysteine on a protein reacts with the stereoprobe), stereoselectivity (the enantiomer of the stereoprobe hit does not react with the cysteine), and sufficient potency (>70% engagement at 20 μM in cells) for use in cell biological studies that include inactive enantiomeric compounds and stereoprobe-resistant cysteine mutant proteins as controls. Examples of functional stereoprobes discovered by ABPP include allosteric inhibitors of the NAD-metabolizing enzyme SARM1 22 and the exonuclease TOE1, 25 E3 ligase ligands that can be converted into heterobifunctional degraders, 21 and protein−protein interaction modulators targeting proteasomal regulatory and spliceosome complexes.…”

“…The types of electrophilic compounds investigated to date by ABPP include fragments, , natural products, and, most recently, diversity-oriented synthesis (DOS)-inspired sets of stereochemically defined structures (“stereoprobes”). − While fragment electrophiles provide arguably the broadest initial portrait of covalent binding sites throughout the proteome, the hit compounds emerging from fragment-based screens are often weak in potency (high-μM) and promiscuous in protein interactions and therefore challenging to use in cell biology studies without further optimization . We have found alternatively that electrophilic stereoprobes frequently engage cysteine residues with site specificity (only a single cysteine on a protein reacts with the stereoprobe), stereoselectivity (the enantiomer of the stereoprobe hit does not react with the cysteine), and sufficient potency (>70% engagement at 20 μM in cells) for use in cell biological studies that include inactive enantiomeric compounds and stereoprobe-resistant cysteine mutant proteins as controls.…”