A rational blueprint for the design of chemically-controlled protein switches

Small-molecule responsive protein switches are crucial components to control synthetic cellular activities. However, the repertoire of small-molecule protein switches is insufficient for many applications, including those in the translational spaces, where properties such as safety, immunogenicity, drug half-life, and drug side-effects are critical. Here, we present a computational protein design strategy to repurpose drug-inhibited protein-protein interactions as OFF- and ON-switches. The designed binders and drug-receptors form chemically-disruptable heterodimers (CDH) which dissociate in the presence of small molecules. To design ON-switches, we converted the CDHs into a multi-domain architecture which we refer to as activation by inhibitor release switches (AIR) that incorporate a rationally designed drug-insensitive receptor protein. CDHs and AIRs showed excellent performance as drug responsive switches to control combinations of synthetic circuits in mammalian cells. This approach effectively expands the chemical space and logic responses in living cells and provides a blueprint to develop new ON- and OFF-switches.

a) Background signal of SEAP reporter in the absence of the CDH-TF system. HEK239T cells were transfected with SEAP reporter S132 and treated with 1 µM Drugs 12 hours post transfection.
Quantification of SEAP was performed 24 hours after drug treatment. b) Drug specificity of CDH-TFs.
Quantification of SEAP activity of three CDHs with DMSO (negative control; dark color), specific (designed drug induced OFF switches; light color) and unspecific drug treatment (testing for possible unintended drug induced effects; gray). c) Dynamic regulation of SEAP expression mediated by CDHs-TF in modes of ON-OFF. The control samples (Ctrl) were transfected with the SEAP reporter S132 and constitutive Gal4-Rel65 expression plasmid S108 (pCMV-Gal4-Rel65-pA). During the OFFtime period shown in gray box, CDH-TF cells were cultured with 500 nM of the corresponding drugs while Ctrl cells were treated with same concentration of DMSO, drugs/DMSO were replenished every 12 hours at time points of 36h, 48h and 60h. Cells were split at 36h changing medium to with/without drugs respectively. d) Dynamic regulation of SEAP expression mediated by CDHs-TF in modes of OFF-ON. Time point 0 was set 12 hours post transfection to start the intermittent drug treatment shown in gray boxes, CDH-TF cells were cultured with 500 nM of the corresponding drugs while Ctrl cells were treated with same concentration of DMSO. Drugs/DMSO were refreshed at time points of 0h, 12h, 24h. Cells were split at 36h changing medium to without/with drugs respectively. e) Background signal of SEAP reporter in the absence of the CDH-GEMS system. HEK293T cells were transfected with pLS566 SEAP reporter plasmids and treated with drugs. Quantification of SEAP was

PDB code 7AYE
*Values in parentheses are for highest-resolution shell. ** CC1/2 refers to Pearson's correlation coefficients (CC) between intensity estimates from half data sets.
Positive_unbound: Score of the iBcl-XL mutant in its unbound (apo) state.
Negative_unbound: Score of iBcl-XL mutant in its unbound (apo) state, based on the crystal structure of Drug-1:Bcl-XL. Positive_unbound: Score of the iBcl2 mutant in its unbound (apo) state.