Chemically responsive protein switches for the precise control of biological activities
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Abstract
Controlling the proximity or interaction of proteins with small molecules enables researchers to chemically regulate cellular functions. Here, we leveraged CATCHFIRE (chemically assisted tethering of chimera by fluorogenic induced recognition) – a technology enabling to chemically induce dimerization in a reversible manner – to create chemically responsive proteins switches for the precise and reversible control of various biological activities. CATCHFIRE allowed us to chemically induce the assembly and thus function of various split enzymes – including luciferases, proteases, DNA recombinases. We extended this approach to develop CATCH-ON, a chemically inducible gene expression system relying on the chemically induced dimerization of the DNA-binding domain GAL4 and the truncated transcription factor p65Δ. CATCH-ON allowed us to precisely regulate the expression of cellular enzymes such as proteases, DNA recombinases, or suicide switches, as well as to control the secretion of therapeutically relevant proteins such as insulin. We showed that the CATCH-ON system is fast-acting, reversible, titratable, non-toxic and compatible with other chemically induced dimerization systems, opening exciting possibilities for its application in basic research, biotechnology and cell therapy.
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Chemically responsive protein switches for the precise control of biological activities
This work presents a versatile suite of chemically responsive protein switches built on the CATCHFIRE dimerization system, pairing a fluorogenic molecular glue with split protein reconstitution to control luciferases, proteases, recombinases, transcription factors, and secreted proteins. The central design insight of coupling a dimerizer that reports its own assembly via fluorescence activation with two-hybrid-style switching is elegant, and the breadth of applications demonstrated in a single study is impressive. CATCH-ON v2 stands out for combining high dynamic range with near-undetectable basal activity, and the orthogonality of CATCHFIRE with rapamycin and ABA-based CID systems is carefully validated. While the composability this orthogonality …
Chemically responsive protein switches for the precise control of biological activities
This work presents a versatile suite of chemically responsive protein switches built on the CATCHFIRE dimerization system, pairing a fluorogenic molecular glue with split protein reconstitution to control luciferases, proteases, recombinases, transcription factors, and secreted proteins. The central design insight of coupling a dimerizer that reports its own assembly via fluorescence activation with two-hybrid-style switching is elegant, and the breadth of applications demonstrated in a single study is impressive. CATCH-ON v2 stands out for combining high dynamic range with near-undetectable basal activity, and the orthogonality of CATCHFIRE with rapamycin and ABA-based CID systems is carefully validated. While the composability this orthogonality enables extends across the full toolkit, it’s especially powerful for the split protease applications, where unique cleavage sequence specificity could potentially allow for direct wiring into cascades, logic gates, and signaling circuits.
You rightly note that the road from proof of concept to therapeutic implementation is long. In that vein, I have a few questions:
CATCH-ON v2 relies on p65Δ as its activation domain, and match550 is described as having no off-target biological activity. Although you used p65Δ without the Rel homology domain, do you think residual interactions with endogenous NF-κB pathway components could persist? Therapeutic applications here would largely operate in immune cells where NF-κB signaling is central. More broadly, has the cellular footprint of match550 addition been assessed beyond viability, for example at the transcriptomic, proteomic, or metabolic level? This matters both for the off-target selectivity claim and for any application where CATCHFIRE might be used to produce a defined perturbation whose downstream effects need to be cleanly attributable to the intended target.
All demonstrations use bicistronic P2A constructs that enforce 1:1 stoichiometry between the two FIRE-tagged components. This is a clean solution for self-contained genetic switches, but many envisioned applications involving biologically relevant protein payloads with independent folding, stability, and localization properties would not be amenable to P2A linkage or would have different effective expression even with bicistronic transcription. Have you evaluated system performance under conditions where stoichiometry is not enforced, and do you have a sense for how sensitive the dynamic range and basal leakiness are to expression ratio imbalance?
Several panels in Figure 5 show detectable basal activity in DMSO conditions, most notably for UAS-driven CRE. Basal recombinase activity has qualitatively different consequences than for other outputs, since even low-level activity produces irreversible genomic edits. How do you think about the tolerance for basal CRE activity in therapeutic or in vivo contexts, and do you see optimization of split protein self-complementation as the primary path to reducing it?
Finally, all experiments here are in HeLa cells. Have you tested or do you plan to test CATCHFIRE in other cellular contexts, whether primary immune cells relevant to the CAR-T applications or non-mammalian systems where a titratable, orthogonal inducible switch would be broadly useful as a tool?
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