Chemically induced proximity in biology and medicine

Abstract

Proximity, or the physical closeness of molecules, is a pervasive regulatory mechanism in biology. For example, most posttranslational modifications such as phosphorylation, methylation, and acetylation promote proximity of molecules to play deterministic roles in cellular processes. To understand the role of proximity in biologic mechanisms, chemical inducers of proximity (CIPs) were developed to synthetically model biologically regulated recruitment. Chemically induced proximity allows for precise temporal control of transcription, signaling cascades, chromatin regulation, protein folding, localization, and degradation, as well as a host of other biologic processes. A systematic analysis of CIPs in basic research, coupled with recent technological advances utilizing CRISPR, distinguishes roles of causality from coincidence and allows for mathematical modeling in synthetic biology. Recently, induced proximity has provided new avenues of gene therapy and emerging advances in cancer treatment.

Fig. 1.. The evolution of systems for CIPs.

Protein targets and chemical ligands are shown for CIP systems. Proteins are represented as ribbon diagrams from available crystal structures, with endogenous monomeric functions indicated. Chemical ligands are represented in bound conformations docked with protein targets and also represented separately as individual structures. Gray shading of the chemical structures is divided to annotate specific structural moieties associated with molecular recognition of annotated protein targets. CIP systems are represented from left to right in approximate order of development. Me, methyl group; R, linker moiety.

Fig. 2.. Modeling reaction kinetics associated with systems of induced proximity.

(A) The differential concentration with respect to time is explained by the changes in the rate of diffusion and the binding kinetics of the dimerizer system. (B) Changes in concentrations of monomeric and dimeric complexes are dependent on rate of reaction and rate of diffusion, defined by the distance to the site of recruitment. With no chemical induction or high K_d, formation of ternary complexes is determined by the rate of diffusion, as ku(x, t) approaches zero. With chemical induction and low K_d, induced ternary-complex formation is strongly dependent on the rate of the reaction ku(x,t), which dominates the rate of diffusion. Direct-fusion systems are exclusively localized at the site of recruitment as the reaction rate approaches infinity and dominates the rate of diffusion. In protein-dimerizer interactions, these complexes are designated by *. (C) Thermodynamic contributions to chemically induced dimerization include minimizing translational and rotational entropy. Multistate binding equilibria associated with initial binding of a bifunctional dimerizer molecule (hexagons) to respective targets by forming unstable binary complexes that form composite surfaces and rapidly assemble ternary complexes. Arrows show direction of movement or rotation. (D) Kinetics of ternary-complex assembly can be described by three-body binding equilibria.

Fig. 3.. Chemical induction of proximity is sufficient for the regulation of diverse cellular processes.

Induced proximity has been shown to regulate initiation of transcription, signaling cascades, chromatin dynamics, proteasomal degradation, and subcellular localization. (A) Induced proximity has been systematically explored to bypass Tcell antigen receptor activation and for synthetic induction of a variety of signaling cascades. (B) CIPs have been developed for rapid induction of transcriptional activation (VP16) and repression (KRAB or HP1) using DNA binding domains (DBDs) as well as CIP of split–CRISPR-Cas proteins or CIP recruitment of activators or repressors through CRISPR-Cas9 systems. (C) CIP has been used for rapid protein localization, including nuclear import and export, localization to components of the secretory pathway, synaptic vesicles, and mitochondria. (D) Rapid proximity-based protein degradation is achieved through bifunctional molecule–mediated recruitment of E3 ubiquitin ligase complexes (complex composed of E2, ROC1, CUL4A, DDB1, and CRBN). With a related approach, auxin can induce ubiquitin-mediated degradation through recruitment of the TIR1-Cul1 complex. (E) Induced proximity has been used for rapid induction of activated chromatin states through recruitment of ATP-dependent remodeling complexes and induction of repressive chromatin states through HP1-mediated heterochromatin formation.

Fig. 4.. Ubiquitin ligase complexes rapidly degrade oncogenic protein targets.

(A) The CBRN-CUL4A ubiquitin ligase complex can be recruited to BCR-ABL and BRD4 with thalidomide conjugated to bosutinib and JQ1, respectively. Induced proximity rapidly degrades these targets, which are known to drive chronic myelogenous leukemia (BCR-ABL) and acute myeloid leukemia (BRD4). (B) The VHL ligand fused to desatinib or JQ1 can efficiently recruit the VHL-Cullin2 ubiquitin ligase complex to ABL and BRD4, respectively. In each case, rapid degradation of the oncogenic targets results in inhibition of cancer growth.

Fig. 5.. CAR T cell therapeutic applications of CIPs.

(A) Engineered safety switches using ATTAC systems with AP1903. (B) An inactive engineered CAR T cell receptor and (C) an active engineered CAR T cell receptor binding its cognate antigen. (D) AP1903-induced caspase dimerization and activation allows for rapid apoptosis of CAR T cells to prevent complications that may arise from transplant.