Rational discovery of molecular glue degraders via scalable chemical profiling

The basal affinity between DDB1 and the CDK12-CycK complex is relatively weak (~50 μM), and addition of the glue degrader dramatically enhanced this interaction.

“interestingly, while the basal affinity between DDB1 and the cDK12-cycK complex is relatively weak (~50 µM), addition of the glue degrader dramatically enhanced this interaction (Mayor-Ruiz et al. 2020).”

Binding of the molecular glue to the active site of CDK12 enhances dimerization between CDK12 and CycK and promotes the formation of a quaternary complex with the CUL4 adaptor protein DDB1.

“Binding of the molecular glue to the active site of cDK12 enhances dimerization between cDK12 and cycK and also promotes the formation of a quaternary complex with the cul4 adaptor protein DDB1 (lv et al. 2020;Mayor-Ruiz et al. 2020;Słabicki et al. 2020a).”

Phenotypic screening in UBE2M-deficient cancer cells established a scalable drug screening platform that identified novel molecular glue degraders for multiple protein targets including cyclin K.

“using this system, researchers established a scalable drug screening platform that identified novel molecular glue degraders for multiple protein targets including cyclin K (cycK) (Mayor-Ruiz et al. 2020).”

Two independent studies discovered structurally distinct CycK degraders.

“Notably, two independent studies also discovered structurally distinct cycK degraders (lv et al. 2020;Mayor-Ruiz et al. 2020)”

A panel of CDK12 inhibitors act as MGs to enable a neo-morphic interaction between CDK12 and DDB1, inducing CRL4-catalyzed Cyclin K ubiquitination and degradation.

“a panel of CDK12 inhibitors have been shown to act as MGs to enable a neo-morphic interaction between CDK12 and DDB1, inducing CRL4-catalyzed Cyclin K ubiquitination and degradation27–29.”

Mayor-Ruiz et al. examined 2000 cytotoxic compounds between normal and CRL-inactivated cells to identify several compounds whose cytotoxicity requires CRL activity.

“Mayor-Ruiz et al. examined 2000 cytotoxic compounds between normal and CRL-inactivated cells to identify several compounds whose cytotoxicity requires CRL activity.”

Two studies identified CDK12 inhibitors that induce CDK12-DDB1 interactions to trigger CCNK degradation.

“While we are preparing our work for publication, two studies identified CDK12 inhibitors that induce CDK12-DDB1 interactions to trigger CCNK degradation (Mayor-Ruiz et al., 2020; Słabicki et al., 2020).”

Additional MGDs have been reported that induce degradation of proteins devoid of obvious ligand-binding pockets, such as RBM39 or cyclin K.

“Additional MGDs have been reported, which induce the degradation of proteins devoid of obvious ligand-binding pockets, such as RBM3954−57 or cyclin K (CycK).58−60”

Both studies converge on a shared mechanism of action where the identified compounds directly glue the CDK12-CycK complex to DDB1, prompting CycK degradation in the absence of a dedicated substrate receptor.

“Both studies converge on a shared mechanism of action where the identified compounds directly glue the CDK12-CycK complex to DDB1, thus prompting CycK degradation in the absence of a dedicated substrate receptor.”

Cell-viability-based phenotypic assays have led to the identification of several MGDs that induce the degradation of CycK.

“Cell-viability-based phenotypic assays have also led to the identification of several MGDs that induce the degradation of CycK.”

Systematic screening efforts have revealed high glue hit rates in drug-like collections, suggesting that glue-like traits may be more abundant than previously anticipated.

“In addition, systematic screening efforts have revealed high glue hit rates in drug-like collections,59 suggesting that glue-like traits may be more abundant than previously anticipated.”

In 2020, three groups independently reported the discovery of structurally different CycK MG degraders.

“In 2020, three groups independently reported the discovery of structurally different CycK MG degraders (Fig. 7 and 8).”

dCeMM2/3/4 were identified as dependent on functional CRLs to exert their toxicity.

“In addition, three small-molecule drugs (dCeMM2/3/4) were identified as dependent on functional CRLs to exert their toxicity.”

dCeMM2/3/4 were proved to be CycK MG degraders.

“Through an extensive and orthogonal effort aimed at dual target-E3 identification, these chemical entities were proved to be CycK MG degraders.154”

The sulfonamide scaffold dCeMM1 hijacks CRL4 DCAF15 to selectively degrade RBM39.

“First, a novel sulfonamide scaffold (dCeMM1) was found to hijack CRL4 DCAF15 to selectively degrade RBM39, thus validating the chemical profiling strategy.”

All the studies converged on a mechanism in which drug binding to the active site of CDK12 (and presumably CDK13) strengthens dimerization between CDK12-CycK and the CRL adaptor DDB1, in the absence of a dedicated substrate receptor.

“Importantly, all the studies converged on a unifying and unprecedented mechanism of action: drug binding to the active site of the usual CycK partner CDK12 (and, presumably, the paralog CDK13) strengthened the dimerization between CDK12-CycK and the CRL adaptor DDB1, in the absence of a dedicated substrate receptor.”

Exhaustive mechanistic characterizations have revealed unprecedented modes of action to elicit productive TPD, such as rewiring a partial CRL complex or inducing protein polymerization.

“Exhaustive mechanistic characterizations have revealed unprecedented modes of action to elicit productive TPD, such as rewiring a partial CRL complex 134,153,154 or inducing protein polymerization.133”

Molecular glue degraders deplete CCNK by promoting an interaction between CCNK and the CUL4 adaptor protein DDB1 via CDK12, thereby presenting CCNK for ubiquitination and degradation.

“Recently, molecular glue degraders that deplete CCNK were identified (Lv et al., 2020;Mayor-Ruiz et al., 2020;S1abicki et al., 2020).These compounds act by promoting an interaction between CCNK and the CUL4 adaptor protein DDB1 via CDK12, thereby presenting CCNK for ubiquitination and degradation.”

Additional, structurally different, molecular glue degraders of CCNK/CDK12 have been reported.

“Recently, additional, but structurally different, molecular glue degraders of CCNK/CDK12 have been reported (Lv et al., 2020;Mayor-Ruiz et al., 2020;S1abicki et al., 2020).”

These molecular glue degraders induce complex formation between CDK12/CCNK and the CUL4 adaptor protein DDB1, presenting CCNK for ubiquitination and degradation via DDB1-CUL4-RBX1 ubiquitin ligases.

“These molecules induce complex formation between CDK12/CCNK and the CUL4 adaptor protein DDB1, presenting CCNK for ubiquitination and degradation via DDB1-CUL4-RBX1 ubiquitin ligases.”

CR8 directly co-opts DDB1 as an E3 ligase for the degradation of cyclin K (CCNK).

“a unique class of cyclin-dependent kinase 12 (CDK12) inhibitors, exemplified by CR8, which, unexpectedly, has been shown to directly co-opt DDB1 as an E3 ligase for the degradation of cyclin K (CCNK)84–86.”

Four separate classes of compounds were found to have molecular glue-like properties in potentiating the interaction of DDB1 and CDK12, resulting in CCNK degradation.

“In fact, not only CR8, but four separate classes of compounds, each discovered by a different screening method, were found to have molecular glue-like properties in potentiating the interaction of DDB1 and CDK12, resulting in CCNK degradation84–87.”

Simonetta and colleagues prospectively identified and performed rational structure-guided optimization of drug-like small molecules that enhance the interaction between SCFβ-TrCP and mutant forms of β-catenin.

“Simonetta and colleagues186 prospectively identified and performed subsequent rational structure-guided optimization of drug-like small molecules that profoundly enhance the interaction between SCFβ–TrCP and such mutant forms of β-catenin.”