Can the molecular adhesive degradation agent usher in a breakthrough after PROTAC?

Many diseases are caused by the accumulation of specific pathogenic proteins, so most drugs often use a "key-lock" design concept that blocks their activity by binding directly into the bound pocket of the target proteinHowever, there are a lot of proteins, the use of this conventional design method can not achieve the target, we call the "non-drug" target, but with the rise of PROTAC technology, especially the recent release of clinical results, let us see the hope of targeting these "non-drug targets"However, PROTAC targets most lying at intracellular proteins, which appear to be so hard for most extracellular proteinsIn fact, there are a variety of protein degradation pathways in the cell, ubiquitin-protease is only one of them, in addition to the ability to degrade extracellular protein autophagy, lysosome degradation and so onRecently, scientists from the Massachusetts Institute of Technology, Harvard University and others published a new molecular adhesive degradation agent, CR8, in Nature, which showed us how to build more of these unique compounds as potential treatments for a variety of diseases by analyzing the details of the mechanism of CR8CR8 was originally designed to inhibit compounds that play an important role in cell growth, known as cell cycle protein-dependent kinase (CDK)Using their bioinformatics method, the researchers found that the cell-killing activity of CR8 was associated with levels of the E3 ubiquitin-connected enzyme complex DDB1First, in order to identify small molecules that degrade proteins by E3 ubiquitin-mediated enzymes, the researchers correlated the drug sensitivity data of 4,518 clinical and preclinical drugs tested by 578 cancer cell lines with the respective mRNA levels of 499 E3 connecting enzymes, and used CRISPR-mediated E3 connecting enzyme components to inactivate to verify the correlation between the connectivee expression and drug toxicityIn quantitative proteins (more than 8,000), cell cycle protein K is the only protein that always shows decreased abundance after the addition of CR8, CR8 does not change the level of cell cycle protein K mRNA, and CR8-induced cell cycle protein K degradation can inhibit the action of e1 unophinary activation enzyme (using MLN7243), inhibitcullin adenosine (MLN4924) and protein receptors (MG132) to inhibit the effects of the enzyme (MG132)These show that CR8 is rapid protease degradation of the cell cycle protein K by the activity of cullin-RING ubiquitin containing DDB1 (Figure e)in vitro ubiquitization tests have confirmed that the CUL4A-RBX1-DDB1 connecting core alone is sufficient to drive the powerful ubiquitization of cell cycle protein KIn the absence of drugs, weak interactions between CDK12-cyclinK and DDB1 can still be detected in vitro, but CR8 enhances the formation of complexesThe quantitative results of the interaction show that CR8 stimulates the combination between CDK12-cyclin K and DDB1 in the 100-500 nM range, indicating that CR8's participation CDK12-cyclinK was raised through DDB1 to the CUL4-RBX1-DDB1 connecting enzyme core, and CR8 sufficiently tightened the complex to drive CR8-induced Kin K degradationother teams have studied a drug with a structure similar to CR8 and found that it does not cause the cell cycle protein K to degradeThe only structural difference between the two compounds is the protruding chemical part of the argonThe team concluded that this part was sufficient to make CR8 work like a molecular glue degradation agentThe findings suggest that the chemical modification of the outer part of the inhibitor can transform it into a molecular adhesive degradation agent that targets specific proteins finally, to examine how CUL4-mediated cell cycle protein K degradation gained CR8 cytotoxicity compared to non-degradable CDK inhibition, the researchers analyzed the wild HEK293T-Cas9 cells, as well as the PREtreatment of CR8 toxicity in cells that could cause DCAF overexpression or DDB1 gene deletion The results showed that MLN4924's total inhibition of the activity of cullin-RING connecting enzymes had only a small effect on cell vitality, but resulted in reduced sensitivity to CR8, indicating that cullin-RING's incartation acting connectivase actually contributed to CR8 toxicity The overexpression of the substrate receptor CRBN also affects the sensitivity of cells to CR8 and reduces the degradation of cell cycle protein K As expected, CR8-induced cell cycle protein K degradation depends on DDB1 and, consistently, CR8 (rather than other CDK inhibitors) has ten times lower cytotoxicity in cells lacking DDB1 "We have shown that conventional kinase inhibitors can be used and converted into molecular glue degradation agents by connecting specific chemical groups," said Study co-author Benjamin Ebert, director of the Department of Medical Oncology at the Dana-Farber Cancer Institute These findings show that the use of small molecule-induced kinase inactivation of the protein-protein interface provides a way to improve drug selectivity, and proves that modifying the surface exposure area of the target small molecule is a reasonable strategy that can be used to develop a molecular glue degradation agent for a given protein target Looking here, it's a thought of a recent article published in JMC, when researchers studied Bcl6 inhibitors but discovered compounds that degrade Bcl6 the researchers used fluorescent polarization (FP) analysis and HIGH-throughput screening of HIT compounds 2 and 3 (Table 1), which had a weaker price (IC50 to 100 m) Due to poor solubility hindering the acquisition of ligand-binding X-ray structure, in order to solve this problem, a less aromatic ring analogue with reduced lipolateability was further prepared Compounds 4 and 5 exhibit activity comparable to previously obtained compounds and improve solubility (Table 1) to determine the X-ray structure analysis of protein crystals, it is found that the compound binds at the dipolymer interface of the BTB domain of BCL6 (Figures A and B) 4 and 5 have two interaction characteristics: the hydrogen bonds of the skeleton molybdenum from NH to Met51 and the cyanide part inserted into the gap between Tyr58 and Asn21 on the protein surface, resulting in a critical hydrophobic interaction between the side chains of cyanide and Tyr58 Compound 4 forms an additional interaction with glu115's main chain NH and the main chain NH of His116 through the oxygen atoms of its benzodiazepine nucleus The cmconioscoise 5 interacts only with weak hydrophobic interactions with the skeleton of the residual 53-55, indicating that 5 of the chlorpyrifos provides most of the binding affinity of the compound , the researchers hypothesized that the combination of benzodiazepine core with 5 chlorocyanol in combination would lead to increased potency It is gratifying to note that the combined affinity of the resulting compound 6 (CCT365386) has indeed improved to 10 m Since then, the researchers have continued to study improving the efficacy and solubility of the compound, however, in the study, it was found that the SU-DHL4 and OCI-Ly1 cells processed by compound 24b were consistent with compound-mediated BCL6 degradation, and Western analysis of protein lysates showed a decrease in concentration dependence on BCL6 protein levels In view of the Bcl6 degradation capability of the replacement base expression of the different R3 positions in the above figure, taking into account the catalytic action of small molecule degradations, the need for full continuous occupation of BCL6, and the possibility of being active in lower concentrationcompounds, the researchers decided to focus on the optimization of this new BCL6 degradation, with the aim of identifying compounds with appropriate physical and pharmacokinetic properties, and eventually discovered CCT369260 By analyzing the position of protein binding that causes the argon-based protein, which has a compound degradation effect, they found that the group is also in the surface exposure area of the dipolymer interface researchers speculated on a potential mechanism by which these molecules promote protein degradation, possibly by "hydrophobic markers" that bind to target proteins and appear on the surface in the form of "hydrophobic spots", mimicking some of the unfolded protein states and then targeting degradation Another hypothesis is that the combination of protein surfaces and degradation agents creates "neo-substrates" or "neo-Degron" that can be identified by effectos that can be degraded by E3 ubiquitin sases or other proteases These two literatures suggest that modifying the structure of molecules in the protein-protein interface or target-binding pocket surface exposure area is expected to reveal new molecular glioprotein degradation agents, but the question of how to reasonably design and select those fragments to transform small molecular inhibitors into degradation agents is still a problem to be solved Related paper: s1 Miko aj Sabicki et al The CDK cat cr8 acts as a molecular glue that's the sqe-vres cyclin K, Nature (2020) DOI: 10.1038/s41586-020-2374-x .2 Benjamin R Bellenie, et al Achieving In Vivo Target through The Discovery and Optimization of The Management of Benzimidazolone BCL6ers, DoI: 10.1021/acs.jmedchem.9b02076.