Targeted Protein Degradation through Recruitment of the CUL4 Complex Adaptor Protein DDB1

Targeted protein degradation has arisen as a powerful therapeutic modality for eliminating proteins. Thus far, most heterobifunctional proteolysis targeting chimeras (PROTACs) have utilized recruiters against substrate receptors of Cullin RING E3 ubiquitin ligases, such as cereblon and VHL. However, previous studies have surprisingly uncovered molecular glue degraders that exploit a CUL4 adaptor protein DDB1 to degrade neosubstrate proteins. Here, we sought to investigate whether DDB1 recruiters can be discovered that can be exploited for PROTAC applications. We utilized activity-based protein profiling and cysteine chemoproteomic screening to identify a covalent recruiter that targets C173 on DDB1 and exploited this recruiter to develop PROTACs against BRD4 and androgen receptor (AR). We demonstrated that the BRD4 PROTAC results in selective degradation of the short BRD4 isoform over the long isoform in a proteasome, NEDDylation, and DDB1-dependent manner. We also demonstrated degradation of AR with the AR PROTAC in prostate cancer cells. Our study demonstrated that covalent chemoproteomic approaches can be used to discover recruiters against Cullin RING adapter proteins and that these recruiters can be used for PROTAC applications to degrade neo-substrates

Targeted Protein Degradation through Recruitment of the CUL4 Complex Adaptor Protein DDB1

Targeted protein degradation has arisen as a powerful therapeutic modality for eliminating proteins. Thus far, most heterobifunctional proteolysis targeting chimeras (PROTACs) have utilized recruiters against substrate receptors of Cullin RING E3 ubiquitin ligases, such as cereblon and VHL. However, previous studies have surprisingly uncovered molecular glue degraders that exploit a CUL4 adaptor protein DDB1 to degrade neosubstrate proteins. Here, we sought to investigate whether DDB1 recruiters can be discovered that can be exploited for PROTAC applications. We utilized activity-based protein profiling and cysteine chemoproteomic screening to identify a covalent recruiter that targets C173 on DDB1 and exploited this recruiter to develop PROTACs against BRD4 and androgen receptor (AR). We demonstrated that the BRD4 PROTAC results in selective degradation of the short BRD4 isoform over the long isoform in a proteasome, NEDDylation, and DDB1-dependent manner. We also demonstrated degradation of AR with the AR PROTAC in prostate cancer cells. Our study demonstrated that covalent chemoproteomic approaches can be used to discover recruiters against Cullin RING adapter proteins and that these recruiters can be used for PROTAC applications to degrade neo-substrates

Targeted Protein Degradation through Recruitment of the CUL4 Complex Adaptor Protein DDB1

Targeted protein degradation has arisen as a powerful therapeutic modality for eliminating proteins. Thus far, most heterobifunctional proteolysis targeting chimeras (PROTACs) have utilized recruiters against substrate receptors of Cullin RING E3 ubiquitin ligases, such as cereblon and VHL. However, previous studies have surprisingly uncovered molecular glue degraders that exploit a CUL4 adaptor protein DDB1 to degrade neosubstrate proteins. Here, we sought to investigate whether DDB1 recruiters can be discovered that can be exploited for PROTAC applications. We utilized activity-based protein profiling and cysteine chemoproteomic screening to identify a covalent recruiter that targets C173 on DDB1 and exploited this recruiter to develop PROTACs against BRD4 and androgen receptor (AR). We demonstrated that the BRD4 PROTAC results in selective degradation of the short BRD4 isoform over the long isoform in a proteasome, NEDDylation, and DDB1-dependent manner. We also demonstrated degradation of AR with the AR PROTAC in prostate cancer cells. Our study demonstrated that covalent chemoproteomic approaches can be used to discover recruiters against Cullin RING adapter proteins and that these recruiters can be used for PROTAC applications to degrade neo-substrates

Structural and biophysical comparisons of the pomalidomide- and CC-220-induced interactions of SALL4 with cereblon.

The design of cereblon-binding molecular glues (MGs) that selectively recruit a desired protein while excluding teratogenic SALL4 is an area of significant interest when designing therapeutic agents. Previous studies show that SALL4 is degraded in the presence of IKZF1 degraders pomalidomide, and to a lesser extent by CC-220. To expand our understanding of the molecular basis for the interaction of SALL4 with cereblon, we performed biophysical and structural studies demonstrating that SALL4 zinc finger domains one and two (ZF1-2) interact with cereblon (CRBN) in a unique manner. ZF1 interacts with the N-terminal domain of cereblon and ZF2 binds as expected in the C-terminal IMiD-binding domain. Both ZF1 and ZF2 contribute to the potency of the interaction of ZF1-2 with CRBN:MG complexes and the affinities of SALL4 ZF1-2 for the cereblon:CC-220 complex are less potent than for the corresponding pomalidomide complex. Structural analysis provides a rationale for understanding the reduced affinity of SALL4 for cereblon in the presence of CC-220, which engages both ZF1 and ZF2. These studies further our understanding of the molecular glue-mediated interactions of zinc finger-based proteins with cereblon and may provide structural tools for the prospective design of compounds with reduced binding and degradation of SALL4

Structural and biophysical comparisons of the pomalidomide- and CC-220-induced interactions of SALL4 with cereblon

The design of cereblon-binding molecular glues (MGs) that selectively recruit a desired protein while excluding teratogenic SALL4 is an area of significant interest when designing therapeutic agents. Previous studies show that SALL4 is degraded in the presence of IKZF1 degraders pomalidomide, and to a lesser extent by CC-220. To expand our understanding of the molecular basis for the interaction of SALL4 with cereblon, we performed biophysical and structural studies demonstrating that SALL4 zinc finger domains one and two (ZF1-2) interact with cereblon (CRBN) in a unique manner. ZF1 interacts with the N-terminal domain of cereblon and ZF2 binds as expected in the C-terminal IMiD-binding domain. Both ZF1 and ZF2 contribute to the potency of the interaction of ZF1-2 with CRBN:MG complexes and the affinities of SALL4 ZF1-2 for the cereblon:CC-220 complex are less potent than for the corresponding pomalidomide complex. Structural analysis provides a rationale for understanding the reduced affinity of SALL4 for cereblon in the presence of CC-220, which engages both ZF1 and ZF2. These studies further our understanding of the molecular glue-mediated interactions of zinc finger-based proteins with cereblon and may provide structural tools for the prospective design of compounds with reduced binding and degradation of SALL4

Structural and Biological Basis of Small Molecule Inhibition of Escherichia coli LpxD Acyltransferase Essential for Lipopolysaccharide Biosynthesis

LpxD, acyl-ACP-dependent N-acyltransferase, is the third enzyme of lipid A biosynthesis in Gram-negative bacteria. A recent probe-based screen identified several compounds, including 6359-0284 (compound 1), that inhibit the enzymatic activity of Escherichia coli (E. coli) LpxD. Here, we use these inhibitors to chemically validate LpxD as an attractive antibacterial target. We first found that compound 1 was oxidized in solution to the more stable aromatized tetrahydro-pyrazolo-quinolinone compound 1o. From the Escherichia coli strain deficient in efflux, we isolated a mutant that was less susceptible to compound 1o and had an lpxD missense mutation (Gly268Cys), supporting the cellular on-target activity. Using surface plasma resonance, we showed direct binding to E. coli LpxD for compound 1o and other reported LpxD inhibitors in vitro. Furthermore, we determined eight cocrystal structures of E. coli LpxD/inhibitor complexes. These costructures pinpointed the 4′-phosphopantetheine binding site as the common ligand binding hotspot, where hydrogen bonds to Gly269 and/or Gly287 were important for inhibitor binding. In addition, the LpxD/compound 1o costructure rationalized the reduced activity of compound 1o in the LpxDGly268Cys mutant. Moreover, we obtained the LpxD structure in complex with a previously reported LpxA/LpxD dual targeting peptide inhibitor, RJPXD33, providing structural rationale for the unique dual targeting properties of this peptide. Given that the active site residues of LpxD are conserved in multidrug resistant Enterobacteriaceae, this work paves the way for future LpxD drug discovery efforts combating these Gram-negative pathogens