Masoud Vedadi

DCAF1 is a donut shaped WD40 repeat protein and a substrate receptor of two distinct E3 ligases. In HIV-1 infection, the viral protein Vpr binds to the top surface of DCAF1, changes its substrate specificity to degrade human proteins involved in antiviral activities, enabling HIV-1 to replicate. We hypothesized that artificial top-to-top self-dimerization of DCAF1 could result in DCAF1 loss-of-function and blocking of the Vpr-DCAF1 interaction. We designed and synthesized seven compounds we call SDIPTACs (Self-dimerization Induced Proximity Targeting Chimeras) which artificially induce DCAF1 self-dimerization through the Vpr interaction surface. Interestingly, SDIPTACs C8 and C9, inhibited Vpr-dependent HIV replication in CD4⁺ T cells. Biophysical data and crystal structures of four DCAF1-SDIPTAC-DCAF1 ternary complexes revealed more details on DCAF1-DCAF1 complex stability, conformation, and their contribution to compound efficacy. Using SDIPTACs with unique mechanism may therefore be an efficient strategy in the development of future therapeutics for HIV infection and other diseases.

Human RNA capping is critical for mRNA splicing, protection of RNA from 5′ exonucleases in the cytoplasm, and targeting to the ribosome. Human RNMT, CMTR1, and CMTR2 are RNA methyltransferases involved in the RNA capping process. They play a significant role in the proliferation and differentiation of embryonic stem cells and have been implicated in cancer. Substrate specificities of human RNA capping methyltransferases have been somewhat explored in a few studies. Here, we report on a comprehensive, systematic, and quantitative assessment of their substrate specificities along with SARS‐CoV‐2 counterparts, nsp14 and nsp16. We discovered novel cooperative activities of human enzymes. We designed and synthesized various RNA substrates with defined patterns of methylation to systematically assess the dependency or cooperativity of their activities using radiometric assays followed by mass spectrometry to verify RNA methylation status. We have tested all five enzymes in parallel against these substrates and determined kinetic parameters. Our data not only indicate that the catalytic activities of human RNMT, CMTR1, and CMTR2 are distinct and nonoverlapping, but also provide a novel quantitative assessment of their activities, indicating how and to what extent these proteins affect each other's function. Unlike nsp14 and nsp16, their functions are not necessarily sequential, but show significant cooperativity. Altogether, our data provide a comprehensive understanding of substrate specificities of human RNA capping methyltransferases, enabling the development of potential future anticancer therapeutics and assessment of antiviral therapeutics' selectivity.

Epigenetically targeted therapies, especially those inhibiting S-adenosyl-l-methionine (SAM)-dependent methylations of DNA, mRNA, and histones, have advanced rapidly in cancer treatment. However, these therapies remain underexplored for antibiotic development, despite the growing threat of antimicrobial resistance. Here, we screened a focused library of SAM analogs against the DNA adenine methyltransferase CamA specific to the enteric pathogen Clostridioides difficile. At the same time, we examined six other adenine methyltransferases, including two bacterial DNA methyltransferases and four human RNA methyltransferases having distinct RNA substrates. Compound 113 selectively inhibited CamA (IC₅₀ = 0.15 μM). In addition, compound 67 inhibited Caulobacter crescentus CcrM (IC₅₀ = 1.8 μM), which has orthologs present in pathogens such as Brucella, while compounds 77 and 37 inhibited the human RNA methyltransferase complexes MettL3-MettL14 and MettL5-Trm112, respectively, at 7–8 μM concentrations. These results provide chemical probes for exploring the role of CamA in sporulation and colonization with potential as antivirulence agents against C. difficile infection. Our study also introduces the first chemical probes for inhibiting bacterial CcrM and human MettL5, each of which plays key roles in their respective hosts.

Proteolysis-targeting chimeras (PROTACs) have been explored for the degradation of drug targets for more than two decades. However, only a handful of E3 ligase substrate receptors have been efficiently used. Downregulation and mutation of these receptors would reduce the effectiveness of such PROTACs. We recently developed potent ligands for DCAF1, a substrate receptor of EDVP and CUL4 E3 ligases. Here, we focus on DCAF1 toward the development of PROTACs for WDR5, a drug target in various cancers. We report four DCAF1-based PROTACs with endogenous and exogenous WDR5 degradation effects and high-resolution crystal structures of the ternary complexes of DCAF1-PROTAC-WDR5. The structures reveal detailed insights into the interaction of DCAF1 with various WDR5-PROTACs, indicating a significant role of DCAF1 loops in providing needed surface plasticity, and reflecting the mechanism by which DCAF1 functions as a substrate receptor for E3 ligases with diverse sets of substrates.

Proteolysis-targeting chimeras (PROTACs) have been explored for the degradation of drug targets, particularly undruggable proteins, for more than two decades. They have been employed by various groups successfully, however only a handful of E3 ligase substrate receptors such as CRBN, VHL, MDM2, and IAP have been efficiently used. Downregulation and mutation of these receptors would reduce the effectiveness of PROTACs, thus necessitating the addition of new substrate receptors and E3 ligases to the repertoire to avoid resistance. We recently developed potent ligands for DCAF1, a substrate receptor of EDVP and CUL4 E3 ligases with diverse substrate specificity. Here, we focused on DCAF1 toward the development of PROTACs for WDR5, a drug target in various cancers. We report three DCAF1-based PROTACs with endogenous and exogenous WDR5 degradation effects and solved the first high-resolution crystal structures of DCAF1-PROTAC-WDR5 ternary complexes. The structures reveal detailed insights into the interaction of DCAF1 with various WDR5-PROTACs, including the finding that PROTACs can compress like a spring to similar effective lengths to keep DCAF1 and WDR5 within interaction distance of each other. Furthermore, specific DCAF1 loops play a significant role in providing surface plasticity needed to interact with different PROTAC-WDR5 complexes, reflecting the mechanism by which DCAF1 functions as a substrate receptor for E3 ligases with diverse sets of substrates.

Workflow used for identifying inhibitors of the SARS-CoV-2 non-structural protein 14 (nsp14), a key player in viral RNA methylation.

The DNA damage binding protein 1 (DDB1) is an essential component of protein complexes involved in DNA damage repair and the ubiquitin-proteasome system (UPS) for protein degradation. As an adaptor protein specific to Cullin-RING E3 ligases, DDB1 binds different receptors that poise protein substrates for ubiquitination and subsequent degradation by the 26S proteasome. Examples of DDB1-binding protein receptors are Cereblon (CRBN) and the WD-repeat containing DDB1- and CUL4-associated factors (DCAFs). Cognate substrates of CRBN and DCAFs are involved in cancer-related cellular processes or are mimicked by viruses to reprogram E3 ligases for the ubiquitination of antiviral host factors. Thus, disrupting interactions of DDB1 with receptor proteins might be an effective strategy for anticancer and antiviral drug discovery. Here, we developed fluorescence polarization (FP)-based peptide displacement assays that utilize full-length DDB1 and fluorescein isothiocyanate (FITC)-labeled peptide probes derived from the specific binding motifs of DDB1 interactors. A general FP-based assay condition applicable to diverse peptide probes was determined and optimized. Mutagenesis and biophysical analyses were then employed to identify the most suitable peptide probe. The FITC-DCAF15 L49A peptide binds DDB1 with a dissociation constant of 68 nM and can be displaced competitively by unlabeled peptides at sub-μM to low nM concentrations. These peptide displacement assays can be used to screen small molecule libraries to identify novel modulators that could specifically antagonize DDB1 interactions toward development of antiviral and cancer therapeutics.

A chemical probe (PFI-7) for the Gid4 subunit of the human E3 ligase CTLH degradation complex.