Development of Peptide Displacement Assays to Screen for Antagonists of DDB1 Interactions.

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.

Probing the CRL4DCAF12 interactions with MAGEA3 and CCT5 di-Glu C-terminal degrons.

Damaged DNA-binding protein-1 (DDB1)- and CUL4-associated factor 12 (DCAF12) serves as the substrate recognition component within the Cullin4-RING E3 ligase (CRL4) complex, capable of identifying C-terminal double-glutamic acid degrons to promote the degradation of specific substrates through the ubiquitin proteasome system. Melanoma-associated antigen 3 (MAGEA3) and T-complex protein 1 subunit epsilon (CCT5) proteins have been identified as cellular targets of DCAF12. To further characterize the interactions between DCAF12 and both MAGEA3 and CCT5, we developed a suite of biophysical and proximity-based cellular NanoBRET assays showing that the C-terminal degron peptides of both MAGEA3 and CCT5 form nanomolar affinity interactions with DCAF12 in vitro and in cells. Furthermore, we report here the 3.17 Šcryo-EM structure of DDB1-DCAF12-MAGEA3 complex revealing the key DCAF12 residues responsible for C-terminal degron recognition and binding. Our study provides new insights and tools to enable the discovery of small molecule handles targeting the WD40-repeat domain of DCAF12 for future proteolysis targeting chimera design and development.

ChemRAP uncovers specific mRNA translation regulation via RNA 5' phospho-methylation.

5'-end modifications play key roles in determining RNA fates. Phospho-methylation is a noncanonical cap occurring on either 5'-PPP or 5'-P ends. We used ChemRAP, in which affinity purification of cellular proteins with chemically synthesized modified RNAs is coupled to quantitative proteomics, to identify 5'-Pme "readers". We show that 5'-Pme is directly recognized by EPRS, the central subunit of the multisynthetase complex (MSC), through its linker domain, which has previously been involved in key noncanonical EPRS and MSC functions. We further determine that the 5'-Pme writer BCDIN3D regulates the binding of EPRS to specific mRNAs, either at coding regions rich in MSC codons, or around start codons. In the case of LRPPRC (leucine-rich pentatricopeptide repeat containing), a nuclear-encoded mitochondrial protein associated with the French Canadian Leigh syndrome, BCDIN3D deficiency abolishes binding of EPRS around its mRNA start codon, increases its translation but ultimately results in LRPPRC mislocalization. Overall, our results suggest that BCDIN3D may regulate the translation of specific mRNA via RNA-5'-Pme.

The co-crystal structure of Cbl-b and a small-molecule inhibitor reveals the mechanism of Cbl-b inhibition.

Cbl-b is a RING-type E3 ubiquitin ligase that is expressed in several immune cell lineages, where it negatively regulates the activity of immune cells. Cbl-b has specifically been identified as an attractive target for cancer immunotherapy due to its role in promoting an immunosuppressive tumor environment. A Cbl-b inhibitor, Nx-1607, is currently in phase I clinical trials for advanced solid tumor malignancies. Using a suite of biophysical and cellular assays, we confirm potent binding of C7683 (an analogue of Nx-1607) to the full-length Cbl-b and its N-terminal fragment containing the TKBD-LHR-RING domains. To further elucidate its mechanism of inhibition, we determined the co-crystal structure of Cbl-b with C7683, revealing the compound's interaction with both the TKBD and LHR, but not the RING domain. Here, we provide structural insights into a novel mechanism of Cbl-b inhibition by a small-molecule inhibitor that locks the protein in an inactive conformation by acting as an intramolecular glue.

Discovery of a First-in-Class Small-Molecule Ligand for WDR91 Using DNA-Encoded Chemical Library Selection Followed by Machine Learning.

WD40 repeat-containing protein 91 (WDR91) regulates early-to-late endosome conversion and plays vital roles in endosome fusion, recycling, and transport. WDR91 was recently identified as a potential host factor for viral infection. We employed DNA-encoded chemical library (DEL) selection against the WDR domain of WDR91, followed by machine learning to predict ligands from the synthetically accessible Enamine REAL database. Screening of predicted compounds identified a WDR91 selective compound 1, with a KD of 6 ± 2 µM by surface plasmon resonance. The co-crystal structure confirmed the binding of 1 to the WDR91 side pocket, in proximity to cysteine 487, which led to the discovery of covalent analogues 18 and 19. The covalent adduct formation for 18 and 19 was confirmed by intact mass liquid chromatography-mass spectrometry. The discovery of 1, 18, and 19, accompanying structure-activity relationship, and the co-crystal structures provide valuable insights for designing potent and selective chemical tools against WDR91 to evaluate its therapeutic potential.

Discovery of a 53BP1 Small Molecule Antagonist Using a Focused DNA-Encoded Library Screen.

Methyl-lysine reader p53 binding protein 1 (53BP1) is a central mediator of DNA break repair and is associated with various human diseases, including cancer. Thus, high-quality 53BP1 chemical probes can aid in further understanding the role of 53BP1 in genome repair pathways. Herein, we utilized focused DNA-encoded library screening to identify the novel hit compound UNC8531, which binds the 53BP1 tandem Tudor domain (TTD) with an IC50 of 0.47 ± 0.09 µM in a TR-FRET assay and Kd values of 0.85 ± 0.17 and 0.79 ± 0.52 µM in ITC and SPR, respectively. UNC8531 was cocrystallized with the 53BP1 TTD to guide further optimization efforts, leading to UNC9512. NanoBRET and 53BP1-dependent foci formation experiments confirmed cellular target engagement. These results show that UNC9512 is a best-in-class small molecule 53BP1 antagonist that can aid further studies investigating the role of 53BP1 in DNA repair, gene editing, and oncogenesis.

Discovery of Nanomolar DCAF1 Small Molecule Ligands.

DCAF1 is a substrate receptor of two distinct E3 ligases (CRL4DCAF1 and EDVP), plays a critical physiological role in protein degradation, and is considered a drug target for various cancers. Antagonists of DCAF1 could be used toward the development of therapeutics for cancers and viral treatments. We used the WDR domain of DCAF1 to screen a 114-billion-compound DNA encoded library (DEL) and identified candidate compounds using similarity search and machine learning. This led to the discovery of a compound (Z1391232269) with an SPR KD of 11 µM. Structure-guided hit optimization led to the discovery of OICR-8268 (26e) with an SPR KD of 38 nM and cellular target engagement with EC50 of 10 µM as measured by cellular thermal shift assay (CETSA). OICR-8268 is an excellent tool compound to enable the development of next-generation DCAF1 ligands toward cancer therapeutics, further investigation of DCAF1 functions in cells, and the development of DCAF1-based PROTACs.

Chemical Genetics Screen Identifies COPB2 Tool Compounds That Alters ER Stress Response and Induces RTK Dysregulation in Lung Cancer Cells.

Activating mutations in the epidermal growth factor receptor (EGFR) are common driver mutations in non-small cell lung cancer (NSCLC). First, second and third generation EGFR tyrosine kinase inhibitors (TKIs) are effective at inhibiting mutant EGFR NSCLC, however, acquired resistance is a major issue, leading to disease relapse. Here, we characterize a small molecule, EMI66, an analog of a small molecule which we previously identified to inhibit mutant EGFR signalling via a novel mechanism of action. We show that EMI66 attenuates receptor tyrosine kinase (RTK) expression and signalling and alters the electrophoretic mobility of Coatomer Protein Complex Beta 2 (COPB2) protein in mutant EGFR NSCLC cells. Moreover, we demonstrate that EMI66 can alter the subcellular localization of EGFR and COPB2 within the early secretory pathway. Furthermore, we find that COPB2 knockdown reduces the growth of mutant EGFR lung cancer cells, alters the post-translational processing of RTKs, and alters the endoplasmic reticulum (ER) stress response pathway. Lastly, we show that EMI66 treatment also alters the ER stress response pathway and inhibits the growth of mutant EGFR lung cancer cells and organoids. Our results demonstrate that targeting of COPB2 with EMI66 presents a viable approach to attenuate mutant EGFR signalling and growth in NSCLC.

Structure and Function of Protein Arginine Methyltransferase PRMT7.

PRMT7 is a member of the protein arginine methyltransferase (PRMT) family, which methylates a diverse set of substrates. Arginine methylation as a posttranslational modification regulates protein-protein and protein-nucleic acid interactions, and as such, has been implicated in various biological functions. PRMT7 is a unique, evolutionarily conserved PRMT family member that catalyzes the mono-methylation of arginine. The structural features, functional aspects, and compounds that inhibit PRMT7 are discussed here. Several studies have identified physiological substrates of PRMT7 and investigated the substrate methylation outcomes which link PRMT7 activity to the stress response and RNA biology. PRMT7-driven substrate methylation further leads to the biological outcomes of gene expression regulation, cell stemness, stress response, and cancer-associated phenotypes such as cell migration. Furthermore, organismal level phenotypes of PRMT7 deficiency have uncovered roles in muscle cell physiology, B cell biology, immunity, and brain function. This rapidly growing information on PRMT7 function indicates the critical nature of context-dependent functions of PRMT7 and necessitates further investigation of the PRMT7 interaction partners and factors that control PRMT7 expression and levels. Thus, PRMT7 is an important cellular regulator of arginine methylation in health and disease.

Development of LM98, a Small-Molecule TEAD Inhibitor Derived from Flufenamic Acid.

The YAP-TEAD transcriptional complex is responsible for the expression of genes that regulate cancer cell growth and proliferation. Dysregulation of the Hippo pathway due to overexpression of TEAD has been reported in a wide range of cancers. Inhibition of TEAD represses the expression of associated genes, demonstrating the value of this transcription factor for the development of novel anti-cancer therapies. We report herein the design, synthesis and biological evaluation of LM98, a flufenamic acid analogue. LM98 shows strong affinity to TEAD, inhibits its autopalmitoylation and reduces the YAP-TEAD transcriptional activity. Binding of LM98 to TEAD was supported by 19 F-NMR studies while co-crystallization experiments confirmed that LM98 is anchored within the palmitic acid pocket of TEAD. LM98 reduces the expression of CTGF and Cyr61, inhibits MDA-MB-231 breast cancer cell migration and arrests cell cycling in the S phase during cell division.

Affinity-matured HLA class II dimers for robust staining of antigen-specific CD4+ T cells.

Peptide-major histocompatibility complex (pMHC) multimers enable the detection of antigen-specific T cells in studies ranging from vaccine efficacy to cancer immunotherapy. However, this technology is unreliable when applied to pMHC class II for the detection of CD4+ T cells. Here, using a combination of molecular biological and immunological techniques, we cloned sequences encoding human leukocyte antigen (HLA)-DP, HLA-DQ and HLA-DR molecules with enhanced CD4 binding affinity (with a Kd of 8.9 ± 1.1 µM between CD4 and affinity-matured HLA-DP4) and produced affinity-matured class II dimers that stain antigen-specific T cells better than conventional multimers in both in vitro and ex vivo analyses. Using a comprehensive library of dimers for HLA-DP4, which is the most frequent HLA allele in many ancestry groups, we mapped 103 HLA-DP4-restricted epitopes derived from diverse tumor-associated antigens and cloned the cognate T-cell antigen receptor (TCR) genes from in vitro-stimulated CD4+ T cells. The availability of affinity-matured class II dimers across HLA-DP, HLA-DQ and HLA-DR alleles will aid in the investigation of human CD4+ T-cell responses.

A First-in-Class, Highly Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 6.

Protein arginine methyltransferase 6 (PRMT6) catalyzes monomethylation and asymmetric dimethylation of arginine residues in various proteins, plays important roles in biological processes, and is associated with multiple cancers. To date, a highly selective PRMT6 inhibitor has not been reported. Here we report the discovery and characterization of a first-in-class, highly selective allosteric inhibitor of PRMT6, (R)-2 (SGC6870). (R)-2 is a potent PRMT6 inhibitor (IC50 = 77 ± 6 nM) with outstanding selectivity for PRMT6 over a broad panel of other methyltransferases and nonepigenetic targets. Notably, the crystal structure of the PRMT6-(R)-2 complex and kinetic studies revealed (R)-2 binds a unique, induced allosteric pocket. Additionally, (R)-2 engages PRMT6 and potently inhibits its methyltransferase activity in cells. Moreover, (R)-2's enantiomer, (S)-2 (SGC6870N), is inactive against PRMT6 and can be utilized as a negative control. Collectively, (R)-2 is a well-characterized PRMT6 chemical probe and a valuable tool for further investigating PRMT6 functions in health and disease.

Discovery of a First-in-Class Protein Arginine Methyltransferase 6 (PRMT6) Covalent Inhibitor.

Protein arginine methyltransferase 6 (PRMT6) plays important roles in several biological processes associated with multiple cancers. Well-characterized potent, selective, and cell-active PRMT6 inhibitors are invaluable tools for testing biological and therapeutic hypotheses. Although there are several known reversible PRMT6 inhibitors, covalent PRMT6 inhibitors have not been reported. Based on a cocrystal structure of PRMT6-MS023 (a type I PRMT inhibitor), we discovered the first potent and cell-active irreversible PRMT6 inhibitor, 4 (MS117). The covalent binding mode of compound 4 to PRMT6 was confirmed by mass spectrometry and kinetic studies and by a cocrystal structure. Compound 4 did not covalently modify other closely related PRMTs, potently inhibited PRMT6 in cells, and was selective for PRMT6 over other methyltransferases. We also developed two structurally similar control compounds, 5 (MS167) and 7 (MS168). We provide these valuable chemical tools to the scientific community for further studying PRMT6 physiological and pathophysiological functions.

HMCES Functions in the Alternative End-Joining Pathway of the DNA DSB Repair during Class Switch Recombination in B Cells.

HMCES (5hmC binding, embryonic stem cell-specific-protein), originally identified as a protein capable of binding 5-hydroxymethylcytosine (5hmC), an epigenetic modification generated by TET proteins, was previously reported to covalently crosslink to DNA at abasic sites via a conserved cysteine. We show here that Hmces-deficient mice display normal hematopoiesis without global alterations in 5hmC. HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells, and HMCES deficiency leads to a significant defect in CSR. HMCES mediates Alt-EJ through its SOS-response-associated-peptidase domain (SRAPd), a function that requires DNA binding but is independent of its autopeptidase and DNA-crosslinking activities. We show that HMCES is recruited to switch regions of the immunoglobulin locus and provide a potential structural basis for the interaction of HMCES with long DNA overhangs generated by Alt-EJ during CSR. Our studies provide further evidence for a specialized role for HMCES in DNA repair.

Discovery of a chemical probe for PRDM9.

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases.

Direct interaction between the PRDM3 and PRDM16 tumor suppressors and the NuRD chromatin remodeling complex.

Aberrant isoform expression of chromatin-associated proteins can induce epigenetic programs related to disease. The MDS1 and EVI1 complex locus (MECOM) encodes PRDM3, a protein with an N-terminal PR-SET domain, as well as a shorter isoform, EVI1, lacking the N-terminus containing the PR-SET domain (ΔPR). Imbalanced expression of MECOM isoforms is observed in multiple malignancies, implicating EVI1 as an oncogene, while PRDM3 has been suggested to function as a tumor suppressor through an unknown mechanism. To elucidate functional characteristics of these N-terminal residues, we compared the protein interactomes of the full-length and ΔPR isoforms of PRDM3 and its closely related paralog, PRDM16. Unlike the ΔPR isoforms, both full-length isoforms exhibited a significantly enriched association with components of the NuRD chromatin remodeling complex, especially RBBP4. Typically, RBBP4 facilitates chromatin association of the NuRD complex by binding to histone H3 tails. We show that RBBP4 binds to the N-terminal amino acid residues of PRDM3 and PRDM16, with a dissociation constant of 3.0 µM, as measured by isothermal titration calorimetry. Furthermore, high-resolution X-ray crystal structures of PRDM3 and PRDM16 N-terminal peptides in complex with RBBP4 revealed binding to RBBP4 within the conserved histone H3-binding groove. These data support a mechanism of isoform-specific interaction of PRDM3 and PRDM16 with the NuRD chromatin remodeling complex.

Wild type beta-2 microglobulin and DE loop mutants display a common fibrillar architecture.

Beta-2 microglobulin (ß2m) is the protein responsible for a pathologic condition known as dialysis related amyloidosis. In recent years an important role has been assigned to the peptide loop linking strands D and E (DE loop) in determining ß2m stability and amyloid propensity. Several mutants of the DE loop have been studied, showing a good correlation between DE loop geometrical strain, protein stability and aggregation propensity. However, it remains unclear whether the aggregates formed by wild type (wt) ß2m and by the DE loop variants are of the same kind, or whether the mutations open new aggregation pathways. In order to address this question, fibrillar samples of wt and mutated ß2m variants have been analysed by means of atomic force microscopy and infrared spectroscopy. The data here reported indicate that the DE loop mutants form aggregates with morphology and structural organisation very similar to the wt protein. Therefore, the main effect of ß2m DE loop mutations is proposed to stem from the different stabilities of the native fold. Considerations on the structural role of the DE loop in the free monomeric ß2m and as part of the Major Histocompatibility Complex are also presented.

Class I major histocompatibility complex, the trojan horse for secretion of amyloidogenic ß2-microglobulin.

To form extracellular aggregates, amyloidogenic proteins bypass the intracellular quality control, which normally targets unfolded/aggregated polypeptides. Human D76N ß2-microglobulin (ß2m) variant is the prototype of unstable and amyloidogenic protein that forms abundant extracellular fibrillar deposits. Here we focus on the role of the class I major histocompatibility complex (MHCI) in the intracellular stabilization of D76N ß2m. Using biophysical and structural approaches, we show that the MHCI containing D76N ß2m (MHCI76) displays stability, dissociation patterns, and crystal structure comparable with those of the MHCI with wild type ß2m. Conversely, limited proteolysis experiments show a reduced protease susceptibility for D76N ß2m within the MHCI76 as compared with the free variant, suggesting that the MHCI has a chaperone-like activity in preventing D76N ß2m degradation within the cell. Accordingly, D76N ß2m is normally assembled in the MHCI and circulates as free plasma species in a transgenic mouse model.