The voltage-gated potassium channel family (Kv) constitutes the most diverse class of ion channels in the nervous system. Dipeptidyl peptidase 10 (DPP10) is an inactive peptidase that modulates the electrophysiological properties, cell-surface expression and subcellular localization of voltage-gated potassium channels. As a consequence, DPP10 malfunctioning is associated with neurodegenerative conditions like Alzheimer and fronto-temporal dementia, making this protein an attractive drug target. In this work, we report the crystal structure of DPP10 and compare it to that of DPP6 and DPP4. DPP10 belongs to the S9B serine protease subfamily and contains two domains with two distinct folds: a β-propeller and a classical α/β-hydrolase fold. The catalytic serine, however, is replaced by a glycine, rendering the protein enzymatically inactive. Difference in the entrance channels to the active sites between DPP10 and DPP4 provide an additional rationale for the lack of activity. We also characterize the DPP10 dimer interface focusing on the alternative approach for designing drugs able to target protein-protein interactions.

Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation.

Many cancers show aberrant silencing of gene expression and overexpression of histone methyltransferases. The histone methyltransferases (HKMT) EZH2 and EHMT2 maintain the repressive chromatin histone methylation marks H3K27me and H3K9me, respectively, which are associated with transcriptional silencing. Although selective HKMT inhibitors reduce levels of individual repressive marks, removal of H3K27me3 by specific EZH2 inhibitors, for instance, may not be sufficient for inducing the expression of genes with multiple repressive marks.

Therapeutic approach for Alzheimer's disease (AD) is still deficient. To find active compounds from herbal medicine is of interest in the alleviation of AD symptoms. This study aimed to investigate the protective effects of Tanshinone IIA (TIIA) on memory performance and synaptic plasticity in a transgenic AD model at the early phase. 25-100 mg/kg TIIA (intraperitoneal injection, i.p.) was administered to the six-month-old APP and PS1 transgenic mice for 30 consecutive days. After treatment, spatial memory, synaptic plasticity, and related mechanisms were investigated. Our result showed that memory impairment in AD mice was mitigated by 50 and 100 mg/kg TIIA treatments. Hippocampal long-term potentiation was impaired in AD model but rescued by 100 mg/kg TIIA treatment. Mechanically, TIIA treatment reduced the accumulations of beta-amyloid 1-42, C-terminal fragments (CTFs), and p-Tau in the AD model. TIIA did not affect basal BDNF but promoted depolarization-induced BDNF synthesis in the AD mice. Taken together, TIIA repairs hippocampal LTP and memory, likely, through facilitating the clearance of AD-related proteins and activating synaptic BDNF synthesis. TIIA might be a candidate drug for AD treatment.

Chemical inhibition of proteins involved in chromatin-mediated signaling is an emerging strategy to control chromatin compaction with the aim to reprogram expression networks to alter disease states. Protein methyltransferases constitute one of the protein families that participate in epigenetic control of gene expression, and represent a novel therapeutic target class. Recruitment of the protein lysine methyltransferase DOT1L at aberrant loci is a frequent mechanism driving acute lymphoid and myeloid leukemias, particularly in infants, and pharmacological inhibition of DOT1L extends survival in a mouse model of mixed lineage leukemia. A better understanding of the structural chemistry of DOT1L inhibition would accelerate the development of improved compounds. Here, we report that the addition of a single halogen atom at a critical position in the cofactor product S-adenosylhomocysteine (SAH, an inhibitor of SAM-dependent methyltransferases) results in an 8-fold increase in potency against DOT1L, and reduced activities against other protein and non-protein methyltransferases. We solved the crystal structure of DOT1L in complex with Bromo-deaza-SAH and rationalized the observed effects. This discovery reveals a simple strategy to engineer selectivity and potency towards DOT1L into the adenosine scaffold of the cofactor shared by all methyltransferases, and can be exploited towards the development of clinical candidates against mixed lineage leukemia.

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.

The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.

The p53 transcription factor is a critical barrier to pancreatic cancer progression. To unravel mechanisms of p53-mediated tumor suppression, which have remained elusive, we analyzed pancreatic cancer development in mice expressing p53 transcriptional activation domain (TAD) mutants. Surprisingly, the p5353,54 TAD2 mutant behaves as a "super-tumor suppressor," with an enhanced capacity to both suppress pancreatic cancer and transactivate select p53 target genes, including Ptpn14. Ptpn14 encodes a negative regulator of the Yap oncoprotein and is necessary and sufficient for pancreatic cancer suppression, like p53. We show that p53 deficiency promotes Yap signaling and that PTPN14 and TP53 mutations are mutually exclusive in human cancers. These studies uncover a p53-Ptpn14-Yap pathway that is integral to p53-mediated tumor suppression.

Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as a candidate oncogene owing to its overexpression in several types of cancer. Selective PRMT4 inhibitors are useful tools for clarifying the molecular events regulated by PRMT4 and for validating PRMT4 as a therapeutic target. Here, we report the discovery of TP-064, a potent, selective, and cell-active chemical probe of human PRMT4 and its co-crystal structure with PRMT4. TP-064 inhibited the methyltransferase activity of PRMT4 with high potency (half-maximal inhibitory concentration, IC50 < 10 nM) and selectivity over other PRMT family proteins, and reduced arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155; IC50= 340 ± 30 nM) and Mediator complex subunit 12 (MED12; IC50 = 43 ± 10 nM). TP-064 treatment inhibited the proliferation of a subset of multiple myeloma cell lines, with affected cells arrested in G1 phase of the cell cycle. TP-064 and its negative control (TP-064N) will be valuable tools to further investigate the biology of PRMT4 and the therapeutic potential of PRMT4 inhibition.

BACKGROUND MicroRNA-200c (miR-200c) is a short non-coding RNA that has a role in tumorigenesis and cancer progression. The aims of this study were to investigate the role of miR-200c in cell migration and epithelial-mesenchymal transition (EMT) in endometrial carcinoma cells in vitro. MATERIAL AND METHODS Potential direct targets of miR-200c were identified through the TargetScan database. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used study the expression of miR-200c in the endometrial carcinoma cell lines, Ishikawa and JEC, in vitro. Cell migration was studied using transwell assays. Expression of the mesenchymal marker, N-cadherin, the epithelial marker, E-cadherin, the transcription factor, Slug, the BMI-1 protein, AKT, and p-AKT were measured using Western blot. Small interfering RNA (siRNA) was used to silence the BMI-1 gene to study the targeting effect. RESULTS Over-expression of miR-200c in Ishikawa and JEC cells resulted in reduced cell migration and proliferation. Western blot showed that overexpression of miR-200c downregulated the expression of the BMI-1 protein, p-AKT, N-cadherin and Slug, and the expression E-cadherin was upregulated; silencing miR-200c reversed these results. Silencing the BMI-1 gene inhibited EMT and suppressed p-AKT in miR-200c-inhibited endometrial carcinoma cells by increasing E-cadherin expression, reducing the expression of N-cadherin and the EMT-associated transcription factor, Slug. CONCLUSIONS In endometrial carcinoma cells in vitro, miR-200c inhibited EMT by targeting the BMI-1 gene through the p-AKT pathway.

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.

Pathological complete response (pCR) is considered a surrogate endpoint for favorable survival in breast cancer patients treated with neoadjuvant chemotherapy (NAC). Predictive biomarkers of treatment response are crucial for guiding treatment decisions. With the hypothesis that histological information on tumor biopsy images could predict NAC response in breast cancer, we proposed a novel deep learning (DL)-based biomarker that predicts pCR from images of hematoxylin and eosin (H&E)-stained tissue and evaluated its predictive performance.

Survival of motor neuron (SMN) functions in diverse biological pathways via recognition of symmetric dimethylarginine (Rme2s) on proteins by its Tudor domain, and deficiency of SMN leads to spinal muscular atrophy. Here we report a potent and selective antagonist with a 4-iminopyridine scaffold targeting the Tudor domain of SMN. Our structural and mutagenesis studies indicate that both the aromatic ring and imino groups of compound 1 contribute to its selective binding to SMN. Various on-target engagement assays support that compound 1 specifically recognizes SMN in a cellular context and prevents the interaction of SMN with the R1810me2s of RNA polymerase II subunit POLR2A, resulting in transcription termination and R-loop accumulation mimicking SMN depletion. Thus, in addition to the antisense, RNAi and CRISPR/Cas9 techniques, potent SMN antagonists could be used as an efficient tool to understand the biological functions of SMN.

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that interrupt malaria parasite development in the mosquito, thereby blocking onward transmission, and provide a much-needed tool for malaria control and elimination. The parasite surface protein Pfs48/45 is a leading TBV candidate. Here, we isolated and characterized a panel of 81 human Pfs48/45-specific monoclonal antibodies (mAbs) from donors naturally exposed to Plasmodium parasites. Genetically diverse mAbs against each of the three domains (D1-D3) of Pfs48/45 were identified. The most potent mAbs targeted D1 and D3 and achieved >80% transmission-reducing activity in standard membrane-feeding assays, at 10 and 2 μg/mL, respectively. Co-crystal structures of D3 in complex with four different mAbs delineated two conserved protective epitopes. Altogether, these Pfs48/45-specific human mAbs provide important insight into protective and non-protective epitopes that can further our understanding of transmission and inform the design of refined malaria transmission-blocking vaccine candidates.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCF FBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCF FBXO22 . Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.

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.

The 5'-mRNA-cap formation is a conserved process in protection of mRNA in eukaryotic cells, resulting in mRNA stability and efficient translation. In humans, two methyltransferases, RNA cap guanine-N7 methyltransferase (hRNMT) and cap-specific nucleoside-2'-O-methyltransferase 1 (hCMTr1) methylate the mRNA resulting in cap0 (N7mGpppN-RNA) and cap1 (N7mGpppN2'-Om-RNA) formation, respectively. Coronaviruses mimic this process by capping their RNA to evade human immune systems. The coronaviral nonstructural proteins, nsp14 and nsp10-nsp16, catalyze the same reactions as hRNMT and hCMTr1, respectively. These two viral enzymes are important targets for development of inhibitor-based antiviral therapeutics. However, assessing the selectivity of such inhibitors against human corresponding proteins is crucial. Human RNMTs have been implicated in proliferation of cancer cells and are also potential targets for development of anticancer therapeutics. Here, we report the development and optimization of a radiometric assay for hRNMT, full kinetic characterization of its activity, and optimization of the assay for high-throughput screening with a Z-factor of 0.79. This enables selectivity determination for a large number of hits from various screening of coronaviral methyltransferases, and also screening hRNMT for discovery of inhibitors and chemical probes that potentially could be used to further investigate the roles RNMTs play in cancers.

Bromodomains (BRDs) are conserved protein interaction modules which recognize (read) acetyl-lysine modifications, however their role(s) in regulating cellular states and their potential as targets for the development of targeted treatment strategies is poorly understood. Here we present a set of 25 chemical probes, selective small molecule inhibitors, covering 29 human bromodomain targets. We comprehensively evaluate the selectivity of this probe-set using BROMOscan and demonstrate the utility of the set identifying roles of BRDs in cellular processes and potential translational applications. For instance, we discovered crosstalk between histone acetylation and the glycolytic pathway resulting in a vulnerability of breast cancer cell lines under conditions of glucose deprivation or GLUT1 inhibition to inhibition of BRPF2/3 BRDs. This chemical probe-set will serve as a resource for future applications in the discovery of new physiological roles of bromodomain proteins in normal and disease states, and as a toolset for bromodomain target validation.

Many common disease-causing mutations result in loss-of-function (LOF) of the proteins in which they occur. LOF mutations have proven recalcitrant to pharmacologic intervention, presenting a challenge for the development of targeted therapeutics. Polycomb repressive complex 2 (PRC2), which contains core subunits (EZH2, EED, and SUZ12), regulates gene activity by trimethylation of histone 3 lysine 27. The dysregulation of PRC2 catalytic activity by mutations has been implicated in cancer and other diseases. Among the mutations that cause PRC2 malfunction, an I363M LOF mutation of EED has been identified in myeloid disorders, where it prevents allosteric activation of EZH2 catalysis. We describe structure-based design and computational simulations of ligands created to ameliorate this LOF. Notably, these compounds selectively stimulate the catalytic activity of PRC2-EED-I363M over wildtype-PRC2. Overall, this work demonstrates the feasibility of developing targeted therapeutics for PRC2-EED-I363M that act as allosteric agonists, potentially correcting this LOF mutant phenotype.

The lysine methyltransferase SETD8 is the only known methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20). Monomethylation of H4K20 has been implicated in regulating diverse biological processes including the DNA damage response. In addition to H4K20, SETD8 monomethylates non-histone substrates including proliferating cell nuclear antigen (PCNA) and promotes carcinogenesis by deregulating PCNA expression. However, selective inhibitors of SETD8 are scarce. The only known selective inhibitor of SETD8 to date is nahuoic acid A, a marine natural product, which is competitive with the cofactor. Here, we report the discovery of the first substrate-competitive inhibitor of SETD8, UNC0379 (1). This small-molecule inhibitor is active in multiple biochemical assays. Its affinity to SETD8 was confirmed by ITC (isothermal titration calorimetry) and SPR (surface plasmon resonance) studies. Importantly, compound 1 is selective for SETD8 over 15 other methyltransferases. We also describe structure-activity relationships (SAR) of this series.