PRMT1 Inhibition Activates the Interferon Pathway to Potentiate Antitumor Immunity and Enhance Checkpoint Blockade Efficacy in Melanoma.

Despite the immense success of immune checkpoint blockade (ICB) in cancer treatment, many tumors, including melanoma, exhibit innate or adaptive resistance. Tumor-intrinsic T-cell deficiency and T-cell dysfunction have been identified as essential factors in the emergence of ICB resistance. Here, we found that protein arginine methyltransferase 1 (PRMT1) expression was inversely correlated with the number and activity of CD8+ T cells within melanoma specimen. PRMT1 deficiency or inhibition with DCPT1061 significantly restrained refractory melanoma growth and increased intratumoral CD8+ T cells in vivo. Moreover, PRMT1 deletion in melanoma cells facilitated formation of double-stranded RNA derived from endogenous retroviral elements (ERV) and stimulated an intracellular interferon response. Mechanistically, PRMT1 deficiency repressed the expression of DNA methyltransferase 1 (DNMT1) by attenuating modification of H4R3me2a and H3K27ac at enhancer regions of Dnmt1, and DNMT1 downregulation consequently activated ERV transcription and the interferon signaling. Importantly, PRMT1 inhibition with DCPT1061 synergized with PD-1 blockade to suppress tumor progression and increase the proportion of CD8+ T cells as well as IFNγ+CD8+ T cells in vivo. Together, these results reveal an unrecognized role and mechanism of PRMT1 in regulating antitumor T-cell immunity, suggesting PRMT1 inhibition as a potent strategy to increase the efficacy of ICB. SIGNIFICANCE: Targeting PRMT1 stimulates interferon signaling by increasing expression of endogenous retroviral elements and double-stranded RNA through repression of DNMT1, which induces antitumor immunity and synergizes with immunotherapy to suppress tumor progression.

Natural product 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose is a reversible inhibitor of glyceraldehyde 3-phosphate dehydrogenase.

Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancer cell glucose metabolism and plays a crucial role in the activation of various types of immune cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of D-glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate in the 6th critical step in glycolysis. GAPDH exerts metabolic flux control during aerobic glycolysis and therefore is an attractive therapeutic target for cancer and autoimmune diseases. Recently, GAPDH inhibitors were reported to function through common suicide inactivation by covalent binding to the cysteine catalytic residue of GAPDH. Herein, by developing a high-throughput enzymatic screening assay, we discovered that the natural product 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose (PGG) is an inhibitor of GAPDH with Ki = 0.5 µM. PGG blocks GAPDH activity by a reversible and NAD+ and Pi competitive mechanism, suggesting that it represents a novel class of GAPDH inhibitors. In-depth hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis revealed that PGG binds to a region that disrupts NAD+ and inorganic phosphate binding, resulting in a distal conformational change at the GAPDH tetramer interface. In addition, structural modeling analysis indicated that PGG probably reversibly binds to the center pocket of GAPDH. Moreover, PGG inhibits LPS-stimulated macrophage activation by specific downregulation of GAPDH-dependent glucose consumption and lactate production. In summary, PGG represents a novel class of GAPDH inhibitors that probably reversibly binds to the center pocket of GAPDH. Our study sheds new light on factors for designing a more potent and specific inhibitor of GAPDH for future therapeutic applications.

miR­146a reduces depressive behavior by inhibiting microglial activation.

Depression is one of the major psychiatric diseases affecting the quality of life for individuals worldwide. Numerous reports have investigated depression, although its etiology remains to be elucidated. microRNA (miR)­146a is suggested to regulate innate immune and inflammatory responses. However, it is unclear whether miR­146a is involved in depression. Depression model mice were established using lipopolysaccharide­induced depression and chronic unpredictable mild stress, separately. miR­146a mimic and short interfering RNA were used to treat depressed mice. Depression­like behaviors and levels of pro­inflammatory cytokines were measured, while ionized calcium binding adapter molecule 1 (Iba­1) expression in hippocampus was quantified by immunohistochemistry. Neuroinflammatory factor levels in hippocampus were measured by western blotting. BV­2 cells were used to confirm that miR­146a suppressed microglia activation. Compared with control mice, the two depressed mouse models showed clearly decreased sucrose preference and significantly increased immobility time in the forced swimming test and tail suspension test (P<0.05). miR­146a overexpression significantly increased sucrose preference and reduced immobility time in depressed mice (P<0.05). However, total distance traveled in the locomotor activity test did not differ among groups. Compared with controls, expression levels of Iba­1, inducible nitric oxide, IL­1ß, TNF­α, interleukin 1 receptor associated kinase 1 (IRAK1), TNF receptor­associated factor 6 (TRAF6) and phosphorylated NF­κB p65 were significantly increased in depressed mice (P<0.05). miR­146a overexpression effectively inhibited expression of these neuroinflammatory proteins, while miR­146a silencing significantly upregulated their expression (P<0.05). Consistent with these in vivo results, miR­146a mimic treatment inhibited TNF­α, IL­1ß, IRAK1 and TRAF6 expression in BV­2 cells. miR­146a improved depressive behaviors in depressed model mice by inhibiting microglial activation and neuroinflammatory factor expression.

Covalent Inhibitors Allosterically Block the Activation of Rho Family Proteins and Suppress Cancer Cell Invasion.

The Rho family GTPases are crucial drivers of tumor growth and metastasis. However, it is difficult to develop GTPases inhibitors due to a lack of well-characterized binding pockets for compounds. Here, through molecular dynamics simulation of the RhoA protein, a groove around cysteine 107 (Cys107) that is relatively well-conserved within the Rho family is discovered. Using a combined strategy, the novel inhibitor DC-Rhoin is discovered, which disrupts interaction of Rho proteins with guanine nucleotide exchange factors (GEFs) and guanine nucleotide dissociation inhibitors (GDIs). Crystallographic studies reveal that the covalent binding of DC-Rhoin to the Cys107 residue stabilizes and captures a novel allosteric pocket. Moreover, the derivative compound DC-Rhoin04 inhibits the migration and invasion of cancer cells, through targeting this allosteric pocket of RhoA. The study reveals a novel allosteric regulatory site within the Rho family, which can be exploited for anti-metastasis drug development, and also provides a novel strategy for inhibitor discovery toward "undruggable" protein targets.

Discovery of novel CBP bromodomain inhibitors through TR-FRET-based high-throughput screening.

The cAMP-responsive element binding protein (CREB) binding protein (CBP) and adenoviral E1A-binding protein (P300) are two closely related multifunctional transcriptional coactivators. Both proteins contain a bromodomain (BrD) adjacent to the histone acetyl transferase (HAT) catalytic domain, which serves as a promising drug target for cancers and immune system disorders. Several potent and selective small-molecule inhibitors targeting CBP BrD have been reported, but thus far small-molecule inhibitors targeting BrD outside of the BrD and extraterminal domain (BET) family are especially lacking. Here, we established and optimized a TR-FRET-based high-throughput screening platform for the CBP BrD and acetylated H4 peptide. Through an HTS assay against an in-house chemical library containing 20 000 compounds, compound DC_CP20 was discovered as a novel CBP BrD inhibitor with an IC50 value of 744.3 nM. This compound bound to CBP BrD with a KD value of 4.01 µM in the surface plasmon resonance assay. Molecular modeling revealed that DC_CP20 occupied the Kac-binding region firmly through hydrogen bonding with the conserved residue N1168. At the celluslar level, DC_CP20 dose-dependently inhibited the proliferation of human leukemia MV4-11 cells with an IC50 value of 19.2 µM and markedly downregulated the expression of the c-Myc in the cells. Taken together, the discovery of CBP BrD inhibitor DC_CP20 provides a novel chemical scaffold for further medicinal chemistry optimization and a potential chemical probe for CBP-related biological function research. In addition, this inhibitor may serve as a promising therapeutic strategy for MLL leukemia by targeting CBP BrD protein.

Mitochondria-targeted cytochrome P450 2E1 induces oxidative damage and augments alcohol-mediated oxidative stress.

The ethanol-inducible cytochrome P450 2E1 (CYP2E1) is also induced under different pathological and physiological conditions. Studies including ours have shown that CYP2E1 is bimodally targeted to both the endoplasmic reticulum (microsomes) (mc CYP2E1) and mitochondria (mt CYP2E1). In this study we investigated the role of mtCYP2E1 in ethanol-mediated oxidative stress in stable cell lines expressing predominantly mt CYP2E1 or mc CYP2E1. The ER+ mutation (A2L, A9L), which increases the affinity of the nascent protein for binding to the signal recognition particle, preferentially targets CYP2E1 to the endoplasmic reticulum. The Mt+ (L17G) and Mt++ (I8R, L11R, L17R) mutant proteins, showing progressively lower affinity for signal recognition particle binding, were targeted to mitochondria at correspondingly higher levels. The rate of GSH depletion, used as a measure of oxidative stress, was higher in cells expressing Mt++ and Mt+ proteins as compared with cells expressing ER+ protein. In addition, the cellular level of F(2)-isoprostanes, a direct indicator of oxidative stress, was increased markedly in Mt++ cells after ethanol treatment. Notably, expression of Mt++ CYP2E1 protein in yeast cells caused more severe mitochondrial DNA damage and respiratory deficiency than the wild type or ER+ proteins as tested by the inability of cells to grow on glycerol or ethanol. Additionally, liver mitochondria from ethanol-fed rats containing high mt CYP2E1 showed higher levels of F(2)-isoprostane production. These results strongly suggest that mt CYP2E1 induces oxidative stress and augments alcohol-mediated cell/tissue injury.

PPIase domain of trigger factor acts as auxiliary chaperone site to assist the folding of protein substrates bound to the crevice of trigger factor.

Trigger factor (TF) is the first chaperone encountered by nascent chains in bacteria, which consists of two modules: peptidyl-prolyl-cis/trans-isomerase (PPIase) domain and a crevice built by both N- and C-terminal domains. While the crevice is suggested to provide a protective space over the peptide exit site of ribosome for nascent polypeptides to fold, it remains unclear whether PPIase domain is directly involved in assisting protein folding. Here, we introduced structural change into different regions of TF, and investigated their influence on the chaperone function of TF in assisting the folding of various substrate proteins, including oligomeric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and monomeric carbonic anhydrase II (CA II) and lysozyme. Results showed that structural disturbances by site-specific mutations in the PPIase active site or by deletion of the PPIase domain from TF affected the chaperone activity of TF toward CA II and GAPDH but had no effect on TF-assisted lysozyme refolding, suggesting PPIase domain is involved in assisting the folding of substrates larger than lysozyme. Mutants with the structural disturbances in the crevice totally lost the chaperone activity toward all the substrates we used in this investigation. These results provide further evidence to confirm that the crevice is the major chaperone site of TF, and the hydrophobic pocket in PPIase domain acts as an auxiliary site to assist the folding of substrate proteins bound to the crevice in a substrate-dependent manner, which is beneficial for TF to provide appropriate assistance for protein folding by changing protective space and binding affinity.