P300/CBP inhibition sensitizes mantle cell lymphoma to PI3Kδ inhibitor idelalisib.

Mantle cell lymphoma (MCL) is a lymphoproliferative disorder lacking reliable therapies. PI3K pathway contributes to the pathogenesis of MCL, serving as a potential target. However, idelalisib, an FDA-approved drug targeting PI3Kδ, has shown intrinsic resistance in MCL treatment. Here we report that a p300/CBP inhibitor, A-485, could overcome resistance to idelalisib in MCL cells in vitro and in vivo. A-485 was discovered in a combinational drug screening from an epigenetic compound library containing 45 small molecule modulators. We found that A-485, the highly selective catalytic inhibitor of p300 and CBP, was the most potent compound that enhanced the sensitivity of MCL cell line Z-138 to idelalisib. Combination of A-485 and idelalisib remarkably decreased the viability of three MCL cell lines tested. Co-treatment with A-485 and idelalisib in Maver-1 and Z-138 MCL cell xenograft mice for 3 weeks dramatically suppressed the tumor growth by reversing the unsustained inhibition in PI3K downstream signaling. We further demonstrated that p300/CBP inhibition decreased histone acetylation at RTKs gene promoters and reduced transcriptional upregulation of RTKs, thereby inhibiting the downstream persistent activation of MAPK/ERK signaling, which also contributed to the pathogenesis of MCL. Therefore, additional inhibition of p300/CBP blocked MAPK/ERK signaling, which rendered maintaining activation to PI3K-mTOR downstream signals p-S6 and p-4E-BP1, thus leading to suppression of cell growth and tumor progression and eliminating the intrinsic resistance to idelalisib ultimately. Our results provide a promising combination therapy for MCL and highlight the potential use of epigenetic inhibitors targeting p300/CBP to reverse drug resistance in tumor.

Inhibition of Autophagy by a Small Molecule through Covalent Modification of the LC3 Protein.

The autophagic ubiquitin-like protein LC3 functions through interactions with LC3-interaction regions (LIRs) of other autophagy proteins, including autophagy receptors, which stands out as a promising protein-protein interaction (PPI) target for the intervention of autophagy. Post-translational modifications like acetylation of Lys49 on the LIR-interacting surface could disrupt the interaction, offering an opportunity to design covalent small molecules interfering with the interface. Through screening covalent compounds, we discovered a small molecule modulator of LC3A/B that covalently modifies LC3A/B protein at Lys49. Activity-based protein profiling (ABPP) based evaluations reveal that a derivative molecule DC-LC3in-D5 exhibits a potent covalent reactivity and selectivity to LC3A/B in HeLa cells. DC-LC3in-D5 compromises LC3B lipidation in vitro and in HeLa cells, leading to deficiency in the formation of autophagic structures and autophagic substrate degradation. DC-LC3in-D5 could serve as a powerful tool for autophagy research as well as for therapeutic interventions.

Discovery of alkoxy benzamide derivatives as novel BPTF bromodomain inhibitors via structure-based virtual screening.

Bromodomain PHD finger transcription factor (BPTF), a bromodomain-containing protein, plays a crucial role in the regulation of downstream gene expression through the specific recognition of lysine acetylation on bulk histones. The dysfunction of BPTF is closely involved with the development and progression of many human diseases, especially cancer. Therefore, BPTF bromodomain has become a promising drug target for epigenetic cancer therapy. However, unlike BET family inhibitors, few BPTF bromodomain inhibitors have been reported. In this study, by integrating docking-based virtual screening with biochemical analysis, we identified a novel selective BPTF bromodomain inhibitor DCB29 with the IC50 value of 13.2 ±â€¯1.6 µM by homogenous time-resolved fluorescence resonance energy transfer (HTRF) assays. The binding between DCB29 and BPTF was confirmed by NMR and SPR. Molecular docking disclosed that DCB29 occupied the pocket of acetylated H4 peptide substrate and provided detailed SAR explanations for its derivatives. Collectively, DCB29 presented great potential as a powerful tool for BPTF-related biological research and further medicinal chemistry optimization.

Discovery of novel BET inhibitors by drug repurposing of nitroxoline and its analogues.

The BET family of bromodomain-containing proteins (BRDs) is believed to be a promising drug target for therapeutic intervention in a number of diseases including cancer, inflammation and cardiovascular diseases. Hence, there is a great demand for novel chemotypes of BET inhibitors. The drug repurposing strategy offers great benefits to find inhibitors with known safety and pharmacokinetic profiles, thus increasing medicinal chemists' interest in recent years. Using the drug repurposing strategy, a BRD4-specific score based virtual screening campaign on an in-house drug library was conducted followed by the ALPHA screen assay test. Nitroxoline, an FDA-approved antibiotic, was identified to effectively disrupt the interaction between the first bromodomain of BRD4 (bromodomain-containing protein 4) and acetylated H4 peptide with IC50 of 0.98 µM. Nitroxoline inhibited all BET family members with good selectivity against non-BET bromodomain-containing proteins, thus it is defined as a selective BET inhibitor. Based on the crystal structure of the nitroxoline-BRD4_BD1 complex, the mechanism of action as well as BET specificity of nitroxoline were determined. Since the anticancer activity of nitroxoline against MLL leukemia, one of the BET related diseases, has not been studied before, we tested whether nitroxoline might serve as a potential repurposing drug candidate for MLL leukemia. Nitroxoline effectively inhibited the proliferation of MLL leukemia cells by inducing cell cycle arrest and apoptosis. The profound efficacy is, at least in part, due to the inhibition of BET and downregulation of target gene transcription. Our discovery of nitroxoline as a BET inhibitor suggests potential application of nitroxoline and its derivatives for clinical translation in BET family related diseases.

Structural insights on mouse L-threonine dehydrogenase: A regulatory role of Arg180 in catalysis.

Mouse L-threonine dehydrogenase (mTDH), which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and mediates threonine catabolism, plays pivotal roles in both powerful biosynthesis and signaling in mouse stem cells and has a regulatory residue Arg180. Here we determined three crystal structures of mTDH: wild-type (WT) in the apo form; in complex with NAD(+) and a substrate analog, glycerol, or with only NAD(+); as well as the R180K variant with NAD(+). This is the first description of a structure for mammalian SDR-type TDH. Structural comparison revealed the structural basis for SDR-type TDH catalysis remains strictly conserved in bacteria and mammals. Kinetic enzyme assays, and isothermal titration calorimetry (ITC) measurements indicated the R180K mutation has little effect on NAD(+) binding affinity, whereas affects the substrate's affinity for the enzyme. The crystal structure of R180K with NAD(+), biochemical and spectroscopic studies suggested that the R180K mutant should bind NAD(+) in a similar way and have a similar folding to the WT. However, the R180K variant may have difficulty adopting the closed form due to reduced interaction of residue 180 with a loop which connects a key position for mTDH switching between the closed and open forms in mTDH catalysis, and thereby exhibited a significantly decreased kcat/Km value toward the substrate, L-Thr. In sum, our results suggest that activity of GalE-like TDH can be regulated by remote interaction, such as hydrogen bonding and hydrophobic interaction around the Arg180 of mTDH.

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.

Spermine is a natural suppressor of AR signaling in castration-resistant prostate cancer.

In castration-resistant prostate cancer (CRPC), clinical response to androgen receptor (AR) antagonists is limited mainly due to AR-variants expression and restored AR signaling. The metabolite spermine is most abundant in prostate and it decreases as prostate cancer progresses, but its functions remain poorly understood. Here, we show spermine inhibits full-length androgen receptor (AR-FL) and androgen receptor splice variant 7 (AR-V7) signaling and suppresses CRPC cell proliferation by directly binding and inhibiting protein arginine methyltransferase PRMT1. Spermine reduces H4R3me2a modification at the AR locus and suppresses AR binding as well as H3K27ac modification levels at AR target genes. Spermine supplementation restrains CRPC growth in vivo. PRMT1 inhibition also suppresses AR-FL and AR-V7 signaling and reduces CRPC growth. Collectively, we demonstrate spermine as an anticancer metabolite by inhibiting PRMT1 to transcriptionally inhibit AR-FL and AR-V7 signaling in CRPC, and we indicate spermine and PRMT1 inhibition as powerful strategies overcoming limitations of current AR-based therapies in CRPC.

L-5-hydroxytryptophan promotes antitumor immunity by inhibiting PD-L1 inducible expression.

BACKGROUND: The repression or downregulation of programmed death-ligand 1 (PD-L1) can release its inhibition of T cells and activate antitumor immune responses. Although PD-1 and PD-L1 antibodies are promising treatments for diverse tumor types, their inherent disadvantages and immune-related adverse events remain significant issues. The development of small molecule inhibitors targeting the interaction surface of PD-1 and PD-L1 has been reviving, yet many challenges remain. To address these issues, we aimed to find small molecules with durable efficacy and favorable biosafety that alter PD-L1 surface expression and can be developed into a promising alternative and complementary therapy for existing anti-PD-1/PD-L1 therapies. METHODS: Cell-based screen of 200 metabolic molecules using a high-throughput flow cytometry assay of PD-L1 surface expression was conducted, and L-5-hydroxytryptophan (L-5-HTP) was found to suppress PD-L1 expression induced by interferon gamma (IFN-γ). Inhibition of PD-L1 induction and antitumor effect of L-5-HTP were evaluated in two syngeneic mouse tumor models. Flow cytometry was performed to investigate the change in the tumor microenvironment caused by L-5-HTP treatment. RESULTS: We discovered that L-5-HTP suppressed IFN-γ-induced PD-L1 expression in tumor cells transcriptionally, and this effect was directly due to itself. Mechanistically, L-5-HTP inhibited IFN-γ-induced expression of RTK ligands and thus suppressed phosphorylation-mediated activation of RTK receptors and the downstream MEK/ERK/c-JUN signaling cascade, leading to decreased PD-L1 induction. In syngeneic mouse tumor models, treatment with 100 mg/kg L-5-HTP (intraperitoneal) inhibited PD-L1 expression and exhibited antitumor effect. L-5-HTP upregulated the ratio of granzyme B+ CD8+ activated cytotoxic T cells. An intact immune system and PD-L1 expression was critical for L-5-HTP to exert its antitumor effects. Furthermore, L-5-HTP acted synergistically with PD-1 antibody to improve anticancer effect. CONCLUSION: Our study illustrated L-5-HTP's inhibitory effect on PD-L1 induction stimulated by IFN-γ in tumor cells and also provided insight into repurposing L-5-HTP for use in tumor immunotherapy.

A potent PGK1 antagonist reveals PGK1 regulates the production of IL-1ß and IL-6.

Glycolytic metabolism enzymes have been implicated in the immunometabolism field through changes in metabolic status. PGK1 is a catalytic enzyme in the glycolytic pathway. Here, we set up a high-throughput screen platform to identify PGK1 inhibitors. DC-PGKI is an ATP-competitive inhibitor of PGK1 with an affinity of K d = 99.08 nmol/L. DC-PGKI stabilizes PGK1 in vitro and in vivo, and suppresses both glycolytic activity and the kinase function of PGK1. In addition, DC-PGKI unveils that PGK1 regulates production of IL-1ß and IL-6 in LPS-stimulated macrophages. Mechanistically, inhibition of PGK1 with DC-PGKI results in NRF2 (nuclear factor-erythroid factor 2-related factor 2, NFE2L2) accumulation, then NRF2 translocates to the nucleus and binds to the proximity region of Il-1ß and Il-6 genes, and inhibits LPS-induced expression of these genes. DC-PGKI ameliorates colitis in the dextran sulfate sodium (DSS)-induced colitis mouse model. These data support PGK1 as a regulator of macrophages and suggest potential utility of PGK1 inhibitors in the treatment of inflammatory bowel disease.

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 trisubstituted nicotinonitrile derivatives as novel human GCN5 inhibitors through AlphaScreen-based high throughput screening.

The general control nonrepressed protein 5 (GCN5) is an important target for drug design and drug discovery largely owing to its pathogenic role in malignancies. Chemical probes that target GCN5 have been developed in recent decades, but their potencies are still unsatisfactory. In this study, through an in-house developed AlphaScreen-based high throughput screening platform, radioactive acetylation assays and 2D-similarity based analogue searching, we discovered DC_HG24-01 as the novel hGCN5 inhibitor with the IC50 value of 3.1 ± 0.2 µM. Further docking studies suggested that DC_HG24-01 could occupy the binding pocket of acetyl-CoA cofactor, which laid the foundation for the development of more potent hGCN5 inhibitors in the future. At the cellular level, DC_HG24-01 could retard cell proliferation and block the acetylation of H3K14 leading to cell apoptosis and cell cycle arrest at the G1 phase in MV4-11 cell lines. Taken together, the discovery of DC_HG24-01 may serve as a good starting point to accelerate the development of more potent hGCN5 inhibitors through further structural decoration and provide new insight into the pharmacological treatment of leukemia.

Discovery and biological evaluation of vinylsulfonamide derivatives as highly potent, covalent TEAD autopalmitoylation inhibitors.

Transcriptional enhancer associated domain family members (TEADs) are the most important downstream effectors that play the pivotal role in the development, regeneration and tissue homeostasis. Recent biochemical studies have demonstrated that TEADs could undergo autopalmitoylation that is indispensable for its function making the lipid-binding pocket an attractive target for chemical intervention. Herein, through structure-based virtual screen and rational medicinal chemistry optimization, we identified DC-TEADin02 as the most potent, selective, covalent TEAD autopalmitoylation inhibitor with the IC50 value of 197 ±â€¯19 nM while it showed minimal effect on TEAD-YAP interaction. Further biochemical counter-screens demonstrate the specific thiol reactivity and selectivity of DC-TEADin02 over the kinase family, lipid-binding proteins and epigenetic targets. Notably, DC-TEADin02 inhibited TEADs transcription activity leading to downregulation of YAP-related downstream gene expression. Taken together, our findings proved the validity of modulating transcriptional output in the Hippo signaling pathway through irreversible chemical interventions of TEADs autopalmitoylation activity, which may serve as a qualified chemical tool for TEADs palmitoylation-related studies in the future.

iASPP-PP1 complex is required for cytokinetic abscission by controlling CEP55 dephosphorylation.

Cytokinesis is the last step of cell division and is concluded by the abscission of the intercellular bridge that connects two daughter cells. The tight regulation of cytokinesis completion is essential because cytokinesis failure is associated with various human diseases. Here, we report that iASPP, a member of the apoptosis-stimulating proteins of p53 (ASPP) family, is required for proper cell division. iASPP depletion results in abnormal midbody structure and failed cytokinesis. We used protein affinity purification methods to identify the functional partners of iASPP. We found that iASPP associates with centrosomal protein of 55 kDa (CEP55), an important cytokinetic abscission regulator. Mechanically, iASPP acts as a PP1-targeting subunit to facilitate the interaction between PP1 and CEP55 and to remove PLK1-mediated Ser436 phosphorylation in CEP55 during late mitosis. The latter step is critical for the timely recruitment of CEP55 to the midbody. The present observations revealed a previously unrecognized function of iASPP in cytokinesis. This function, in turn, likely contributes to the roles of iASPP in tumor development and genetic diseases.