Discovery of a Potent, Selective, and Cell-Active SPIN1 Inhibitor.

The methyl-lysine reader protein SPIN1 plays important roles in various human diseases. However, targeting methyl-lysine reader proteins has been challenging. Very few cellularly active SPIN1 inhibitors have been developed. We previously reported that our G9a/GLP inhibitor UNC0638 weakly inhibited SPIN1. Here, we present our comprehensive structure-activity relationship study that led to the discovery of compound 11, a dual SPIN1 and G9a/GLP inhibitor, and compound 18 (MS8535), a SPIN1 selective inhibitor. We solved the cocrystal structure of SPIN1 in complex with 11, confirming that 11 occupied one of the three Tudor domains. Importantly, 18 displayed high selectivity for SPIN1 over 38 epigenetic targets, including G9a/GLP, and concentration dependently disrupted the interactions of SPIN1 and H3 in cells. Furthermore, 18 was bioavailable in mice. We also developed 19 (MS8535N), which was inactive against SPIN1, as a negative control of 18. Collectively, these compounds are useful chemical tools to study biological functions of SPIN1.

A Chemical Probe for Tudor Domain Protein Spindlin1 to Investigate Chromatin Function.

Modifications of histone tails, including lysine/arginine methylation, provide the basis of a "chromatin or histone code". Proteins that contain "reader" domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyllysine/arginine reader domains and was identified as a putative oncogene and transcriptional coactivator. Here we report a SPIN1 chemical probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, identified genes which are transcriptionally regulated by SPIN1 in squamous cell carcinoma and suggest that SPIN1 may have a role in cancer related inflammation and/or cancer metastasis.

Discovery of a Potent and Selective Fragment-like Inhibitor of Methyllysine Reader Protein Spindlin 1 (SPIN1).

By screening an epigenetic compound library, we identified that UNC0638, a highly potent inhibitor of the histone methyltransferases G9a and GLP, was a weak inhibitor of SPIN1 (spindlin 1), a methyllysine reader protein. Our optimization of this weak hit resulted in the discovery of a potent, selective, and cell-active SPIN1 inhibitor, compound 3 (MS31). Compound 3 potently inhibited binding of trimethyllysine-containing peptides to SPIN1, displayed high binding affinity, was highly selective for SPIN1 over other epigenetic readers and writers, directly engaged SPIN1 in cells, and was not toxic to nontumorigenic cells. The crystal structure of the SPIN1-compound 3 complex indicated that it selectively binds tudor domain II of SPIN1. We also designed a structurally similar but inactive compound 4 (MS31N) as a negative control. Our results have demonstrated for the first time that potent, selective, and cell-active fragment-like inhibitors can be generated by targeting a single tudor domain.

Synthesis, Characterization and Immunological Evaluation of Self-Adjuvanting Group†A Streptococcal Vaccine Candidates Bearing Various Lipidic Adjuvanting Moieties.

Four group A streptococcal glycolipopeptide vaccine candidates with different lipidic adjuvanting moieties were prepared and characterized. The immunogenicity of the compounds was evaluated by macrophage and dendritic cell uptake studies and by in vivo quantification of systemic IgG antibody by ELISA. Three of the candidates showed significant induction of the IgG response.

Evaluation of Lipopeptides as Toll-like Receptor 2 Ligands.

BACKGROUND: Peptide-based vaccines are considered to be the next generation of modern immunizations, as they are safe, easy to produce and well-defined. However, due to their weak immunogenic effect, it is important to first develop an appropriate adjuvant for peptide-based vaccines. OBJECTIVE: The aim of this work was to synthesize a series of four adjuvanting moieties as alkyne derivatives, incorporating dipalmitoyl serine (DPS), 1,3-diglyceride (DG), two hexadecane lipoamino acids (diLAA), and 2,3-dipalmitoyl-S-glycerylcysteine (Pam2Cys). Next aim was to synthesize and attach the azide derivative of biotinylated J14 peptide (model B-cell epitope) to the alkynes through copper- catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction. Final aim was to test the ability of the final biotin labeled conjugates to directly interact with in vitro expressed TLR2 and 8 using AlphaScreen proximity assay. METHOD: All of the peptides were synthesized by manual stepwise solid phase peptide synthesis (SPPS) on rink amide MBHA resin using HATU/DIPEA Fmoc-chemistry. The target compounds were synthesized in a solution phase using CuAAC reaction. RESULTS: Pam2Cys analogue bound to TLR2 as expected. Analogues of DPS and C16-LAA showed also affinity to TLR2, while it did not bind to the control protein (TLR8), demonstrating ability of the DPS and C16-LAA to be recognized by TLR2. CONCLUSION: Four alkyne derivatives of lipids were successfully synthesized and coupled to a biotinylated J14 peptide to give a series of self-adjuvanting ligands. These ligands showed different affinity to TLR2 upon testing by AlphaScreen assay. The DPS derivative showed the most promising affinity in comparison to the standard TLR2 agonist, Pam2Cys.

Convergent synthetic methodology for the construction of self-adjuvanting lipopeptide vaccines using a novel carbohydrate scaffold.

A novel convergent synthetic strategy for the construction of multicomponent self-adjuvanting lipopeptide vaccines was developed. A tetraalkyne-functionalized glucose derivative and lipidated Fmoc-lysine were prepared by novel efficient and convenient syntheses. The carbohydrate building block was coupled to the self-adjuvanting lipidic moiety (three lipidated Fmoc-lysines) on solid support. Four copies of a group A streptococcal B cell epitope (J8) were then conjugated to the glyco-lipopeptide using a copper-catalyzed cycloaddition reaction. The approach was elaborated by the preparation of a second vaccine candidate which incorporated an additional promiscuous T-helper epitope.

Design, synthesis and characterisation of mannosylated ovalbumin lipid core peptide self-adjuvanting vaccine delivery system.

Peptide-based vaccine delivery can be hampered by rapid peptidase activity and poor inherent immunogenicity. The self-adjuvanting lipid core peptide system (LCP) has been shown to confer improved stability and immunogenicity on peptide epitopes of group A Streptococcus, Chlamydia, hookworm, and malaria pathogens. However, various diseases, including cancer, still require targeted delivery of their vaccine candidates. For this reason, we have selected two model peptides (ovalbumin CD4(+) and/or CD8(+) T cell epitopes), and incorporated two or four copies of either epitope into our LCP vaccine. Optimised glycosylation of ovalbumin peptides yielded 46 % when microwave-assisted double coupling with 2 eq of carbohydrate derivative, activated by N,N-diisopropylethylamine and (O-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, was performed. All ovalbumin peptides were successfully synthesised and purified in 11-55 % yields by Fmoc- or Boc-chemistry using solid-phase peptide synthesis. The mannosylated ovalbumin peptides were nontoxic to human erythrocytes in haemolytic assay (<2 % haemolysis) and showed increased (up to 20-fold) stability in plasma.

COMPARE analysis of the toxicity of an iminoquinone derivative of the imidazo[5,4-f]benzimidazoles with NAD(P)H:quinone oxidoreductase 1 (NQO1) activity and computational docking of quinones as NQO1 substrates.

Synthesis and cytotoxicity of imidazo[5,4-f]benzimidazolequinones and iminoquinone derivatives is described, enabling structure-activity relationships to be obtained. The most promising compound (an iminoquinone derivative) has undergone National Cancer Institute (NCI) 60 cell line (single and five dose) screening, and using the NCI COMPARE program, has shown correlation to NQO1 activity and to other NQO1 substrates. Common structural features suggest that the iminoquinone moiety is significant with regard to NQO1 specificity. Computational docking into the active site of NQO1 was performed, and the first comprehensive mitomycin C (MMC)-NQO1 docking study is presented. Small distances for hydride reduction and high binding affinities are characteristic of MMC and of iminoquinones showing correlations with NQO1 via COMPARE analysis. Docking also indicated that the presence of a substituent capable of hydrogen bonding to the His194 residue is important in influencing the orientation of the substrate in the NQO1 active site, leading to more efficient reduction.

One-pot double intramolecular homolytic aromatic substitution routes to dialicyclic ring fused imidazobenzimidazolequinones and preliminary analysis of anticancer activity.

Bu(3)SnH/1,1'-azobis(cyclohexanecarbonitrile) (ACN)-mediated five, six, and seven-membered double alkyl radical cyclizations onto imidazo[5,4-f]benzimidazole and imidazo[4,5-f]benzimidazole are described. The quinone derivatives evaluated show selective toxicity towards human cervical (HeLa) and prostate (DU145) cancer cell lines (with negligible toxicity towards a normal human cell line, GM00637). Only the Fremy oxidation of the 6-aminoimidazo[5,4-f]benzimidazole gave iminoquinone, which showed high specificity towards the prostate cancer cell line (DU145).