Exogenous pyruvate represses histone gene expression and inhibits cancer cell proliferation via the NAMPT-NAD+-SIRT1 pathway.

Pyruvate is a glycolytic metabolite used for energy production and macromolecule biosynthesis. However, little is known about its functions in tumorigenesis. Here, we report that exogenous pyruvate inhibits the proliferation of different types of cancer cells. This inhibitory effect of pyruvate on cell growth is primarily attributed to its function as a signal molecule to repress histone gene expression, which leads to less compact chromatin and misregulation of genome-wide gene expression. Pyruvate represses histone gene expression by inducing the expression of NAD+ biosynthesis enzyme, nicotinamide phosphoribosyltransferase (NAMPT) via myocyte enhancer factor 2C (MEF2C), which then increases NAD+ levels and activates the histone deacetylase activity of SIRT1. Chromatin immunoprecipitation analysis indicates that pyruvate enhances SIRT1 binding at histone gene promoters where it reduces histone acetylation. Although pyruvate delays cell entry into S phase, pyruvate represses histone gene expression independent of cell cycle progression. Moreover, we find that administration of pyruvate reduces histone expression and retards tumor growth in xenograft mice without significant side effects. Using tissues from cervical and lung cancer patients, we find intracellular pyruvate concentrations inversely correlate with histone protein levels. Together, we uncover a previously unknown function of pyruvate in regulating histone gene expression and cancer cell proliferation.

Global profiling of functional histidines in live cells using small-molecule photosensitizer and chemical probe relay labelling.

Recent advances in chemical proteomics have focused on developing chemical probes that react with nucleophilic amino acid residues. Although histidine is an attractive candidate due to its importance in enzymatic catalysis, metal binding and protein-protein interaction, its moderate nucleophilicity poses challenges. Its modification is frequently influenced by cysteine and lysine, which results in poor selectivity and narrow proteome coverage. Here we report a singlet oxygen and chemical probe relay labelling method that achieves high selectivity towards histidine. Libraries of small-molecule photosensitizers and chemical probes were screened to optimize histidine labelling, enabling histidine profiling in live cells with around 7,200 unique sites. Using NMR spectroscopy and X-ray crystallography, we characterized the reaction mechanism and the structures of the resulting products. We then applied this method to discover unannotated histidine sites key to enzymatic activity and metal binding in select metalloproteins. This method also revealed the accessibility change of histidine mediated by protein-protein interaction that influences select protein subcellular localization, underscoring its capability in discovering functional histidines.

Spatiotemporal-resolved protein networks profiling with photoactivation dependent proximity labeling.

Enzymatic-based proximity labeling approaches based on activated esters or phenoxy radicals have been widely used for mapping subcellular proteome and protein interactors in living cells. However, activated esters are poorly reactive which leads to a wide labeling radius and phenoxy radicals generated by peroxide treatment may disturb redox-sensitive pathways. Herein, we report a photoactivation-dependent proximity labeling (PDPL) method designed by genetically attaching photosensitizer protein miniSOG to a protein of interest. Triggered by blue light and tunned by irradiation time, singlet oxygen is generated, thereafter enabling spatiotemporally-resolved aniline probe labeling of histidine residues. We demonstrate its high-fidelity through mapping of organelle-specific proteomes. Side-by-side comparison of PDPL with TurboID reveals more specific and deeper proteomic coverage by PDPL. We further apply PDPL to the disease-related transcriptional coactivator BRD4 and E3 ligase Parkin, and discover previously unknown interactors. Through over-expression screening, two unreported substrates Ssu72 and SNW1 are identified for Parkin, whose degradation processes are mediated by the ubiquitination-proteosome pathway.

Discovery and optimization of RO-85, a novel drug-like, potent, and selective P2X3 receptor antagonist.

Despite the extensive literature describing the role of the ATP-gated P2X(3) receptors in a variety of physiological processes the potential of antagonists as therapeutic agents has been limited by the lack of drug-like selective molecules. In this paper we report the discovery and optimization of RO-85, a novel drug-like, potent and selective P2X(3) antagonist. High-throughput screening of the Roche compound collection identified a small hit series of heterocyclic amides from a large parallel synthesis library. Rapid optimization, facilitated by high-throughput synthesis, focusing on increasing potency and improving drug-likeness resulted in the discovery of RO-85.

Design, synthesis, and biological evaluation of new monoamine reuptake inhibitors with potential therapeutic utility in depression and pain.

Two new series of monoamine triple reuptake inhibitors (TRIs) have been discovered through scaffold homologation of our recently reported series of 3,3-disubstituted pyrrolidine TRIs. The regioisomeric 2- and 3-ketopyrrolidines demonstrated high levels of potency against all three monoamine transporters as well as good human in vitro stability, low drug-drug interaction potential and a decreased propensity for hERG channel binding. Representative compounds from these series displayed good in vivo pharmacokinetics and high monoamine receptor occupancies which are indicators of good brain penetration.

Identification and SAR of novel diaminopyrimidines. Part 2: The discovery of RO-51, a potent and selective, dual P2X(3)/P2X(2/3) antagonist for the treatment of pain.

The purinoceptor subtypes P2X(3) and P2X(2/3) have been shown to play a pivotal role in models of various pain conditions. Identification of a potent and selective dual P2X(3)/P2X(2/3) diaminopyrimidine antagonist RO-4 prompted subsequent optimization of the template. This paper describes the SAR and optimization of the diaminopyrimidine ring and particularly the substitution of the 2-amino group. The discovery of the highly potent and drug-like dual P2X(3)/P2X(2/3) antagonist RO-51 is presented.

Identification and SAR of novel diaminopyrimidines. Part 1: The discovery of RO-4, a dual P2X(3)/P2X(2/3) antagonist for the treatment of pain.

P2X purinoceptors are ligand-gated ion channels whose endogenous ligand is ATP. Both the P2X(3) and P2X(2/3) receptor subtypes have been shown to play an important role in the regulation of sensory function and dual P2X(3)/P2X(2/3) antagonists offer significant potential for the treatment of pain. A high-throughput screen of the Roche compound collection resulted in the identification of a novel series of diaminopyrimidines; subsequent optimization resulted in the discovery of RO-4, a potent, selective and drug-like dual P2X(3)/P2X(2/3) antagonist.

Novel 3,3-disubstituted pyrrolidines as selective triple serotonin/norepinephrine/dopamine reuptake inhibitors.

A series of 3,3-disubstituted pyrrolidine monoamine triple reuptake inhibitors were discovered. Analogues with low nanomolar potency, good human in vitro microsomal stability and in vitro permeability, and low drug-drug interaction potential are described. One example showed in vivo anti-depressant-like effects in the mouse tail suspension assay with a minimum effective dose of 30 mg/kg i.p.

Reciprocal Regulation of Metabolic Reprogramming and Epigenetic Modifications in Cancer.

Cancer cells reprogram their metabolism to meet their demands for survival and proliferation. The metabolic plasticity of tumor cells help them adjust to changes in the availability and utilization of nutrients in the microenvironment. Recent studies revealed that many metabolites and metabolic enzymes have non-metabolic functions contributing to tumorigenesis. One major function is regulating epigenetic modifications to facilitate appropriate responses to environmental cues. Accumulating evidence showed that epigenetic modifications could in turn alter metabolism in tumors. Although a comprehensive understanding of the reciprocal connection between metabolic and epigenetic rewiring in cancer is lacking, some conceptual advances have been made. Understanding the link between metabolism and epigenetic modifications in cancer cells will shed lights on the development of more effective cancer therapies.

Anti-tumor activity of metformin: from metabolic and epigenetic perspectives.

Metformin has been used to treat type 2 diabetes for over 50 years. Epidemiological, preclinical and clinical studies suggest that metformin treatment reduces cancer incidence in diabetes patients. Due to its potential as an anti-cancer agent and its low cost, metformin has gained intense research interest. Its traditional anti-cancer mechanisms involve both indirect and direct insulin-dependent pathways. Here, we discussed the anti-tumor mechanism of metformin from the aspects of cell metabolism and epigenetic modifications. The effects of metformin on anti-cancer immunity and apoptosis were also described. Understanding these mechanisms will shed lights on application of metformin in clinical trials and development of anti-cancer therapy.