Liqiang Chen

A single-amino acid substitution in West Nile virus 2K peptide between NS4A and NS4B confers resistance to lycorine, a flavivirus inhibitor

Abstract Lycorine potently inhibits flaviviruses in cell culture. At 1.2-μM concentration, lycorine reduced viral titers of West Nile (WNV), dengue, and yellow fever viruses by 102- to 104-fold. However, the compound did not inhibit an alphavirus (Western equine encephalitis virus) or a rhabdovirus (vesicular stomatitis virus), indicating a selective antiviral spectrum. The compound exerts its antiviral activity mainly through suppression of viral RNA replication. A Val→Met substitution at the 9th amino acid position of the viral 2K peptide (spanning the endoplasmic reticulum membrane between NS4A and NS4B proteins) confers WNV resistance to lycorine, through enhancement of viral RNA replication. Initial chemistry synthesis demonstrated that modifications of the two hydroxyl groups of lycorine can increase the compounds potency, while reducing its cytotoxicity. Taken together, the results have established lycorine as a flavivirus inhibitor for antiviral development. The lycorine-resistance results demonstrate a direct role of the 2K peptide in flavivirus RNA synthesis.

Medicinal chemistry of sirtuin inhibitors

As members of Class III histone deacetylases (HDACs), sirtuins use stoichiometric nicotinamide adenine dinucleotide (NAD(+)) to remove the acetyl group from N-acetyl-lysines of histones or non-histone proteins. Sirtuins have been implicated in metabolic diseases, cancer, and neurodegenerative diseases, constituting a promising target for drug discovery. While the early sirtuin inhibitors mimicked NAD(+) or substrate peptides, high-throughput and in silico screenings have identified a wide range of core structures, many of which have been subjected to medicinal chemistry efforts. This review outlines inhibitor chemotypes, and their chemical modifications and biological evaluations, highlighting strategies to enhance inhibitory activity and selectivity among isoforms.

Triazole-Linked Inhibitors of Inosine Monophosphate Dehydrogenase from Human and Mycobacterium tuberculosis

The modular nature of nicotinamide adenine dinucleotide (NAD)-mimicking inosine monophsophate dehydrogenase (IMPDH) inhibitors has prompted us to investigate novel mycophenolic adenine dinucleotides (MAD) in which 1,2,3-triazole linkers were incorporated as isosteric replacements of the pyrophosphate linker. Synthesis and evaluation of these inhibitors led to identification of low nanomolar inhibitors of human IMPDH and more importantly the first potent inhibitor of IMPDH from Mycobacterium tuberculosis (mtIMPDH). Computational studies of these IMPDH enzymes helped rationalize the observed structure-activity relationships. Additionally, the first cloning, expression, purification and characterization of mtIMPDH is reported.

Discovery of potent and selective sirtuin 2 (SIRT2) inhibitors using a fragment-based approach.

Sirtuin 2 (SIRT2) is one of the sirtuins, a family of NAD(+)-dependent deacetylases that act on a variety of histone and non-histone substrates. Accumulating biological functions and potential therapeutic applications have drawn interest in the discovery and development of SIRT2 inhibitors. Herein we report our discovery of novel SIRT2 inhibitors using a fragment-based approach. Inspired by the purported close binding proximity of suramin and nicotinamide, we prepared two sets of fragments, namely, the naphthylamide sulfonic acids and the naphthalene-benzamides and -nicotinamides. Biochemical evaluation of these two series provided structure-activity relationship (SAR) information, which led to the design of (5-benzamidonaphthalen-1/2-yloxy)nicotinamide derivatives. Among these inhibitors, one compound exhibited high anti-SIRT2 activity (48 nM) and excellent selectivity for SIRT2 over SIRT1 and SIRT3. In vitro, it also increased the acetylation level of α-tubulin, a well-established SIRT2 substrate, in both concentration- and time-dependent manners. Further kinetic studies revealed that this compound behaves as a competitive inhibitor against the peptide substrate and most likely as a noncompetitive inhibitor against NAD(+). Taken together, these results indicate that we have discovered a potent and selective SIRT2 inhibitor whose novel structure merits further exploration.

Nicotinamide adenine dinucleotide based therapeutics.

Nicotinamide adenine dinucleotide (NAD), generally considered a key component involved in redox reactions, has been found to participate in an increasingly diverse range of cellular processes, including signal transduction, DNA repair, and post-translational protein modifications. In recent years, medicinal chemists have become interested in the therapeutic potential of molecules affecting interactions of NAD with NAD-dependent enzymes. Also, enzymes involved in de novo biosynthesis, salvage pathways, and down-stream utilization of NAD have been extensively investigated and implicated in a wide variety of diseases. These studies have bolstered NAD-based therapeutics as a new avenue for the discovery and development of novel treatments for medical conditions ranging from cancer to aging. Industrial and academic groups have produced structurally diverse molecules which target NAD metabolic pathways, with some candidates advancing into clinical trials. However, further intensive structural, biological, and medical studies are needed to facilitate the design and evaluation of new generations of NAD-based therapeutics. At this time, the field of NAD-therapeutics is most likely at a stage similar to that of the early successful development of protein kinase inhibitors, where analogs of ATP (a more widely utilized metabolite than NAD) began to show selectivity against target enzymes. This review focuses on key representative opportunities for research in this area, which extends beyond the scope of this article.

Multi-Targeted Histone Deacetylase Inhibitors in Cancer Therapy

The heterogeneous nature of cancer requires a comprehensive approach for attacking the multiple mechanisms underlying the initiation and progression of cancers. Histone deacetylase inhibitors (HDACi) have emerged as a new class of anticancer agents, targeting the biological processes including cell cycle, apoptosis and differentiation. Studies have revealed that HDACi are synergistic with diverse classes of anticancer therapies including targeted therapeutics and conventional anticancer agents. Extensive medicinal chemistry efforts have yielded a wide range of chemical structures, indicative of the structural flexibility of HDACi. These findings have supported a strategy to generate multi-targeted HDACi by combining structural features from HDACi and other anticancer agents. HDACi can also be connected to a motif that allows for a selective delivery. Highlighting current examples, this brief review focuses on the rational design of multi-targeted inhibitors based on the examination and manipulation of chemical structures.

Dual inhibitors of inosine monophosphate dehydrogenase and histone deacetylase based on a cinnamic hydroxamic acid core structure

Small molecules that act on multiple biological targets have been proposed to combat the drug resistance commonly observed for cancer chemotherapy. By combining the structural features of known inhibitors of inosine monophosphate dehydrogense (IMPDH) and histone deacetylase (HDAC), dual inhibitors of IMPDH and HDAC based on the scaffold of cinnamic hydroxamic acid (CHA) have been designed, synthesized, and evaluated in biological assays. Key features, including the linker length, linker functionality, substitution position, and interacting groups, have been explored. Their individual contribution to the inhibitory activities against human IMPDH1 and IMPDH2 as well as HDAC has been assessed.

mTORC1 Coordinates Protein Synthesis and Immunoproteasome Formation via PRAS40 to Prevent Accumulation of Protein Stress

Reduction of translational fidelity often occurs in cells with high rates of protein synthesis, generating defective ribosomal products. If not removed, such aberrant proteins can be a major source of cellular stress causing human diseases. Here, we demonstrate that mTORC1 promotes the formation of immunoproteasomes for efficient turnover of defective proteins and cell survival. mTORC1 sequesters precursors of immunoproteasome β subunits via PRAS40. When activated, mTORC1 phosphorylates PRAS40 to enhance protein synthesis and simultaneously to facilitate the assembly of the β subunits for forming immunoproteasomes. Consequently, the PRAS40 phosphorylations play crucial roles in clearing aberrant proteins that accumulate due to mTORC1 activation. Mutations of RAS, PTEN, and TSC1, which cause mTORC1 hyperactivation, enhance immunoproteasome formation in cells and tissues. Those mutations increase cellular dependence on immunoproteasomes for stress response and survival. These results define a mechanism by which mTORC1 couples elevated protein synthesis with immunoproteasome biogenesis to protect cells against protein stress.

Hydroxamic acids block replication of hepatitis C virus.

Intrigued by the role of protein acetylation in hepatitis C virus (HCV) replication, we tested known histone deacetylase (HDAC) inhibitors and a focused library of structurally simple hydroxamic acids for inhibition of a HCV subgenomic replicon. While known HDAC inhibitors with varied inhibitory profiles proved to be either relatively toxic or ineffective, structure-activity relationship (SAR) studies on cinnamic hydroxamic acid and benzo[b]thiophen-2-hydroxamic acid gave rise to compounds 22 and 53, which showed potent and selective anti-HCV activity and therefore are promising starting points for further structural optimization and mechanistic studies.

Intracellular nicotinamide adenine dinucleotide promotes TNF-induced necroptosis in a sirtuin-dependent manner.

Cellular necrosis has long been regarded as an incidental and uncontrolled form of cell death. However, a regulated form of cell death termed necroptosis has been identified recently. Necroptosis can be induced by extracellular cytokines, pathogens and several pharmacological compounds, which share the property of triggering the formation of a RIPK3-containing molecular complex supporting cell death. Of interest, most ligands known to induce necroptosis (including notably TNF and FASL) can also promote apoptosis, and the mechanisms regulating the decision of cells to commit to one form of cell death or the other are still poorly defined. We demonstrate herein that intracellular nicotinamide adenine dinucleotide (NAD+) has an important role in supporting cell progression to necroptosis. Using a panel of pharmacological and genetic approaches, we show that intracellular NAD+ promotes necroptosis of the L929 cell line in response to TNF. Use of a pan-sirtuin inhibitor and shRNA-mediated protein knockdown led us to uncover a role for the NAD+-dependent family of sirtuins, and in particular for SIRT2 and SIRT5, in the regulation of the necroptotic cell death program. Thus, and in contrast to a generally held view, intracellular NAD+ does not represent a universal pro-survival factor, but rather acts as a key metabolite regulating the choice of cell demise in response to both intrinsic and extrinsic factors.