Karunakaran A. Kalesh

High-throughput discovery of Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) inhibitors using click chemistry.

A approximately 3500-member library of bidentate inhibitors against protein tyrosine phosphatases (PTPs) was rapidly assembled using click chemistry. Subsequent high-throughput screening had led to the discovery of highly potent (K(i) as low as 150 nM) and selective MptpB inhibitors, some of which represent the most potent MptpB inhibitors developed to date.

Global profiling of protein lipidation using chemical proteomic technologies

Highlights • Protein lipidation is an essential modification (PTM) in all forms of life.• Key modifications include acylation, prenylation, cholesterylation and GPI anchors.• Global profiling of this class of PTM is beyond the scope of standard technologies.• Metabolic chemical tagging can reveal the full scope of protein lipidation in vivo.• Chemical proteomics will have a deep impact on understanding of protein lipidation.

Key cell signaling pathways modulated by zerumbone: Role in the prevention and treatment of cancer

Phytochemicals and their synthetic derivatives are making a significant contribution in modern drug discovery programs by targeting several human diseases, including cancer. Most of these natural compounds are often multitargeted in nature, which is generally a very desirable property for cancer therapy, as carcinomas typically involve dysregulation of multiple genes and associated cell-signaling pathways at various stages of initiation, progression and metastasis. Additionally, these natural agents generally have lower side-effects, are readily available and hence are cost effective. One such natural compound is zerumbone, a cyclic eleven-membered sesquiterpene, isolated from the tropical plant Zingiber zerumbet Smith that has attracted great attention recently for its potent anticancer activities in several tumor models. This review summarizes the data based on various in vitro and in vivo studies related to the effects of zerumbone on numerous pivotal molecular targets in cancer and its reported chemopreventive/therapeutic effects in different models of cancer.

Target identification of natural and traditional medicines with quantitative chemical proteomics approaches

Natural and traditional medicines, being a great source of drugs and drug leads, have regained wide interests due to the limited success of high-throughput screening of compound libraries in the past few decades and the recent technology advancement. Many drugs/bioactive compounds exert their functions through interaction with their protein targets, with more and more drugs showing their ability to target multiple proteins, thus target identification has an important role in drug discovery and biomedical research fields. Identifying drug targets not only furthers the understanding of the mechanism of action (MOA) of a drug but also reveals its potential therapeutic applications and adverse side effects. Chemical proteomics makes use of affinity chromatography approaches coupled with mass spectrometry to systematically identify small molecule-protein interactions. Although traditional affinity-based chemical proteomics approaches have made great progress in the identification of cellular targets and elucidation of MOAs of many bioactive molecules, nonspecific binding remains a major issue which may reduce the accuracy of target identification and may hamper the drug development process. Recently, quantitative proteomics approaches, namely, metabolic labeling, chemical labeling, or label-free approaches, have been implemented in target identification to overcome such limitations. In this review, we will summarize and discuss the recent advances in the application of various quantitative chemical proteomics approaches for the identification of targets of natural and traditional medicines.

An improved mechanism-based cross-linker for multiplexed kinase detection and inhibition in a complex proteome.

Protein phosphorylation is the most prevalent event in cell signaling. Despite decades of intensive research, there exist several major challenges in the field of phosphoproteomics: 1) how does one identify new kinase–substrate pairs in the ever-expanding phosphosignaling cascades, 2) how can one detect multiple kinases in their native environment, and subsequently 3) look for potent and selective small-molecule inhibitors? Existing biological and chemical methods have offered invaluable tools for the identification of phosphorylated proteins, as well as sites of phosphorylation. Shokat et al. recently developed a mechanism-based cross-linker, OPA-AD (1, Figure 1), that potentially allows researchers to use known phosphoproteins/phosphopeptides to identify their upstream kinases. To detect kinases in their native environments, protein-based biosensors have routinely been used but offer limited success. Imperiali et al. recently developed a homogeneous fluorescence-based assay that enables multiplexed kinase detection in cell lysates. Activity-based probes (ABPs), based on either reversible small-molecule kinase inhibitors or irreversible ATP analogues, have also demonstrated good utilities in large-scale kinase detection and identification. None of these kinase-detecting methods, however, has thus far been expanded to the screening and identification of inhibitors against specific kinase–substrate pairs in their native states. Herein, we report an improved mechanism-based cross-linker, NDA-AD (2, Figure 1), based on the originally reported 1, for multiplexed detection and inhibition of kinase–substrate pairs in a complex proteome. When 1 was initially used in a complex proteome, we unACHTUNGTRENNUNGexpectedly discovered that it produces a large number of nonspecific cross-linking bands (Figure 1C and in the Supporting Information) in addition to the one corresponding to the desired kinase–pseudosubstrate pair (i.e. , a kinase peptide substrate in which the S/T/Y phosphorylation site was replaced by cysteine). This severely limits its potential applications. Thus we aimed to modify the highly reactive o-phthaldialdehyde (OPA) moiety in 1 and make it compatible with proteomic experiments. Consequently, 2 was designed to covalently trap the transient kinase–substrate–ATP ternary complex formed during the phosphorylation. With an adenosine moiety guiding 2 to the ATP-binding pocket of a kinase, the naphthalene2,3-dicarboxaldehyde (NDA) group serves as a bifunctional chemical that cross-links the proximal catalytic lysine residue (from the kinase) and the cysteine residues (from the pseudosubstrate); this generates a stable isoindole linkage between the kinase–substrate pair (Figure 1B). We reasoned that NDA would cross-link kinase–substrate pairs more specifically than OPA, because of its more desirable chemical properties and better structural fit in the kinase active site. In the current study with 2 we demonstrate, for the first time, that 1) the cross-linking strategy is compatible with both tyrosine and serine/threonine kinases, 2) it can be used to cross-link the ACHTUNGTRENNUNGdesired kinase–substrate pairs in a crude proteome with high specificity and sensitivity, 3) multiplexed detection of kinases in their native environment is possible, and 4) screening of potent and selective inhibitors of a given kinase–substrate pair can be done in a complex proteome. We first assessed whether 2 could serve as a general mechanism-based cross-linker for both tyrosine and serine/threonine kinases. The cross-linking reactions were tested with a set of six purified kinases, of which three are Tyr kinases (Csk, Src, and Abl) and the other three Ser/Thr kinases (Erk1, Erk2, and Pka). All kinases were recombinantly expressed and tested to ensure their purity as well as enzymatic activities. Fluoresceinlabeled, cysteine-containing kinase pseudosubstrates were chemically synthesized based on their known peptide substrate sequences (Table 1). As shown in Figure 2A, incubation of each of the six kinases, regardless of whether they are Tyr or Ser/Thr kinases, with their cognate pseudosubstrates in the presence of 2 led to the successful cross-linking of the kinase–substrate complex, as indicated by a fluorescent band on the SDS-PAGE. All three components (i.e. , kinase, pseudosubstrate, and 2) were necessary as labeling was not observed in the absence of any of them. No cross-linking was seen with heat-denatured kinases; this indicates that cross-linking was dependent on the active conformation of kinases. To test the tolerance of the cross-linking towards exogenous thiols or amines, the Pka–PKAtide pair was incubated with 2 with increasing concentrations of b-mercaptoethanol (BME) or lysine. Similar to previous reports with OPA-AD, NDA-AD guided cross-linking reactions were not affected by 200-fold excess of exogenous thiols or 1000-fold excess of exogenous amines (Supporting Information). Competition experiments were performed with ATP and LRRASLG-OH (a Pka peptide substrate) ; a 1000-fold excess of either ATP or LRRASLG-OH was necessary to completely block the cross-linking. To assess whether the degree of cross-linking depends proportionally on the availa[a] K. Liu, Prof. Dr. S. Q. Yao Deptartment of Biological Sciences, National University of Singapore 14 Science Drive 4, Singapore 117557 (Singapore) Fax: (+65)67791691 E-mail : chmyaosq@nus.edu.sg [b] K. A. Kalesh, L. Bing Ong, Prof. Dr. S. Q. Yao Department of Chemistry, National University of Singapore 3 Science Drive 3, Singapore 117543 (Singapore) [c] Prof. Dr. S. Q. Yao NUS MedChem Program of the Office of Life Sciences National University of Singapore 3 Science Drive 3, Singapore 17543 (Singapore) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

A capillary electrophoresis method to explore the self-assembly of a novel polypeptide ligand with quantum dots

Polyhistidine peptides are effective ligands to coat quantum dots (QDs). It is known that both the number of histidine (His) residues repeats and their structural arrangements in a peptide ligand play important roles in the assembly of the peptide onto CdSe/ZnS QDs. However, due to steric hindrance, a peptide sequence with more than six His residue tandem repeats would hardly coordinate well with Zn2+ in the QD shell to further enhance the binding affinity. To solve this problem, a His‐containing peptide ligand, ATTO 590‐E2G (NH)6 (ATTO‐NH), was specifically designed and synthesized for assembly with QDs. With sequential injection of QDs and ATTO‐NH into the capillary electrophoresis with fluorescence detection, strong Förster resonance energy transfer phenomenon between the QDs and the ATTO 590 dye was observed, indicating efficient self‐assembly of the novel peptide onto the QDs to form ATTO‐NH capped QDs inside the capillary. The binding stability of the ligand onto the QD was then systematically investigated by titrating with imidazole, His, and a his‐tag containing competitive peptide. It is believed that this new in‐capillary assay significantly reduced the sample consumption and the analysis time. By functionalizing QDs with certain metal cation‐specific group fused peptide ligand, the QD‐based probes could be even extended to the online detection of metal cations for monitoring environment in the future.

Artemisinin as an anticancer drug: Recent advances in target profiling and mechanisms of action

Artemisinin and its derivatives (collectively termed as artemisinins) are among the most important and effective antimalarial drugs, with proven safety and efficacy in clinical use. Beyond their antimalarial effects, artemisinins have also been shown to possess selective anticancer properties, demonstrating cytotoxic effects against a wide range of cancer types both in vitro and in vivo. These effects appear to be mediated by artemisinin‐induced changes in multiple signaling pathways, interfering simultaneously with multiple hallmarks of cancer. Great strides have been taken to characterize these pathways and to reveal their anticancer mechanisms of action of artemisinin. Moreover, encouraging data have also been obtained from a limited number of clinical trials to support their anticancer property. However, there are several key gaps in knowledge that continue to serve as significant barriers to the repurposing of artemisinins as effective anticancer agents. This review focuses on important and emerging aspects of this field, highlighting breakthroughs in unresolved questions as well as novel techniques and approaches that have been taken in recent studies. We discuss the mechanism of artemisinin activation in cancer, novel and significant findings with regards to artemisinin target proteins and pathways, new understandings in artemisinin‐induced cell death mechanisms, as well as the practical issues of repurposing artemisinin. We believe these will be important topics in realizing the potential of artemisinin and its derivatives as safe and potent anticancer agents.

Nanoparticles-chemistry, new synthetic approaches, gas phase clustering and novel applications

In this paper, an overview of the synthesis, chemistry and applications of nanosystems carried out in our laboratory is presented. The discussion is divided into four sections, namely (a) chemistry of nanoparticles, (b) development of new synthetic approaches, (c) gas phase clusters and (d) device structures and applications. In ‘chemistry of nanoparticles’ we describe a novel reaction between nanoparticles of Ag and Au with halocarbons. The reactions lead to the formation of various carbonaceous materials and metal halides. In ‘development of new synthetic approaches’ our one-pot methodologies for the synthesis of core-shell nanosystems of Au, Ag and Cu protected with TiO2 and ZrO2 as well as various polymers are discussed. Some results on the interaction of nanoparticles with biomolecules are also detailed in this section. The third section covers the formation of gas phase aggregates/clusters of thiol-protected sub-nanoparticles. Laser desorption of H2MoO4, H2WO4, MoS2, and WS2 giving novel clusters is discussed. The fourth section deals with the development of simple devices and technologies using nanomaterials described above.

Recent Advances in Synthesis and Identification of Cyclic Peptides for Bioapplications

Cyclic peptides, owing to their good stability, high resistance to exo- and to some extent endo-peptidases, enhanced binding affinity and selectivity towards target biomolecules, are actively investigated as biochemical tools and therapeutic agents. In this review, we discuss various commonly utilized synthetic strategies for cyclic peptides and peptoids (peptidomimetics), their important screening methods to identify the bioactive cyclic peptides and peptoids such as combinatorial beadbased peptide library, phage display, mRNA display etc. and recent advances in their applications as bioactive compounds. Lastly, we also make a summary and provide an outlook of the research area.