Rajavel Srinivasan

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.

Cell-permeable small molecule probes for site-specific labeling of proteinsElectronic supplementary information (ESI) available: experimental details and characterization of compounds. See http://www.rsc.org/suppdata/cc/b3/b309196a/

We have successfully synthesized a number of small molecule probes designed for site-specific labeling of N-terminal cysteine-containing proteins expressed in live cells. Their utility for site-specific, covalent modifications of proteins was successfully demonstrated with purified proteins in vitro, and with live bacterial cells in vivo.

Site-specific covalent labeling of proteins inside live cells using small molecule probes

The study of dynamic movement and interactions of proteins inside living cells in real time is critical for a better understanding of cellular mechanisms and functions in molecular detail. Genetically encoded fusions to fluorescent protein(s) (FP) have been widely used for this purpose [Annu. Rev. Biochem. 1998, 67, 509-544]. To obviate some of the drawbacks associated with the use of FPs [Curr. Opin. Biotechnol. 2005, 16, 1-6; Nat. Methods2006, 3, 591-596], we report a small molecule-based approach that exploits the unique reactivity between the cysteine residue at the N-terminus of a target protein and cell-permeable, thioester-based small molecule probes resulting in site-specific, covalent tagging of proteins. This approach has been demonstrated by the in vivo labeling of proteins in both bacterial and mammalian systems thereby making it potentially useful for future bioimaging applications.

Activity‐Based Fingerprinting of Proteases

In the post-genomic era, the characterization of enzyme activity patterns is more meaningful than enzyme identification. This is because the so-called substrate fingerprint of an enzyme reveals the type of chemical entities accepted by the enzyme as its potential substrates, and thereby helps in a better understanding of its catalytic mechanism and properties. Similarly, the unique pattern generated for an unknown enzyme by using a set of known substrates can be used to delineate its identity. With the aid of standard analytical tools, traditional fingerprinting experiments use a whole spectrum of substrates and/or their analogues on a target enzyme to create quantitative and reproducible profiles directly related to the enzyme’s activity. Different classes of enzymes have been studied in this fashion, including cytochrome P450, protein kinases, and hydrolytic enzymes. In recent years, much effort has been expended in developing microarray-based bioassays. If adopted for fingerprinting experiments, they could potentially provide a powerful platform by allowing the simultaneous analysis of thousands of enzymatic reactions on a single chip with very small sample volumes, while retaining a good degree of detection sensitivity. Proteases, one of the largest groups of enzymes that are important therapeutic targets of major human diseases, have been the focus of new enzyme-assay developments in recent years. Activity-based profiling (ABP), originally developed by Cravatt et al. , allows the proteases present in a crude proteome to be studied on the basis of their enzymatic activities rather than their relative abundance. ABP works by using either mechanismor affinity-based chemical probes that can be covalently attached to different classes of enzymes, thus providing a versatile tool for large-scale protease identification, characterization, and even fingerprinting experiments. We recently investigated a new class of ABP probes that target all major classes of proteases by their properties as enzyme substrates, rather than as inhibitors. For this reason, we expect that these probes will be more suitable for protease fingerprinting experiments than existing ones. We now report the chemical synthesis of a full set of these probes and their use in activity-based fingerprinting of proteases in gel-based experiments, as well as their potential application in microarraybased enzyme assays. A total of 16 probes were synthesized (Scheme 1), each containing a common p-aminomandelic acid moiety and a unique recognition head consisting of an N-acetylated amino acid that mimics the P1 position in a protease substrate. The amide bond between the two groups imitates the scissile bond in the protease substrate. A fluorescent reporter group, Cy3, was attached to the other end of p-aminomandelic acid. Upon proteolytic cleavage of the scissile bond, the probe releases the amino acid head group to generate a highly reactive quinolimine methide, which subsequently reacts covalently with the protease (that cleaves it) and renders it detectable (Scheme 1, left). One potential limitation of our enzyme-fingerprinting approach is that, since only P-site residues can be incorporated into the probe design, the approach might only be suitable for profiling proteases that possess P-site specificities. To make the 16 probes, commercially available p-nitrophenylacetic acid (17) was treated with thionyl chloride in methanol to afford the methyl ester 18 in 93% yield. Subsequently, the benzylic proton in 18 was brominated by N-Bromosuccinimide (NBS; 85% yield) to generate 19. This was followed by conversion to the corresponding benzyl alcohol 21 in two steps. Reduction of the nitro group in 21 with 10% Pd/C in the presence of H2 gave intermediate 22 in excellent yield (93%). A number of acylating reagents were tested in order to optimize the subsequent coupling reaction between the aromatic amine on 22 and a properly protected amino acid (both N-a-Boc and N-aFmoc amino acids were used), and it was found that O-(7azabenzotriazole-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU) consistently gave the best yield (80–88% on average). By using the optimized method, 16 different amino acids were used to generate 16 versions of compound 23 in which each compound differed by its amino acid side chain. After deprotection of the N-a-Fmoc or N-a-Boc group, the resulting amino acids were acylated with acetic anhydride in N,N-diisopropylethylamine (DIEA) to afford 24. The hydrolytic cleavage of the methyl ester in 24 was achieved by using LiOH solution in nearly quantitative yield to furnish 25. The final 16 probes, represented by the single-letter codes of their amino acid (Table 1), were subsequently obtained in three steps by conversion of the benzylic OH group in 25 to the corresponding fluoride with (diethylamino)sulfur trifluoride (DAST) at 0 8C, and attachment of a reporter Cy3 dye via a hydrophilic linker. The average yield of these three steps combined for all sixteen probes was approximately 50%. The labeling experiments were first performed with four of the 16 probes synthesized F, K, Y, and W, and four commercially available proteases, trypsin, actinase E, b-chymotrypsin, and a-chymotrypsin. Both geland microarray-based labeling experiments were carried out (Figure 1). Control experiments were run with nonprotease proteins including bovine serum albumin (BSA), alkaline phosphatase, lysozyme, and lipase; but no labeling was observed even after prolonged incubation of these proteins with the probes (see Supporting Information and ref. [6]). As shown in Figure 1 (top), all four proteases were positively labeled by the different substrate-based probes, with [a] R. Srinivasan, X. Huang, S. L. Ng, Prof. Dr. S. Q. Yao Department of Chemistry, National University of Singapore 3 Science Drive 3, Singapore 117543 (Republic of Singapore) Fax: (+65)677-91691 E-mail : chmyaosq@nus.edu.sg [b] Prof. Dr. S. Q. Yao Department of Biological Sciences, National University of Singapore 14 Science Drive 4, Singapore 117543 (Republic of Singapore) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Solid-phase assembly and in situ screening of protein tyrosine phosphatase inhibitors.

A highly efficient solid-phase strategy for assembly of small molecule inhibitors against protein tyrosine phosphatase 1B (PTP1B) is described. The method is highlighted by its simplicity and product purity. A 70-member combinatorial library of analogues of a known PTP1B inhibitor has been synthesized, which upon direct in situ screening revealed a potent inhibitor ( Ki = 7.0 microM) against PTP1B.

Solid-Phase Synthesis of Azidomethylene Inhibitors Targeting Cysteine Proteases

An efficient strategy for the solid-phase synthesis of azidomethylene inhibitors targeting cysteine proteases is described. The method is highlighted by its compatibility with readily available building blocks, as well as its ability to accommodate different functional groups. A 249-member library has thus far been successfully synthesized, characterized, and screened against Caspase-1, -3 and -7.

Chemical approaches for live cell bioimaging.

We review various advancements in small molecule probes, intein-based labeling methods, and the incorporation of synthetic amino acids into proteins for live cell imaging experiments. Finally, recent developments in quantum dots-based labeling are briefly reviewed.

An amidation/cyclization approach to the synthesis of N-hydroxyquinolinones and their biological evaluation as potential anti-plasmodial, anti-bacterial, and iron(II)-chelating agents

A 26-member library of novel N-hydroxyquinolinone derivatives was synthesized by a one-pot Buchwald-type palladium catalyzed amidation and condensation sequence. The design of these rare scaffolds was inspired from N-hydroxypyridones and 2-quinolinones classes of compounds which have been shown to have rich biological activities. The synthesized compounds were evaluated for their anti-plasmodial and anti-bacterial properties. In addition, these compounds were screened for their iron(II)-chelation properties. Notably, four of these compounds exhibited anti-plasmodial activities comparable to that of the natural product cordypyridone B.

In vivo Imaging of a Bacterial Cell Division Protein Using a Protease- Assisted Small Molecule Labeling Approach

Chemical labeling strategies developed to site specifically label proteins in their native cellular milieu have been sought as attractive approaches to obviate some of the drawbacks associated with the use of genetically encoded fusions to fluorescent proteins (FPs). Though elegant, many of these approaches necessitate the use of large protein domains/tags fused to target protein(s). Like FPs, these protein “tags” can potentially perturb the folding and/or activity of target proteins. Small peptide-recognition sequences are more desirable, but they have lower labeling efficiencies. 4] Other methods such as incorporation of unnatural amino acids and metabolic installation of reporter tags are primarily governed by the ability of the enzyme(s) to tolerate the unnatural motif to be introduced. Nevertheless, metabolic-labeling approaches have been successfully used to study glycoproteins and conjugates, which were previously inaccessible by other labeling techniques. Thus, the need still exists to develop new methods that can facilitate the routine use of small-molecule probes for the in vivo study of protein dynamics. Previously we reported an intein-mediated small-molecule approach for site-specific labeling of N-terminal cysteine-containing proteins. This method, however, had some significant drawbacks, including the relatively large size of intein tag used, as well as slow and uncontrolled self-splicing of the tag, which inevitably led to a longer labeling time. In this communication, we have sought to develop a more efficient labeling strategy and have capitalized on the highly specific tobacco etch virus (TEV) NIa protease. TEV is a 3C-type cysteine protease that recognizes a seven amino acid sequence, E-X-X-Y-XQflS/G (where X is any amino acid; and fl indicates the cleavage site). It has stringent substrate sequence requirement, with absolutely conserved residues at P6, P3, and P1 positions, and as such, has been widely used for removal of affinity tags from proteins both in vitro and in vivo. It was previously shown that mutation of the P1’ residue from S/G to cysteine does not significantly alter the efficiency of TEV cleavage. In our current strategy (Figure 1A), we used TEV for the rapid and controlled intracellular generation of N-terminal cysteine-containing proteins, which were subsequently labeled in a site-specific