Peng-Yu Yang

Activity-Based Proteome Profiling of Potential Cellular Targets of Orlistat − An FDA-Approved Drug with Anti-Tumor Activities

Orlistat, or tetrahydrolipstatin (THL), is an FDA-approved antiobesity drug with potential antitumor activities. Cellular off-targets and potential side effects of Orlistat in cancer therapies, however, have not been extensively explored thus far. In this study, we report the total of synthesis of THL-like protein-reactive probes, in which extremely conservative modifications (i.e., an alkyne handle) were introduced in the parental THL structure to maintain the native biological properties of Orlistat, while providing the necessary functionality for target identification via the bio-orthogonal click chemistry. With these natural productlike, cell-permeable probes, we were able to demonstrate, for the first time, this chemical proteomic approach is suitable for the identification of previously unknown cellular targets of Orlistat. In addition to the expected fatty acid synthase (FAS), we identified a total of eight new targets, some of which were further validated by experiments including Western blotting, recombinant protein expression, and site-directed mutagenesis. Our findings have important implications in the consideration of Orlistat as a potential anticancer drug at its early stages of development for cancer therapy. Our strategy should be broadly useful for off-target identification against quite a number of existing drugs and/or candidates, which are also covalent modifiers of their biological targets.

Identification of a small molecule with activity against drug-resistant and persistent tuberculosis

Significance The global problem of TB has worsened in recent years with the emergence of drug-resistant organisms, and new drugs are clearly needed. In a cell-based high-throughput screen, a small molecule, TCA1, was discovered that has activity against replicating and nonreplicating Mycobacterium tuberculosis. It is also efficacious in acute and chronic rodent models of TB alone or combined with frontline TB drugs. TCA1 functions by a unique mechanism, inhibiting enzymes involved in cell wall and molybdenum cofactor biosynthesis. This discovery represents a significant advance in the search for new agents to treat persistent and drug-resistant TB. A cell-based phenotypic screen for inhibitors of biofilm formation in mycobacteria identified the small molecule TCA1, which has bactericidal activity against both drug-susceptible and -resistant Mycobacterium tuberculosis (Mtb) and sterilizes Mtb in vitro combined with rifampicin or isoniazid. In addition, TCA1 has bactericidal activity against nonreplicating Mtb in vitro and is efficacious in acute and chronic Mtb infection mouse models both alone and combined with rifampicin or isoniazid. Transcriptional analysis revealed that TCA1 down-regulates genes known to be involved in Mtb persistence. Genetic and affinity-based methods identified decaprenyl-phosphoryl-β-D-ribofuranose oxidoreductase DprE1 and MoeW, enzymes involved in cell wall and molybdenum cofactor biosynthesis, respectively, as targets responsible for the activity of TCA1. These in vitro and in vivo results indicate that this compound functions by a unique mechanism and suggest that TCA1 may lead to the development of a class of antituberculosis agents.

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.

Modulation of Fatty Acid Synthase Enzyme Activity and Expression during Hepatitis C Virus Replication

The hepatitis C virus (HCV) induces alterations of host cells to facilitate its life cycle. Fatty acid synthase (FASN) is a multidomain enzyme that plays a key role in the biosynthesis of fatty acids and is upregulated during HCV infection. Herein, we applied activity-based protein profiling (ABPP) that allows for the identification of differentially active enzymes in complex proteomic samples, to study the changes in activity of FASN during HCV replication. For this purpose, we used an activity-based probe based on the FASN inhibitor Orlistat, and observed an increase in the activity of FASN in the presence of a subgenomic and a genomic HCV replicon as well as in chimeric SCID/Alb-uPA mice infected with HCV genotype 1a. To study the molecular basis for this increase in FASN activity, we overexpressed individual HCV proteins in Huh7 cells and observed increased expression and activity of FASN in the presence of core and NS4B, as measured by western blots and ABPP, respectively. Triglyceride levels were also elevated in accordance with FASN expression and activity. Lastly, immunofluorescence and ABPP imaging analyses demonstrated that while the abundance and activity of FASN increases significantly in the presence of HCV, its localization does not change. Together these data suggest that the HCV-induced production of fatty acids and neutral lipids is provided by an increase in FASN abundance and activity that is sufficient to allow HCV propagation without transporting FASN to the replication complexes.

A Small Molecule Promotes Mitochondrial Fusion in Mammalian Cells

Mitochondria are highly dynamic cellular organelles that continuously undergo fission and fusion. This dynamic nature plays a key role in regulating mitochondrial function, and also gives mitochondria their heterogeneous morphology. Disruption of the balance between mitochondrial fusion and fission, especially a shift towards fission, contributes to a variety of human disorders, including neurodegenerative disease, metabolic disease, and ischemia. In addition, fragmented mitochondria are early signs of activation of apoptosis, and fusion of mitochondria by genetic or chemical manipulation has been shown to have an anti-apoptotic effect. Thus, the identification of small molecules that modulate mitochondrial dynamics can provide useful tools to study mitochondrial function and may ultimately lead to new therapeutics. Here, we report the identification and preliminary biological characterization of the small molecule, M1, which significantly restores the mitochondrial tubular network in response to genetically or chemically induced fragmentation. Mitochondrial fusion is a two-step process in which the outer and inner mitochondrial membranes (OMM and IMM, respectively) fuse separately, but in an ordered fashion. The core components of the mitochondrial fusion machinery are the OMM proteins, mitofusin 1 and 2 (Mfn1 and Mfn2), and the IMM protein, optic atrophy 1 (Opa1). Unlike wild-type mouse embryonic fibroblasts (WT MEFs), which mainly have interconnected tubular mitochondria, Mfn1 Knockout (KO) MEFs exhibit severely and uniformly frag-

A peptide aldehyde microarray for high-throughput profiling of cellular events.

Microarrays provide exciting opportunities in the field of large-scale proteomics. With the aim to elucidate enzymatic activity and profiles within native biological samples, we developed a microarray comprising a focused positional-scanning library of enzyme inhibitors. The library was diversified across P(1)-P(4) positions, creating 270 different inhibitor sublibraries which were immobilized onto avidin slides. The peptide aldehyde-based small-molecule microarray (SMM) specifically targeted cysteine proteases, thereby enabling large-scale functional assessment of this subgroup of proteases, within fluorescently labeled samples, including pure proteins, cellular lysates, and infected samples. The arrays were shown to elicit binding fingerprints consistent with those of model proteins, specifically caspases and purified cysteine proteases from parasites (rhodesein and cruzain). When tested against lysates from apoptotic Hela and red blood cells infected with Plasmodium falciparum, clear signatures were obtained that were readily attributable to the activity of constituent proteases within these samples. Characteristic binding profiles were further able to distinguish various stages of the parasite infection in erythrocyte lysates. By converting one of our brightest microarray hits into a probe, putative protein markers were identified and pulled down from within apoptotic Hela lysates, demonstrating the potential of target validation and discovery. Taken together, these results demonstrate the utility of targeted SMMs in dissecting cellular biology in complex proteomic samples.

Design, Synthesis and Biological Evaluation of Potent Azadipeptide Nitrile Inhibitors and Activity‐Based Probes as Promising Anti‐Trypanosoma brucei Agents

Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas disease and African sleeping sickness, respectively. There is an urgent need for the development of new drugs against both diseases due to the lack of adequate cures and emerging drug resistance. One promising strategy for the discovery of small-molecule therapeutics against parasitic diseases has been to target the major cysteine proteases such as cruzain for T. cruzi, and rhodesain/TbCatB for T. brucei. Azadipeptide nitriles belong to a novel class of extremely potent cysteine protease inhibitors against papain-like proteases. We herein report the design, synthesis, and evaluation of a series of azanitrile-containing compounds, most of which were shown to potently inhibit both recombinant cruzain and rhodesain at low nanomolar/picomolar ranges. A strong correlation between the potency of rhodesain inhibition (i.e., target-based screening) and trypanocidal activity (i.e., whole-organism-based screening) of the compounds was observed. To facilitate detailed studies of this important class of inhibitors, selected hit compounds from our screenings were chemically converted into activity-based probes (ABPs), which were subsequently used for in situ proteome profiling and cellular localization studies to further elucidate potential cellular targets (on and off) in both the disease-relevant bloodstream form (BSF) and the insect-residing procyclic form (PCF) of Trypanosoma brucei. Overall, the inhibitors presented herein show great promise as a new class of anti-trypanosome agents, which possess better activities than existing drugs. The activity-based probes generated from this study could also serve as valuable tools for parasite-based proteome profiling studies, as well as bioimaging agents for studies of cellular uptake and distribution of these drug candidates. Our studies therefore provide a good starting point for further development of these azanitrile-containing compounds as potential anti-parasitic agents.

Activity-based protein profiling: recent advances in probe development and applications.

The completion of the human genome sequencing project has provided a wealth of new information regarding the genomic blueprint of the cell. Although, to date, there are roughly 20 000 genes in the human genome, the functions of only a handful of proteins are clear. The major challenge lies in translating genomic information into an understanding of their cellular functions. The recently developed activity‐based protein profiling (ABPP) is an unconventional approach that is complementary for gene expression analysis and an ideal utensil in decoding this overflow of genomic information. This approach makes use of synthetic small molecules that covalently modify a set of related proteins and subsequently facilitates identification of the target protein, enabling rapid biochemical analysis and inhibitor discovery. This tutorial review introduces recent advances in the field of ABPP and its applications.

Chemical Modification and Organelle‐Specific Localization of Orlistat‐Like Natural‐Product‐Based Probes

Orlistat, also known as tetrahydrolipstatin (THL), is an FDA-approved anti-obesity drug with potential anti-cancer activity. Previously, we developed a chemical proteomic approach, based on the Orlistat-like probe (1a) for large-scale identification of unknown cellular targets of Orlistat in human hepatocytes. In this article, we report the chemical synthesis and biological evaluation of an expanded set of Orlistat-like compounds, with the intention to further dissect and manipulate potential cellular targets of Orlistat. In doing so, we carried out proteome-wide activity-based profiling and large-scale pull-down/LCMS analysis of these compounds in live HepG2 cells, and successfully identified many putative cellular targets for Orlistat and its structural analogues. By qualitatively assessing the spectra counts of potential protein hits against each of the seventeen Orlistat analogues, we obtained both common and unique targets of these probes. Our results revealed that subtle structural modifications of Orlistat led to noticeable changes in both the cellular potency and target profiles of the drug. In order to further improve the cellular activity of Orlistat, we successfully applied the well-established AGT/SNAP-tag technology to our cell-permeable, benzylguanine (BG)-containing Orlistat variant (4). We showed that the drug could be delivered and effectively retained in different sub-cellular organelles of living cells. This strategy may provide a general and highly effective chemical tool for the potential sub-cellular targeting of small molecule drugs.

Genetic incorporation of histidine derivatives using an engineered pyrrolysyl-tRNA synthetase.

A polyspecific amber suppressor aminoacyl-tRNA synthetase/tRNA pair was evolved that genetically encodes a series of histidine analogues in both Escherichia coli and mammalian cells. In combination with tRNACUAPyl, a pyrrolysyl-tRNA synthetase mutant was able to site-specifically incorporate 3-methyl-histidine, 3-pyridyl-alanine, 2-furyl-alanine, and 3-(2-thienyl)-alanine into proteins in response to an amber codon. Substitution of His66 in the blue fluorescent protein (BFP) with these histidine analogues created mutant proteins with distinct spectral properties. This work further expands the structural and chemical diversity of unnatural amino acids (UAAs) that can be genetically encoded in prokaryotic and eukaryotic organisms and affords new probes of protein structure and function.