YAP-dependent proliferation by a small molecule targeting annexin A2.

The transcriptional coactivator Yes-associated protein 1 (YAP) orchestrates a proproliferative transcriptional program that controls the fate of somatic stem cells and the regenerative responses of certain tissues. As such, agents that activate YAP may hold therapeutic potential in disease states exacerbated by insufficient proliferative repair. Here we report the discovery of a small molecule, termed PY-60, which robustly activates YAP transcriptional activity in vitro and promotes YAP-dependent expansion of epidermal keratinocytes in mouse following topical drug administration. Chemical proteomics revealed the relevant target of PY-60 to be annexin A2 (ANXA2), a protein that directly associates with YAP at the cell membrane in response to increased cell density. PY-60 treatment liberates ANXA2 from the membrane, ultimately promoting a phosphatase-bound, nonphosphorylated and transcriptionally active form of YAP. This work reveals ANXA2 as a previously undescribed, druggable component of the Hippo pathway and suggests a mechanistic rationale to promote regenerative repair in disease.

New Generation Oxyntomodulin Peptides with Improved Pharmacokinetic Profiles Exhibit Weight Reducing and Anti-Steatotic Properties in Mice.

Oxyntomodulin (OXM) is an intestinal peptide hormone that activates both glucagon-like peptide-1 (GLP-1) and glucagon (GCG) receptors. The natural peptide reduces body weight in obese subjects and exhibits direct acute glucoregulatory effects in patients with type II diabetes. However, the clinical utility of OXM is limited due to its lower in vitro potency and short in vivo half-life. To overcome these issues, we developed stapled, long-acting, and highly potent OXM analogs with balanced activities at both GLP-1 and GCG receptors. The lead molecule O14 exhibits potent and long-lasting effects on glucose control, body weight loss, and reduction of hepatic fat reduction in DIO mice. Importantly, O14 significantly reversed hepatic steatosis; reduced liver weight, total cholesterol, and hepatic triglycerides; and improved markers of liver function in a nonalcoholic steatohepatitis (NASH) mouse model. A symmetrical version of the peptide was also shown to be more efficacious and long-lasting in controlling glucose than semaglutide and the clinical candidate cotadutide in wild-type mice, highlighting the utility of our designs of the dual agonist as a potential new therapy for diabetes and liver diseases.

Photoactivatable Fluorogenic Labeling via Turn-On "Click-Like" Nitroso-Diene Bioorthogonal Reaction.

Fluorogenic labeling enables imaging cellular molecules of interest with minimal background. This process is accompanied with the notable increase of the quantum yield of fluorophore, thus minimizing the background signals from unactivated profluorophores. Herein, the development of a highly efficient and bioorthogonal nitroso-based Diels-Alder fluorogenic reaction is presented and its usefulness is validated as effective and controllable in fluorescent probes and live-cell labeling strategies for dynamic cellular imaging. It is demonstrated that nitroso-based cycloaddition is an efficient fluorogenic labeling tool through experiments of further UV-activatable fluorescent labeling on proteins and live cells. The ability of tuning the fluorescence of labeled proteins by UV-irradiation enables selective activation of proteins of interest in a particular cell compartment at a given time point, while leaving the remaining labeled molecules untouched.

Stapled, Long-Acting Glucagon-like Peptide 2 Analog with Efficacy in Dextran Sodium Sulfate Induced Mouse Colitis Models.

Glucagon-like peptide 2 (GLP-2) is a hormone that has been shown to stimulate intestinal growth and attenuate intestinal inflammation. Despite being efficacious in a variety of animal models of disease, its therapeutic potential is hampered by the short half-life in vivo. We now describe a highly potent, stapled long-acting GLP-2 analog, peptide 10, that has a more than 10-fold longer half-life than teduglutide and improved intestinotrophic and anti-inflammatory effects in mouse models of DSS-induced colitis.

Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria.

Tyrosine phosphorylation is a common protein post-translational modification that plays a critical role in signal transduction and the regulation of many cellular processes. Using a propeptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase-tRNA pair that allows site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray structure of the synthetase reveals a reconfigured substrate-binding site, formed by nonconservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site and determining the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.

Engineering a long-acting, potent GLP-1 analog for microstructure-based transdermal delivery.

Antidiabetic treatments aiming to reduce body weight are currently gaining increased interest. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist administered twice daily via s.c. injection, improves glycemic control, often with associated weight reduction. To further improve the therapeutic efficacy of exendin-4, we have developed a novel peptide engineering strategy that incorporates a serum protein binding motif onto a covalent side-chain staple and applied to the peptide to enhance its helicity and, as a consequence, its potency and serum half-life. We demonstrated that one of the resulting peptides, E6, has significantly improved half-life and glucose tolerance in an oral glucose tolerance test in rodents. Chronic treatment of E6 significantly decreased body weight and fasting blood glucose, improved lipid metabolism, and also reduced hepatic steatosis in diet-induced obese mice. Moreover, the high potency of E6 allowed us to administer this peptide using a dissolvable microstructure-based transdermal delivery system. Pharmacokinetic and pharmacodynamic studies in guinea pigs showed that a single 5-min application of a microstructure system containing E6 significantly improved glucose tolerance for 96 h. This delivery strategy may offer an effective and patient-friendly alternative to currently marketed GLP-1 injectables and can likely be extended to other peptide hormones.

Recombinant thiopeptides containing noncanonical amino acids.

Thiopeptides are a subclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs) with complex molecular architectures and an array of biological activities, including potent antimicrobial activity. Here we report the generation of thiopeptides containing noncanonical amino acids (ncAAs) by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin producer strain of Bacillus cereus .We demonstrate that thiopeptide variants containing ncAAs with bioorthogonal chemical reactivity can be further postbiosynthetically modified with biophysical probes, including fluorophores and photo-cross-linkers. This work allows the site-specific incorporation of ncAAs into thiopeptides to increase their structural diversity and probe their biological activity; similar approaches can likely be applied to other classes of RiPPs.

Design of Potent and Proteolytically Stable Oxyntomodulin Analogs.

Incretin-based peptides are effective therapeutics for treating type 2 diabetes mellitus (T2DM). Oxyntomodulin (OXM), a dual agonist of GLP-1R and GCGR, has shown superior weight loss and glucose lowering effects, compared to single GLP-1R agonists. To overcome the short half-life and rapid renal clearance of OXM, which limit its therapeutic potential, both lipid and PEG modified OXM analogs have been reported. However, these approaches often result in reduced potency or PEG-associated toxicity. Herein, we report a new class of cross-linked OXM analogs that show increased plasma stability and higher potency in activating both GLP-1R and GCGR. Moreover, the extended in vivo half-life results in superior antihyperglycemic activity in mice compared to the wild-type OXM.

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 tRNACUA(Pyl), 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.

Parasite-based screening and proteome profiling reveal orlistat, an FDA-approved drug, as a potential anti Trypanosoma brucei agent.

Trypanosoma brucei is a parasite that causes African sleeping sickness in humans and nagana in livestock and is transmitted by the tsetse fly. There is an urgent need for the development of new drugs against African trypanosomiasis due to the lack of vaccines and effective drugs. Orlistat (also called tetrahydrolipstatin or THL) is an FDA-approved antiobesity drug targeting primarily the pancreatic and gastric lipases within the gastrointestinal tract. It shows potential activities against tumors, mycobacteria, and parasites. Herein, we report the synthesis and evaluation of an expanded set of orlistat-like compounds, some of which showed highly potent trypanocidal activities in both the bloodstream form (BSF) and the procyclic form (PCF) of T. brucei. Subsequent in situ parasite-based proteome profiling was carried out to elucidate potential cellular targets of the drug in both forms. Some newly identified targets were further validated by the labeling of recombinantly expressed enzymes in Escherichia coli lysates. Bioimaging experiments with a selected compound were carried out to study the cellular uptake of the drug in T. brucei. Results indicated that orlistat is much more efficiently taken up by the BSF than the PCF of T. brucei and has clear effects on the morphology of mitochondria, glycosomes, and the endoplasmic reticulum in both BSF and PCF cells. These results support specific effects of orlistat on these organelles and correlate well with our in situ proteome profiling. Given the economic challenges of de novo drug development for neglected diseases, we hope that our findings will stimulate further research towards the conversion of orlistat-like compounds into new trypanocidal drugs.

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.

Proteomic profiling and potential cellular target identification of K11777, a clinical cysteine protease inhibitor, in Trypanosoma brucei.

We report herein the design, synthesis and application of K11777-derived activity-based probes (ABPs) allowing in situ profiling and identification of potential cellular targets of K11777 in Trypanosoma brucei.

Multicolor, one- and two-photon imaging of enzymatic activities in live cells with fluorescently Quenched Activity-Based Probes (qABPs).

Fluorescence imaging provides an indispensable way to locate and monitor biological targets within complex and dynamic intracellular environments. Of the various imaging agents currently available, small molecule-based probes provide a powerful tool for live cell imaging, primarily due to their desirable properties, including cell permeability (as a result of their smaller sizes), chemical tractability (e.g., different molecular structures/designs can be installed), and amenability to imaging a wide variety of biological events. With a few exceptions, most existing small molecule probes are however not suitable for in vivo bioimaging experiments in which high-resolution studies of enzyme activity and localization are necessary. In this article, we reported a new class of fluorescently Quenched Activity-Based Probes (qABPs) which are highly modular, and can sensitively image (through multiple enzyme turnovers leading to fluorescence signal amplification) different types of enzyme activities in live mammalian cells with good spatial and temporal resolution. We have also incorporated two-photon dyes into our modular probe design, enabling for the first time activity-based, fluorogenic two-photon imaging of enzyme activities. This, hence, expands the repertoire of 'smart', responsive probes currently available for live cell bioimaging experiments.

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.

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.

Click-based synthesis and proteomic profiling of lipstatin analogues.

Using click chemistry to enable both structural diversity and proteome profiling within a natural product derived library, two out of nineteen lipstatin analogues showed similar activity to Orlistat against fatty acid synthase (FAS), but with an improved ability to induce tumour cell death.

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.

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.