Constraining and Modifying Peptides Using Pd-Mediated Cysteine Allylation.

Over the past decades, several strategies for inducing and stabilizing secondary structure formation in peptides have been developed to increase their proteolytic stability and their binding affinity to specific interaction partners. Here, we report how our recently introduced chemoselective Pd-catalyzed cysteine allylation reaction can be extended to stapling and how the resulting alkene-containing staples themselves can be further modified to introduce additional probes into such stabilized peptides. The latter is demonstrated by introducing a fluorophore as well as a PEG moiety into different stapled peptides using bioorthogonal thiol-ene and Diels-Alder reactions. Furthermore, we investigated structural implications of our allyl staples when used to replace conformationally relevant disulfide bridges. To this end, we chose a selective binder of integrin α3 ß1 (LXY3), which is only active in its cyclic disulfide form. We replaced the disulfide bridge by different stapling reagents in order to increase stability and binding affinity towards integrin α3 ß1 .

Reaction intensification for biocatalytic production of polyphenolic natural product di-C-ß-glucosides.

Polyphenolic aglycones featuring two sugars individually attached via C-glycosidic linkage (di-C-glycosides) represent a rare class of plant natural products with unique physicochemical properties and biological activities. Natural scarcity of such di-C-glycosides limits their use-inspired exploration as pharmaceutical ingredients. Here, we show a biocatalytic process technology for reaction-intensified production of the di-C-ß-glucosides of two representative phenol substrates, phloretin (a natural flavonoid) and phenyl-trihydroxyacetophenone (a phenolic synthon for synthesis), from sucrose. The synthesis proceeds via an iterative two-fold C-glycosylation of the respective aglycone, supplied as inclusion complex with 2-hydroxypropyl ß-cyclodextrin for enhanced water solubility of up to 50 mmol/L, catalyzed by a kumquat di-C-glycosyltransferase (di-CGT), and it uses UDP-Glc provided in situ from sucrose by a soybean sucrose synthase, with catalytic amounts (≤3 mol%) of UDP added. Time course analysis reveals the second C-glycosylation as rate-limiting (0.4-0.5 mmol/L/min) for the di-C-glucoside production. With internal supply from sucrose keeping the UDP-Glc at a constant steady-state concentration (≥50% of the UDP added) during the reaction, the di-C-glycosylation is driven to completion (≥95% yield). Contrary to the mono-C-glucoside intermediate which is stable, the di-C-glucoside requires the addition of reducing agent (10 mmol/L 2-mercaptoethanol) to prevent its decomposition during the synthesis. Both di-C-glucosides are isolated from the reaction mixtures in excellent purity (≥95%), and their expected structures are confirmed by NMR. Collectively, this study demonstrates efficient glycosyltransferase cascade reaction for flexible use in natural product di-C-ß-glucoside synthesis from expedient substrates.

Small-Molecule Inhibitors Targeting Lipolysis in Human Adipocytes.

Chronically elevated circulating fatty acid levels promote lipid accumulation in nonadipose tissues and cause lipotoxicity. Adipose triglyceride lipase (ATGL) critically determines the release of fatty acids from white adipose tissue, and accumulating evidence suggests that inactivation of ATGL has beneficial effects on lipotoxicity-driven disorders including insulin resistance, steatohepatitis, and heart disease, classifying ATGL as a promising drug target. Here, we report on the development and biological characterization of the first small-molecule inhibitor of human ATGL. This inhibitor, designated NG-497, selectively inactivates human and nonhuman primate ATGL but not structurally and functionally related lipid hydrolases. We demonstrate that NG-497 abolishes lipolysis in human adipocytes in a dose-dependent and reversible manner. The combined analysis of mouse- and human-selective inhibitors, chimeric ATGL proteins, and homology models revealed detailed insights into enzyme-inhibitor interactions. NG-497 binds ATGL within a hydrophobic cavity near the active site. Therein, three amino acid residues determine inhibitor efficacy and species selectivity and thus provide the molecular scaffold for selective inhibition.

Design and synthesis of efficient fluororethylene-peptidomimetic inhibitors of dipeptidyl peptidase III (DPP3).

Dipeptidyl peptidase III (DPP3) is a ubiquitously expressed zinc-dependent peptide cutting enzyme and selectively hydrolyses amide bonds to cleave N-terminal dipeptide fragments off of physiologically important oligopeptides. DPP3 has been found in a multitude of different types of cells and appears to be involved in various physiological processes (e.g. nociception, blood pressure control, protein turnover). Using the slowly converted peptide substrate tynorphin (VVYPW) as starting point, we have replaced the scissile bond with a fluoroethylene bioisostere to design ground state inhibitors, which led to the so far most effective peptide-based inhibitor of DPP3.

A Modular Synthesis of Teraryl-Based α-Helix Mimetics, Part†4: Core Fragments with Two Halide Leaving Groups Featuring Side Chains of Proteinogenic Amino Acids.

Teraryl-based α-helix mimetics have proven to be useful compounds for the inhibition of protein-protein interactions (PPI). We have developed a modular and flexible approach for the synthesis of teraryl-based α-helix mimetics using a benzene core unit featuring two halide leaving groups of differentiated reactivity in the Pd-catalyzed cross-coupling used for teraryl assembly. The use of para-bromo iodoarene core fragments resolved the issue of hydrolysis during cross-coupling that was observed when using triflate as a leaving group. We report a complete set of para-bromoiodoarene core fragments decorated with side chains of all proteinogenic amino acids relevant for PPI (Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr and Val). In order to be compatible with general cross-coupling conditions, some of the nucleophilic side chains had to be provided in a protected form to serve as stable building blocks.

A Modular Synthesis of Teraryl-Based α-Helix Mimetics, Part†5: A Complete Set of Pyridine Boronic Acid Pinacol Esters Featuring Side Chains of Proteinogenic Amino Acids.

Teraryl-based α-helix mimetics have proven to be useful compounds for the inhibition of protein-protein interactions (PPI). We have developed a modular and flexible approach for the synthesis of teraryl-based α-helix mimetics using pyridine containing boronic acid building blocks to increase the water solubility. Following our initial publication in which we have introduced the methodology in combination with sequential Pd-catalyzed cross-coupling for teraryl assembly, we can now report a complete set of pyridine based boronic acid building blocks decorated with side chains of all proteinogenic amino acids relevant for PPI (Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val) to complement the core fragment set. For a representative set of teraryls we have studied the influence of the pyridine rings on the solubility of the assembled oligoarenes.

A Modular Synthesis of Teraryl-Based α-Helix Mimetics, Part†3: Iodophenyltriflate Core Fragments Featuring Side Chains of Proteinogenic Amino Acids.

Teraryl-based α-helix mimetics have proven to be useful compounds for the inhibition of protein-protein interactions (PPI). We have developed a modular and flexible approach for the synthesis of teraryl-based α-helix mimetics using a benzene core unit featuring two leaving groups of differentiated reactivity in the Pd-catalyzed cross-coupling used for teraryl assembly. In previous publications we have introduced the methodology of 4-iodophenyltriflates decorated with the side chains of some of the proteinogenic amino acids. We herein report the core fragments corresponding to the previously missing amino acids Arg, Asn, Asp, Met, Trp and Tyr. Therefore, our set now encompasses all relevant amino acid analogues with the exception of His. In order to be compatible with the triflate moiety, some of the nucleophilic side chains had to be provided in a protected form to serve as stable building blocks. Additionally, cross-coupling procedures for the assembly of teraryls were investigated.

Klebsiella oxytoca enterotoxins tilimycin and tilivalline have distinct host DNA-damaging and microtubule-stabilizing activities.

Establishing causal links between bacterial metabolites and human intestinal disease is a significant challenge. This study reveals the molecular basis of antibiotic-associated hemorrhagic colitis (AAHC) caused by intestinal resident Klebsiella oxytoca Colitogenic strains produce the nonribosomal peptides tilivalline and tilimycin. Here, we verify that these enterotoxins are present in the human intestine during active colitis and determine their concentrations in a murine disease model. Although both toxins share a pyrrolobenzodiazepine structure, they have distinct molecular targets. Tilimycin acts as a genotoxin. Its interaction with DNA activates damage repair mechanisms in cultured cells and causes DNA strand breakage and an increased lesion burden in cecal enterocytes of colonized mice. In contrast, tilivalline binds tubulin and stabilizes microtubules leading to mitotic arrest. To our knowledge, this activity is unique for microbiota-derived metabolites of the human intestine. The capacity of both toxins to induce apoptosis in intestinal epithelial cells-a hallmark feature of AAHC-by independent modes of action, strengthens our proposal that these metabolites act collectively in the pathogenicity of colitis.

Inhibition of ATGL in adipose tissue ameliorates isoproterenol-induced cardiac remodeling by reducing adipose tissue inflammation.

Following cardiac injury, increased adrenergic drive plays an important role in compensating for reduced cardiac function. However, chronic excess adrenergic stimulation can be detrimental to cardiac pathophysiology and can also affect other organs including adipose tissue, leading to increased lipolysis. Interestingly, inhibition of adipose triglyceride lipase (ATGL), a rate-limiting enzyme in lipolysis, in adipocytes ameliorates cardiac dysfunction in a heart failure model. Thus, we investigated whether inhibition of adipocyte ATGL can mitigate the adverse cardiac effects of chronic adrenergic stimulation and explored the underlying mechanisms. To do this, isoproterenol (ISO) was continuously administered to C57Bl/6N mice for 2 wk with or without an ATGL inhibitor (Atglistatin). We found that Atglistatin alleviated ISO-induced cardiac remodeling and reduced ISO-induced upregulation of galectin-3, a marker of activated macrophages and a potent inducer of fibrosis, in white adipose tissue (WAT), heart, and the circulation. To test whether the beneficial effects of Atglistatin occur via inhibition of adipocyte ATGL, adipocyte-specific ATGL knockout (atATGL-KO) mice were utilized for similar experiments. Subsequently, the same cardioprotective effects of atATGL-KO following ISO administration were observed. Furthermore, Atglistatin and atATGL-KO abolished ISO-induced galectin-3 secretion from excised WAT. We further demonstrated that activation of cardiac fibroblasts by the conditioned media of ISO-stimulated WAT is galectin-3-dependent. In conclusion, the inhibition of adipocyte ATGL ameliorated ISO-induced cardiac remodeling possibly by reducing galectin-3 secretion from adipose tissue. Thus, inhibition of adipocyte ATGL might be a potential target to prevent some of the adverse effects of chronic excess adrenergic drive.NEW & NOTEWORTHY The reduction of lipolysis by adipocyte ATGL inhibition ameliorates cardiac remodeling induced by chronic ß-adrenergic stimulation likely via reducing galectin-3 secretion from adipose tissue. Our findings highlight that suppressing lipolysis in adipocytes may be a potential therapeutic target for patients with heart failure whose sympathetic nervous system is activated. Furthermore, galectin-3 might be involved in the mechanisms by which excessive lipolysis in adipose tissues influences remote cardiac pathologies and thus warrants further investigation.

Activity-Based Protein Profiling (ABPP) of Oxidoreductases.

Over the last two decades, activity-based protein profiling (ABPP) has been established as a tremendously useful proteomic tool for measuring the activity of proteins in their cellular context, annotating the function of uncharacterized proteins, and investigating the target profile of small-molecule inhibitors. Unlike hydrolases and other enzyme classes, which exhibit a characteristic nucleophilic residue, oxidoreductases have received much less attention in ABPP. In this minireview, the state of the art of ABPP of oxidoreductases is described and the scope and limitations of the existing approaches are discussed. It is noted that several ABPP probes have been described for various oxidases, but none so far for a reductase, which gives rise to opportunities for future research.

Efficient Entropy-Driven Inhibition of Dipeptidyl Peptidase III by Hydroxyethylene Transition-State Peptidomimetics.

Dipeptidyl peptidase III (DPP3) is a ubiquitously expressed Zn-dependent protease, which plays an important role in regulating endogenous peptide hormones, such as enkephalins or angiotensins. In previous biophysical studies, it could be shown that substrate binding is driven by a large entropic contribution due to the release of water molecules from the closing binding cleft. Here, the design, synthesis and biophysical characterization of peptidomimetic inhibitors is reported, using for the first time an hydroxyethylene transition-state mimetic for a metalloprotease. Efficient routes for the synthesis of both stereoisomers of the pseudopeptide core were developed, which allowed the synthesis of peptidomimetic inhibitors mimicking the VVYPW-motif of tynorphin. The best inhibitors inhibit DPP3 in the low µM range. Biophysical characterization by means of ITC measurement and X-ray crystallography confirm the unusual entropy-driven mode of binding. Stability assays demonstrated the desired stability of these inhibitors, which efficiently inhibited DPP3 in mouse brain homogenate.

Synthesis of Hydrophilic Phosphorus Ligands and Their Application in Aqueous-Phase Metal-Catalyzed Reactions.

Transition metal-catalyzed reactions in aqueous media are experiencing a constant increase in interest. In homogenous catalysis the use of water as a solvent offers advantages in cost, safety, the possibility of two-phase catalysis and simplified separation strategies. In the life sciences, transition metal catalysis in aqueous systems enables the ligation or modification of biopolymers in buffer systems or even in their cellular environment. In biocatalysis, aqueous systems allow the simultaneous use of enzymes and transition metal catalysts in cascade reactions. The use of water-soluble phosphine ligands still represents the most reliable and popular strategy for transferring metal catalysts into the aqueous phase. This review summarizes the recent advancements in this field since 2009 and describes current synthetic strategies for the preparation of hydrophilic phosphines and phosphites. In addition, recent applications of transition metal catalysis in aqueous solvents using these hydrophilic ligands are presented.

Pd/BIPHEPHOS is an Efficient Catalyst for the Pd-Catalyzed S-Allylation of Thiols with High n-Selectivity.

The Pd-catalyzed S-allylation of thiols with stable allylcarbonate and allylacetate reagents offers several advantages over established reactions for the formation of thioethers. We could demonstrate that Pd/BIPHEPHOS is a catalyst system which allows the transition metal-catalyzed S-allylation of thiols with excellent n-regioselectivity. Mechanistic studies showed that this reaction is reversible under the applied reaction conditions. The excellent functional group tolerance of this transformation was demonstrated with a broad variety of thiol nucleophiles (18 examples) and allyl substrates (9 examples), and could even be applied for the late-stage diversification of cephalosporins, which might find application in the synthesis of new antibiotics.

Structure-activity relationship studies for the development of inhibitors of murine adipose triglyceride lipase (ATGL).

High serum fatty acid (FA) levels are causally linked to the development of insulin resistance, which eventually progresses to type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) generalized in the term metabolic syndrome. Adipose triglyceride lipase (ATGL) is the initial enzyme in the hydrolysis of intracellular triacylglycerol (TG) stores, liberating fatty acids that are released from adipocytes into the circulation. Hence, ATGL-specific inhibitors have the potential to lower circulating FA concentrations, and counteract the development of insulin resistance and NAFLD. In this article, we report about structure-activity relationship (SAR) studies of small molecule inhibitors of murine ATGL which led to the development of Atglistatin. Atglistatin is a specific inhibitor of murine ATGL, which has proven useful for the validation of ATGL as a potential drug target.

Metabolic disease and ABHD6 alter the circulating bis(monoacylglycerol)phosphate profile in mice and humans.

Bis(monoacylglycerol)phosphate (BMP) is a phospholipid that is crucial for lipid degradation and sorting in acidic organelles. Genetic and drug-induced lysosomal storage disorders (LSDs) are associated with increased BMP concentrations in tissues and in the circulation. Data on BMP in disorders other than LSDs, however, are scarce, and key enzymes regulating BMP metabolism remain elusive. Here, we demonstrate that common metabolic disorders and the intracellular BMP hydrolase α/ß-hydrolase domain-containing 6 (ABHD6) affect BMP metabolism in mice and humans. In mice, dietary lipid overload strongly affects BMP concentration and FA composition in the liver and plasma, similar to what has been observed in LSDs. Notably, distinct changes in the BMP FA profile enable a clear distinction between lipid overload and drug-induced LSDs. Global deletion of ABHD6 increases circulating BMP concentrations but does not cause LSDs. In humans, nonalcoholic fatty liver disease and liver cirrhosis affect the serum BMP FA composition and concentration. Furthermore, we identified a patient with a loss-of-function mutation in the ABHD6 gene, leading to an altered circulating BMP profile. In conclusion, our results suggest that common metabolic diseases and ABHD6 affect BMP metabolism in mice and humans.

Labeling and Natural Post-Translational Modification of Peptides and Proteins via Chemoselective Pd-Catalyzed Prenylation of Cysteine.

The prenylation of peptides and proteins is an important post-translational modification observed in vivo. We report that the Pd-catalyzed Tsuji-Trost allylation with a Pd/BIPHEPHOS catalyst system allows the allylation of Cys-containing peptides and proteins with complete chemoselectivity and high n/i regioselectivity. In contrast to recently established methods, which use non-native connections, the Pd-catalyzed prenylation produces the natural n-prenylthioether bond. In addition, a variety of biophysical probes such as affinity handles and fluorescent tags can be introduced into Cys-containing peptides and proteins. Furthermore, peptides containing two cysteine residues can be stapled or cyclized using homobifunctional allylic carbonate reagents.

Selective Formation of 4,4'-Biphenols by Anodic Dehydrogenative Cross- and Homo-Coupling Reaction.

A simple and selective electrochemical synthesis by dehydrogenative coupling of unprotected 2,6- or 2,5-substituted phenols to the desired 4,4'-biphenols is reported. Using electricity as the oxidizing reagent avoids pre-functionalization of the starting materials, since a selective activation of the substrates takes place. Without the necessity for metal-catalysts or the use of stoichiometric reagents it is an economic and environmentally friendly transformation. The elaborated electrochemical protocol leads to a broad variety of the desired 4,4'-biphenols in a very simplified manner compared to classical approaches. This is particular the case for the cross-coupled products.

Atglistatin ameliorates functional decline in heart failure via adipocyte-specific inhibition of adipose triglyceride lipase.

Despite advancements in therapies for cardiovascular disease and heart failure (HF), the incidence and prevalence of HF are increasing. Previous work has suggested that inhibiting adipose triglyceride lipase (ATGL) in adipose tissue during HF development may assist in the treatment of HF. The ability to specifically target the adipocyte as a potential treatment for HF is a novel approach that could significantly influence the management of HF in the future. Our objectives were to assess the cardiac structural and functional effects of pharmacological inhibition of ATGL in mice with HF, to assess whether ATGL inhibition works in an adipocyte-autonomous manner, and to determine the role that adiposity and glucose homeostasis play in this HF treatment approach. Using a known ATGL inhibitor, atglistatin, as well as mice with germline deletion of adipocyte-specific ATGL, we tested the effectiveness of ATGL inhibition in mice with pressure overload-induced HF. Here, we show that atglistatin can prevent the functional decline in HF and provide evidence that specifically targeting ATGL in the adipocyte is sufficient to prevent worsening of HF. We further demonstrate that the benefit resulting from atglistatin in HF is not dependent on previously suggested improvements in glucose homeostasis, nor are the benefits derived from increased adiposity. Overall, the results of this study suggest that adipocyte-specific pharmacological inhibition of ATGL may represent a novel therapeutic option for HF. NEW & NOTEWORTHY This work shows for the first time that the adipose triglyceride lipase (ATGL)-specific inhibitor atglistatin can prevent worsening heart failure. Furthermore, using mice with adipocyte-specific ATGL ablation, this study demonstrates that ATGL inhibition works in an adipocyte-autonomous manner to ameliorate a functional decline in heart failure. Overall, this work demonstrates that specifically targeting the adipocyte to inhibit ATGL is a potential treatment for heart failure.

Asymmetric Reductive Carbocyclization Using Engineered Ene Reductases.

Ene reductases from the Old Yellow Enzyme (OYE) family reduce the C=C double bond in α,ß-unsaturated compounds bearing an electron-withdrawing group, for example, a carbonyl group. This asymmetric reduction has been exploited for biocatalysis. Going beyond its canonical function, we show that members of this enzyme family can also catalyze the formation of C-C bonds. α,ß-Unsaturated aldehydes and ketones containing an additional electrophilic group undergo reductive cyclization. Mechanistically, the two-electron-reduced enzyme cofactor FMN delivers a hydride to generate an enolate intermediate, which reacts with the internal electrophile. Single-site replacement of a crucial Tyr residue with a non-protic Phe or Trp favored the cyclization over the natural reduction reaction. The new transformation enabled the enantioselective synthesis of chiral cyclopropanes in up to >99 % ee.

Crystal structure of the Saccharomyces cerevisiae monoglyceride lipase Yju3p.

Monoglyceride lipases (MGLs) are a group of α/ß-hydrolases that catalyze the hydrolysis of monoglycerides (MGs) into free fatty acids and glycerol. This reaction serves different physiological functions, namely in the last step of phospholipid and triglyceride degradation, in mammalian endocannabinoid and arachidonic acid metabolism, and in detoxification processes in microbes. Previous crystal structures of MGLs from humans and bacteria revealed conformational plasticity in the cap region of this protein and gave insight into substrate binding. In this study, we present the structure of a MGL from Saccharomyces cerevisiae called Yju3p in its free form and in complex with a covalently bound substrate analog mimicking the tetrahedral intermediate of MG hydrolysis. These structures reveal a high conservation of the overall shape of the MGL cap region and also provide evidence for conformational changes in the cap of Yju3p. The complex structure reveals that, despite the high structural similarity, Yju3p seems to have an additional opening to the substrate binding pocket at a different position compared to human and bacterial MGL. Substrate specificities towards MGs with saturated and unsaturated alkyl chains of different lengths were tested and revealed highest activity towards MG containing a C18:1 fatty acid.