SUMMARY Protein-protein interactions (PPIs) play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable weak PPI detection in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL’s ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a new class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL’s ability to expedite PPI analysis, assess target specificity and cell permeability, and to reveal off-target effects of PPI modifiers should facilitate development of effective, cell permeable PPI therapeutics and elaboration of diverse biological mechanisms.

A unique natural product termed palmerolide A was recently isolated from the Antarctic marine tunicate Synoicum adareanum by Baker and coworkers. 1 The structure and absolute stereochemistry of palmerolide A, expressed by formula 1 (Scheme 1), was solved by NMR studies and reveals a 20-membered macrolide decorated with a poly-unsaturated N-acyl dienamine side chain distinct from the N-acyl enamine found in salicylihalamide and related natural products. 2 Palmerolide is a differential cytotoxin with an activity profile that correlates to VATPase inhibitors. 1 Subsequent in vitro studies confirmed that palmerolide A is indeed a potent inhibitor of bovine brain VATPase (IC 50 ∼2 nM). 1, 3 Palmerolide was isolated from an organism found in one of the most inaccessible areas of the world, and in conjunction with commercial exploitation prohibited by the 'Antarctic Treaty' will severely limit access to this compound from natural sources. 4 Therefore, total synthesis remains as the only option to assure investigation of palmerolide's promising antitumor properties. Herein, we present our synthetic efforts that led to the conclusion that (-)-palmerolide A is in fact a diastereomer of the proposed structure 1, i.e. structure ent-24 (Scheme 4).We envisioned constructing palmerolide from fragments 2-4 via cross-coupling (3 + 4), followed by esterification with enoic acid 2 -a fragment with a keto-phosphonate positioned to induce macrocyclization via Horner-Wadsworth-Emmons olefination (Scheme 1). A Curtius rearrangement followed by isocyanate-trapping with 2-Me-propenylmagnesium bromide will install the sensitive N-acyl dienamine functionality at the final stages. 5The synthesis of fragment 3 began by a vinylogous Mukaiyama aldol addition of vinylketene silyl N,O-acetal 5 6 to aldehyde 6 7 according to Kobayashi et al. 6 to furnish alcohol 7 in excellent yield and diastereoselectivity (Scheme 2). 8 A Mitsunobu inversion provided the desired syn-isomer 8. Simultaneous reductive cleavage of the auxiliary and the benzoate provided an aldehyde that was homologated with PPh 3 CHCO 2 Me. Cross-coupling partner 3 was thus obtained in 49% overall yield from 5.The synthetic equivalent of fragment 4, alkenylboronate 13, was derived from D-arabitol. Benzylidene acetal formation and oxidative α-diol cleavage yielded aldehyde 9 (Scheme 3). 9 Silylation, Wittig homologation and hydrogenation gave diol 11. Bis-silylether formation and ester reduction then provided aldehyde 12 -a material that was condensed with pinacol dichloromethylboronate (CrCl 2 , LiI) to yield vinylboronate 13. 10 E-mail: jef.debrabander@utsouthwestern.edu. Supporting Information Available: Experimental procedures and characterization data for new compounds (PDF, CIF). This material is available free of charge via the Internet at http://pubs.acs.org. propenylmagnesium bromide to the isocyanate generated from heating acyl azide 21 yielded 22 (76%). NIH Public AccessSelective trimethylsilyl ether deprotection enabled introduction of the carbamate at C11 (23, 95%) 18 ...

The natural product salicylihalamide is a potent inhibitor of the Vacuolar ATPase (V-ATPase), a potential target for antitumor chemotherapy. We generated salicylihalamide-resistant tumor cell lines typified by an overexpansion of lysosomal organelles. We also found that many tumor cell lines upregulate tissue-specific plasmalemmal V-ATPases, and hypothesize that tumors that derive their energy from glycolysis rely on these isoforms to maintain a neutral cytosolic pH. To further validate the potential of V-ATPase inhibitors as leads for cancer chemotherapy, we developed a multigram synthesis of the potent salicylihalamide analog saliphenylhalamide.As products of evolution, natural products are selected for interaction with living systems. As such, an unbiased quest to study their function will inevitably lead to discoveries in biology, potentially with therapeutic implications. 1 By accessing congeners for mode-of-action studies, optimization of potency and pharmacokinetic, toxicological, and metabolic properties, synthesis takes center stage as an enabling tool to execute a natural product-based discovery and development program. 2 Herein, we report our efforts to validate inhibition of the Vacuolar ATPase (V-ATPase), the target of salicylihalamide, as a strategy for cancer chemotherapeutic intervention. This program led to the selection and multigram synthesis of a salicylihalamide analog saliphenylhalamide (2, SaliPhe).The marine metabolite salicylihalamide A (1), 3 the first member of a family of marine and terrestrial metabolites characterized by a signature N-acyl-enamine appended macrocyclic salicylate, has elicited a great deal of interest from the synthetic community 4 -certainly due in part because of their growth-inhibitory activities against cultured human tumor cells and oncogene-transformed cell lines through mechanisms distinct from standard clinical antitumor agents. 5 The cellular target of SaliA remained elusive until after our first total synthesis, 3b when Boyd and coworkers reported that SaliA and other related benzolactone enamides inhibit V-ATPase activity in membrane preparations of mammalian cells, but not V-ATPases from Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript yeast and other fungi -an observation that distinguishes them from previously identified VATPase inhibitors. 6 Our biochemical studies utilizing a reconstituted, fully purified bovine brain V-ATPase confirmed this activity and demonstrated that SaliA binds irreversibly to the trans-mem...

Solid-state fermentation (SSF) was employed to enhance the nutritive values of palm kernel cake (PKC) for poultry feeding. Aspergillus flavus was isolated from local PKC and utilized to increase the mannose content of PKC via the degradation of ß-mannan in PKC; evaluation was done for batch SSF in Erlenmeyer flasks and in a novel laterally aerated moving bed (LAMB) bioreactor. The optimum condition for batch SSF in flasks was 110% initial moisture content, initial pH 6.0, 30 °C, 855 µm particle size, and 120 h of fermentation, yielding 90.91 mg mannose g-1 dry PKC (5.9-fold increase). Batch SSF in the LAMB at the optimum condition yielded 79.61 mg mannose g-1 dry PKC (5.5-fold increase) within just 96 h due to better heat and mass transfer when humidified air flowed radially across the PKC bed. In spite of a compromise of 12% reduction in mannose content when compared with the flasks, the LAMB facilitated good heat and mass transfer, and improved the mannose content of PKC in a shorter fermentation period. These attributes are useful for batch production of fermented PKC feed in an industrial scale.

The therapeutic success of peptidic GLP-1 receptor agonists for treatment of type 2 diabetes mellitus (T2DM) motivated our search for orally bioavailable small molecules that can activate the GLP-1 receptor (GLP-1R) as a well-validated target for T2DM. Here, the discovery and characterization of a potent and selective positive allosteric modulator (PAM) for GLP-1R based on a 3,4,5,6-tetrahydro-1H-1,5-epiminoazocino­[4,5-b]­indole scaffold is reported. Optimization of this series from HTS was supported by a GLP-1R ligand binding model. Biological in vitro testing revealed favorable ADME and pharmacological profiles for the best compound 19. Characterization by in vivo pharmacokinetic and pharmacological studies demonstrated that 19 activates GLP-1R as positive allosteric modulator (PAM) in the presence of the much less active endogenous degradation product GLP1(9–36)­NH2 of the potent endogenous ligand GLP-1(7–36)­NH2. While these data suggest the potential of small molecule GLP-1R PAMs for T2DM treatment, further optimization is still required towards a clinical candidate.