Site-selective, stereocontrolled glycosylation of minimally protected sugars.

The identification of general and efficient methods for the construction of oligosaccharides stands as one of the great challenges for the field of synthetic chemistry1,2. Selective glycosylation of unprotected sugars and other polyhydroxylated nucleophiles is a particularly significant goal, requiring not only control over the stereochemistry of the forming bond but also differentiation between similarly reactive nucleophilic sites in stereochemically complex contexts3,4. Chemists have generally relied on multi-step protecting-group strategies to achieve site control in glycosylations, but practical inefficiencies arise directly from the application of such approaches5-7. Here we describe a strategy for small-molecule-catalyst-controlled, highly stereo- and site-selective glycosylations of unprotected or minimally protected mono- and disaccharides using precisely designed bis-thiourea small-molecule catalysts. Stereo- and site-selective galactosylations and mannosylations of a wide assortment of polyfunctional nucleophiles is thereby achieved. Kinetic and computational studies provide evidence that site-selectivity arises from stabilizing C-H/π interactions between the catalyst and the nucleophile, analogous to those documented in sugar-binding proteins. This work demonstrates that highly selective glycosylation reactions can be achieved through control of stabilizing non-covalent interactions, a potentially general strategy for selective functionalization of carbohydrates.

Unifying Catalysis Framework to Dissect Proteasomal Degradation Paradigms.

Diverging from traditional target inhibition, proteasomal protein degradation approaches have emerged as novel therapeutic modalities that embody distinct pharmacological profiles and can access previously undrugged targets. Small molecule degraders have the potential to catalytically destroy target proteins at substoichiometric concentrations, thus lowering administered doses and extending pharmacological effects. With this mechanistic premise, research efforts have advanced the development of small molecule degraders that benefit from stable and increased affinity ternary complexes. However, a holistic framework that evaluates different degradation modes from a catalytic perspective, including focusing on kinetically favored degradation mechanisms, is lacking. In this Outlook, we introduce the concept of an induced cooperativity spectrum as a unifying framework to mechanistically understand catalytic degradation profiles. This framework is bolstered by key examples of published molecular degraders extending from molecular glues to bivalent degraders. Critically, we discuss remaining challenges and future opportunities in drug discovery to rationally design and phenotypically screen for efficient degraders.

Discovery of Diaminopyrimidine Carboxamide HPK1 Inhibitors as Preclinical Immunotherapy Tool Compounds.

Hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase, is a negative immune regulator of T cell receptor (TCR) and B cell signaling that is primarily expressed in hematopoietic cells. Accordingly, it has been reported that HPK1 loss-of-function in HPK1 kinase-dead syngeneic mouse models shows enhanced T cell signaling and cytokine production as well as tumor growth inhibition in vivo, supporting its value as an immunotherapeutic target. Herein, we present the structurally enabled discovery of novel, potent, and selective diaminopyrimidine carboxamide HPK1 inhibitors. The key discovery of a carboxamide moiety was essential for enhanced enzyme inhibitory potency and kinome selectivity as well as sustained elevation of cellular IL-2 production across a titration range in human peripheral blood mononuclear cells. The elucidation of structure-activity relationships using various pendant amino ring systems allowed for the identification of several small molecule type-I inhibitors with promising in vitro profiles.

Highly Selective ß-Mannosylations and ß-Rhamnosylations Catalyzed by Bis-thiourea.

We report highly ß-selective bis-thioureas-catalyzed 1,2-cis-O-pyranosylations employing easily accessible acetonide-protected donors. A wide variety of alcohol nucleophiles, including complex natural products, glycosides, and amino acids were ß-mannosylated and ß-rhamnosylated successfully using an operationally simple protocol under mild and neutral conditions. Less nucleophilic acceptors such as phenols were also glycosylated efficiently in excellent yields and with high ß-selectivities.

Reductive sp3-sp2 Coupling Reactions Enable Late-Stage Modification of Pharmaceuticals.

Late-stage derivatization of pharmaceutically relevant scaffolds relies on the availability of highly functional-group tolerant reactions. Reactions that increase the sp3 character of molecules enable the pursuit of more selective and well-tolerated pharmaceuticals. Herein, we report the use of sp3-sp2 cross-electrophile reductive couplings to modify a generic ATP-competitive kinase inhibitor with a broad range of primary and secondary alkyl halide coupling partners.

Catalytic activation of glycosyl phosphates for stereoselective coupling reactions.

Glycosyl phosphates are shown to be activated to stereospecific nucleophilic substitution reactions by precisely tailored bis-thiourea catalysts. Enhanced reactivity and scope is observed with phosphate relative to chloride leaving groups. Stronger binding (Km) to the H-bond donor and enhanced reactivity of the complex (kcat) enables efficient catalysis with broad functional group compatibility under mild, neutral conditions.

A Unified Approach to Targeting the Lysosome's Degradative and Growth Signaling Roles.

Lysosomes serve dual roles in cancer metabolism, executing catabolic programs (i.e., autophagy and macropinocytosis) while promoting mTORC1-dependent anabolism. Antimalarial compounds such as chloroquine or quinacrine have been used as lysosomal inhibitors, but fail to inhibit mTOR signaling. Further, the molecular target of these agents has not been identified. We report a screen of novel dimeric antimalarials that identifies dimeric quinacrines (DQ) as potent anticancer compounds, which concurrently inhibit mTOR and autophagy. Central nitrogen methylation of the DQ linker enhances lysosomal localization and potency. An in situ photoaffinity pulldown identified palmitoyl-protein thioesterase 1 (PPT1) as the molecular target of DQ661. PPT1 inhibition concurrently impairs mTOR and lysosomal catabolism through the rapid accumulation of palmitoylated proteins. DQ661 inhibits the in vivo tumor growth of melanoma, pancreatic cancer, and colorectal cancer mouse models and can be safely combined with chemotherapy. Thus, lysosome-directed PPT1 inhibitors represent a new approach to concurrently targeting mTORC1 and lysosomal catabolism in cancer.Significance: This study identifies chemical features of dimeric compounds that increase their lysosomal specificity, and a new molecular target for these compounds, reclassifying these compounds as targeted therapies. Targeting PPT1 blocks mTOR signaling in a manner distinct from catalytic inhibitors, while concurrently inhibiting autophagy, thereby providing a new strategy for cancer therapy. Cancer Discov; 7(11); 1266-83. ©2017 AACR.See related commentary by Towers and Thorburn, p. 1218This article is highlighted in the In This Issue feature, p. 1201.

Autophagy Inhibition Enhances Sunitinib Efficacy in Clear Cell Ovarian Carcinoma.

Clear cell ovarian carcinoma (CCOC) is an aggressive form of epithelial ovarian cancer that exhibits low response rates to systemic therapy and poor patient outcomes. Multiple studies in CCOC have revealed expression profiles consistent with increased hypoxia, and our previous data suggest that hypoxia is correlated with increased autophagy in CCOC. Hypoxia-induced autophagy is a key factor promoting tumor cell survival and resistance to therapy. Recent clinical trials with the molecular-targeted receptor tyrosine kinase (RTK) inhibitor sunitinib have demonstrated limited activity. Here, it was evaluated whether the hypoxia-autophagy axis could be modulated to overcome resistance to sunitinib. Importantly, a significant increase in autophagic activity was found with a concomitant loss in cell viability in CCOC cells treated with sunitinib. Pharmacologic inhibition of autophagy with the lysosomotropic analog Lys05 inhibited autophagy and enhanced sunitinib-mediated suppression of cell viability. These results were confirmed by siRNA targeting the autophagy-related gene Atg5 In CCOC tumor xenografts, Lys05 potentiated the antitumor activity of sunitinib compared with either treatment alone. These data reveal that CCOC tumors have an autophagic dependency and are an ideal tumor histotype for autophagy inhibition as a strategy to overcome resistance to RTK inhibitors like sunitinib.Implications: This study shows that autophagy inhibition enhances sunitinib-mediated cell death in a preclinical model of CCOC. Mol Cancer Res; 15(3); 250-8. ©2017 AACR.

Autophagy gene Atg16L1 prevents lethal T cell alloreactivity mediated by dendritic cells.

Atg16L1 mediates the cellular degradative process of autophagy and is considered a critical regulator of inflammation based on its genetic association with inflammatory bowel disease. Here we find that Atg16L1 deficiency leads to an exacerbated graft-versus-host disease (GVHD) in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Atg16L1-deficient allo-HSCT recipients with GVHD displayed increased T cell proliferation due to increased dendritic cell (DC) numbers and costimulatory molecule expression. Reduced autophagy within DCs was associated with lysosomal abnormalities and decreased amounts of A20, a negative regulator of DC activation. These results broaden the function of Atg16L1 and the autophagy pathway to include a role in limiting a DC-mediated response during inflammatory disease, such as GVHD.

Autophagy inhibitor Lys05 has single-agent antitumor activity and reproduces the phenotype of a genetic autophagy deficiency.

Autophagy is a lysosome-dependent degradative process that protects cancer cells from multiple stresses. In preclinical models, autophagy inhibition with chloroquine (CQ) derivatives augments the efficacy of many anticancer therapies, but CQ has limited activity as a single agent. Clinical trials are underway combining anticancer agents with hydroxychloroquine (HCQ), but concentrations of HCQ required to inhibit autophagy are not consistently achievable in the clinic. We report the synthesis and characterization of bisaminoquinoline autophagy inhibitors that potently inhibit autophagy and impair tumor growth in vivo. The structural motifs that are necessary for improved autophagy inhibition compared with CQ include the presence of two aminoquinoline rings and a triamine linker and C-7 chlorine. The lead compound, Lys01, is a 10-fold more potent autophagy inhibitor than HCQ. Compared with HCQ, Lys05, a water-soluble salt of Lys01, more potently accumulates within and deacidifies the lysosome, resulting in impaired autophagy and tumor growth. At the highest dose administered, some mice develop Paneth cell dysfunction that resembles the intestinal phenotype of mice and humans with genetic defects in the autophagy gene ATG16L1, providing in vivo evidence that Lys05 targets autophagy. Unlike HCQ, significant single-agent antitumor activity is observed without toxicity in mice treated with lower doses of Lys05, establishing the therapeutic potential of this compound in cancer.

Palladium-catalyzed C-CN activation for intramolecular cyanoesterification of alkynes.

Conditions for the C-CN activation and intramolecular cyanoesterification of alkynes to provide butenolides in good to excellent yields are presented. Pd catalysts, high temperatures/short reaction times (microwave irradiation), and Lewis basic solvents minimized competitive decarbonylation. Less sterically encumbered, electron-rich alkynes underwent cyanoesterification with greater ease compared to sterically encumbered, electron-deficient alkynes. The results led to the hypothesis that migratory insertion of the alkyne, rather than C-CN activation, might be the product-determining step.