The pro-drug C13 activates AMPK by two distinct mechanisms.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is expressed in almost all eukaryotic cells. In the canonical activation mechanism, it is activated by increases in AMP:ATP and ADP:ATP ratios that signify declining cellular energy status. Once activated, AMPK phosphorylates numerous targets that promote catabolic pathways generating ATP, while inhibiting anabolic and other processes that consume ATP, thus acting to restore energy homeostasis. Pharmacological agents that activate AMPK have been useful in identifying downstream targets and have potential as drugs for treatment of metabolic disorders such as Type 2 diabetes and non-alcoholic fatty liver disease. One such agent is C13, a pro-drug with a phosphonate bis(isobutyryloxymethyl) ester moiety, with the isobutyryloxymethyl groups increasing membrane permeability. Following cellular uptake, C13 is cleaved to release C2, an AMP analogue and potent AMPK activator that is specific for complexes containing the α1 (but not the α2) catalytic subunit isoform. This has previously been assumed to be the sole mechanism by which C13 activates AMPK, with potential roles for the isobutyryloxymethyl groups being ignored. We now report that, following cleavage from C13, these protective groups are metabolized to formaldehyde, an agent that inhibits mitochondrial function and increases cellular AMP:ATP ratios, thus providing additional AMPK activation by the canonical mechanism.

Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2.

The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.

Traceless Thioacid-Mediated Radical Cyclization of 1,6-Dienes.

Five-membered ring systems are ubiquitous throughout natural products and synthetic therapeutics, and thus, efficient methods to access this essential scaffold are required. Herein, we report the thioacid-mediated, 5-exo-trig cyclization of various 1,6-dienes, with high yields of up to 98%. The labile thioester functionality can be exploited to generate a free thiol residue which can be used as a functional handle or removed entirely to provide the traceless cyclized product.

Glycosylated quantum dots as fluorometric nanoprobes for trehalase.

Trehalase is an important enzyme in the metabolic cascades of many organisms, catalysing the hydrolysis of the disaccharide trehalose. Herein we describe the first examples of fluorometric nanoprobes for detection of trehalase, based on trehalose-functionalised quantum dots (QDs). QDs cross-linked with trehalose form aggregates, which are released upon enzymatic cleavage of the trehalose glycosidic bond proportionally to the enzyme concentration, offering a unique and efficient approach for specific sensing of this biologically important enzyme.

Real-Time Multi-Photon Tracking and Bioimaging of Glycosylated Theranostic Prodrugs upon Specific Enzyme Triggered Release.

Real-time tracking of prodrug uptake, delivery and activation in vivo represents a major challenge for prodrug development. Herein, we demonstrate the use of novel glycosylated theranostics of the cancer pharmacophore Amonafide in highly-selective, enzymatic triggered release. We show that the use of endogenous enzymes for activated release of the therapeutic component can be observed, in real time, and monitored using one and two-photon bioimaging, offering unique insight into the prodrug pharmacokinetic profile. Furthermore, we demonstrate that the potent cytotoxicity of Amonafide is preserved using this targeted approach.

Mycobacterial para-Hydroxybenzoic Acid-Derivatives (pHBADs) and Related Structures Induce Macrophage Innate Memory.

Macrophages are key immune cells for combatting Mycobacterium tuberculosis. However, M. tuberculosis possesses means to evade macrophage bactericidal responses by, for instance, secretion of the immunomodulatory para-hydroxybenzoic acid derivatives (pHBADs). While these molecules have been implicated in inhibiting macrophage responses in an acute context, little is known about their ability to reprogram macrophages via induction of long-term innate memory. Since innate memory has been highlighted as a promising strategy to augment bactericidal immune responses against M. tuberculosis, investigating corresponding immune evasion mechanisms is highly relevant. Our results reveal for the first time that pHBAD I and related molecules (unmethylated pHBAD I and the hexose l-rhamnose) reduce macrophage bactericidal mechanisms in both the short- and the long-term. Moreover, we demonstrate how methyl-p-anisate hinders bactericidal responses soon after exposure yet results in enhanced pro-inflammatory responses in the long-term. This work highlights new roles for these compounds in M. tuberculosis pathogenesis.

Thiyl Radicals: Versatile Reactive Intermediates for Cyclization of Unsaturated Substrates.

Sulfur centered radicals are widely employed in chemical synthesis, in particular for alkene and alkyne hydrothiolation towards thioether bioconjugates. The steadfast radical chain process that enables efficient hydrothiolation has been explored in the context of cascade reactions to furnish complex molecular architectures. The use of thiyl radicals offers a much cheaper and less toxic alternative to the archetypal organotin-based radical methods. This review outlines the development of thiyl radicals as reactive intermediates for initiating carbocyclization cascades. Key developments in cascade cyclization methodology are presented and applications for natural product synthesis are discussed. The review provides a chronological account of the field, beginning in the early seventies up to very recent examples; a span of almost 50 years.