Fat and muscle lipolysis involves functional interactions of adipose triglyceride lipase (ATGL), α-β hydrolase domain-containing protein 5 (ABHD5), and tissue-specific perilipins 1 and 5 (PLIN1 and ...PLIN5). ABHD5 potently activates ATGL, but this lipase-promoting activity is suppressed when ABHD5 is bound to PLIN proteins on lipid droplets. In adipocytes, protein kinase A (PKA) phosphorylation of PLIN1 rapidly releases ABHD5 to activate ATGL, but mechanisms for rapid regulation of PLIN5-ABHD5 interaction in muscle are unknown. Here, we identify synthetic ligands that release ABHD5 from PLIN1 or PLIN5 without PKA activation and rapidly activate adipocyte and muscle lipolysis. Molecular imaging and affinity probe labeling demonstrated that ABHD5 is directly targeted by these synthetic ligands and additionally revealed that ABHD5-PLIN interactions are regulated by endogenous ligands, including long-chain acyl-CoA. Our results reveal a new locus of lipolysis control and suggest ABHD5 ligands might be developed into novel therapeutics that directly promote fat catabolism.
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•Synthetic ABHD5 ligands prevent ABHD5 interaction with PLIN1 or PLIN5•ABHD5 ligands stimulate lipolysis independently of PKA activation•Long-chain acyl-CoAs bind ABHD5 and promote its interaction with PLIN proteins•Synthetic ABHD5 ligands might be exploited for therapeutic benefit
Lipolysis involves interactions between ATGL, ABHD5, and PLIN1 or PLIN5. Sanders et al. identify synthetic ligands that quickly release ABHD5 from PLIN proteins to directly activate fat and muscle lipolysis, bypassing canonical PKA-dependent signaling. Long-chain acyl-CoAs act as endogenous allosteric ligands of ABHD5 to suppress lipolysis.
The enantioconvergent hydroboration of racemic allenylstannane (±)-1 with ( d Ipc)2BH converts both enantiomers of (±)-1 into the enantioenriched crotylborane (S)-E-3. Subsequent crotylboration of ...aldehydes with (S)-E-3 provides (E)-stannyl-homoallylic alcohols 5 in good yields and with excellent enantioselectivity.
Structure based design has been widely used in many drug development programs. In parallel with the evolution of high performance computing systems and versatile molecular modeling programs, ...structure based drug design has become indispensible in many research areas. CYP51 is a proven therapeutic target for anti-fungal drugs. While anti-fungal drugs targeting CYP51 have a long history and a large pool of anti-fungal CYP51 inhibitor therapeutics are now available, structure based design of therapeutic agents targeting CYP51 has only recently been attempted, Here, we present structural features of CYP51 and its complexes formed with lanosterol, azole drugs, and specifically designed inhibitors. In particular, the first x-ray co-crystal structures of fungal CYP51 complexed with lanosterol and itraconazole are compared with co-crystal structures of other protozoal CYP51 enzymes. It is anticipated that comparative analyses of these structures, and other structures that emerge in coming years, will provide clear rationales to address issues in the development of CYP51 drug candidates such as drug resistant, selectivity against other human CYP enzymes, and diversity of CYP51 inhibitors.
Monoclonal antibody h38C2 is a humanized catalytic antibody that has been used to generate various immunoconjugate species such as chemically programmed antibodies, antibody–drug conjugates, and ...antibody–siRNA conjugates. Highly efficient and specific conjugation of h38C2 occurs at its uniquely reactive lysine (Lys) residue buried inside the antibody’s catalytic pocket. We recently reported the rational mutation of this Lys residue at position 99 in the heavy chain variable domain to an arginine (Arg) residue. The Lys99Arg mutation can be site-selectively conjugated with molecules containing a hapten-like triazolyl-phenylglyoxal (TPG) unit. Here we show that this conjugation is facilitated by the unusual pH-sensitive reactivity of the Arg99 residue, consistent with an indirectly measured pK a of 5.2. The Arg99/TPG conjugation holds promise to further expand the versatility of the h38C2 conjugation platform, such as for the generation of antibody conjugates with dual payloads.
Catalytic antibody 38C2 and its humanized version h38C2 harbor a uniquely reactive lysine at the bottom of a 11 Å deep pocket that permits site-specific conjugation of β-diketone-, β-lactam-, and ...heteroaryl methylsulfonyl-functionalized small and large molecules. Various dual variable domain formats pair a tumor-targeting antibody with h38C2 to enable precise, fast, and stable assembly of antibody-drug conjugates (ADCs). Here, we expand the scope of this ADC assembly strategy by mutating h38C2’s reactive lysine to a cysteine. X-ray crystallography of this point mutant, h38C2_K99C, confirmed a deeply buried unpaired cysteine. Probing h38C2_K99C with maleimide, monobromomaleimide, and dibromomaleimide derivatives of a fluorophore revealed highly disparate conjugation efficiencies and stabilities. Dibromomaleimide emerged as a suitable electrophile for the precise, fast, efficient, and stable assembly of ADCs with the h38C2_K99C module. Mass spectrometry indicated the presence of a thio-monobromomaleimide linkage which was further supported by in silico docking studies. Using a dibromomaleimide derivative of the highly potent tubulin polymerization inhibitor monomethyl auristatin F, h38C2_K99C-based ADCs were found to be as potent as h38C2-based ADCs and afford a new assembly route for ADCs with single and dual payloads.
Highly enantio‐ and diastereoselective (Ipc)2BOTf mediated aldol reactions of morpholine carboxamides are described. A wide variety of α‐substituted N‐acyl morpholine carboxamides were successfully ...employed, including α‐bromo, α‐chloro, α‐vinyl and para‐methoxyphenyl morpholine carboxamides which provided the corresponding aldol products in moderate to excellent yields, and generally with high enantio‐ and diastereoselectivities.
On all fours: The title reaction with (Ipc)2BH provides tetrasubstituted enolborinates which undergo aldol reactions with aldehydes to form products with all‐carbon quaternary centers with ...exceptional diastereo‐ and enantioselectivity. A change to the substitution pattern of the starting amide leads to either diastereomer of the α‐methyl‐α‐ethyl‐β‐hydroxy carboxamide (1 or 2).
Chemically programmed bispecific antibodies (biAbs) endow target cell-binding small molecules with the ability to recruit and activate effector cells of the immune system. Here we report a platform ...of chemically programmed biAbs aimed at redirecting cytotoxic T cells to eliminate cancer cells. Two different antibody technologies were merged together to make a novel chemically programmed biAb. This was achieved by combining the humanized anti-hapten monoclonal antibody (mAb) h38C2 with the humanized anti-human CD3 mAb v9 in a clinically investigated diabody format known as Dual-Affinity Re-Targeting (DART). We show that h38C2 × v9 DARTs can readily be equipped with tumor-targeting hapten-derivatized small molecules without causing a systemic response harming healthy tissues. As a proof of concept, we chemically programmed h38C2 × v9 with hapten-folate and demonstrated its selectivity and potency against folate receptor 1 (FOLR1)-expressing ovarian cancer cells in vitro and in vivo. Unlike conventional biAbs, chemically programmed biAbs in DART format are highly modular with broad utility in terms of both target and effector cell engagement. Most importantly, they provide tumor-targeting compounds access to the power of cancer immunotherapy.
Inhibition of cyclin-dependent kinases (CDKs) has evolved as an emerging anticancer strategy. In addition to the cell cycle-regulating CDKs, the transcriptional kinases Cdk12 and Cdk13 have become ...the focus of interest as they mediate a variety of functions, including the transition from transcription initiation to elongation and termination, precursor mRNA splicing, and intronic polyadenylation. Here, we determine the crystal structure of the small molecular inhibitor SR-4835 bound to the Cdk12/cyclin K complex at 2.68 Å resolution. The compound's benzimidazole moiety is embedded in a unique hydrogen bond network mediated by the kinase hinge region with flanking hydroxy groups of the Y815 and D819 side chains. Whereas the SR-4835 head group targets the adenine-binding pocket, the kinase's glycine-rich loop is shifted down toward the activation loop. Additionally, the αC-helix adopts an inward conformation, and the phosphorylated T-loop threonine interacts with all three canonical arginines, a hallmark of CDK activation that is altered in Cdk12 and Cdk13. Dose-response inhibition measurements with recombinant CMGC kinases show that SR-4835 is highly specific for Cdk12 and Cdk13 following a 10-fold lower potency for Cdk10. Whereas other CDK-targeting compounds exhibit tighter binding affinities and higher potencies for kinase inhibition, SR-4835 can be considered a selective transcription elongation antagonist. Our results provide the basis for a rational improvement of SR-4835 toward Cdk12 inhibition and a gain in selectivity over other transcription regulating CDKs.
Cutting costs, cutting corners: In an inexpensive and straightforward synthesis of syn‐propionamide aldols, formation of the Z enolborinate by the hydroboration of 4‐acryloylmorpholine with ...diisopinocampheylborane ((Ipc)2BH) was followed by aldol reactions with achiral and chiral aldehydes to provide syn‐α‐methyl‐β‐hydroxymorpholinecarboxamides with excellent enantio‐ and diastereoselectivity (see scheme; R=alkyl, alkenyl, aryl, heteroaryl).
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