Defining Target Engagement Required for Efficacy In Vivo at the Retinoic Acid Receptor-Related Orphan Receptor C2 (RORγt).

The Th17 pathway has been implicated in autoimmune diseases. The retinoic acid receptor-related orphan receptor C2 (RORγt) is a master regulator of Th17 cells and controls the expression of IL-17A. RORγt is expressed primarily in IL-17A-producing lymphoid cells. Here we describe a virtual screen of the ligand-binding pocket and subsequent screen in a binding assay that identified the 1-benzyl-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-2'-carboxamide scaffold as a starting point for optimization of binding affinity and functional activity guided by structure-based design. Compound 12 demonstrated activity in a mouse PK/PD model and efficacy in an inflammatory arthritis mouse model that were used to define the level and duration of target engagement required for efficacy in vivo. Further optimization to improve ADME and physicochemical properties with guidance from simulations and modeling provided compound 22, which is projected to achieve the level and duration of target engagement required for efficacy in the clinic.

The Angiopoietin-Like Protein 3 and 8 Complex Interacts with Lipoprotein Lipase and Induces LPL Cleavage.

Lipoprotein lipase (LPL) is the key enzyme that hydrolyzes triglycerides from triglyceride-rich lipoproteins. Angiopoietin-like proteins (ANGPTL) 3, 4, and 8 are well-characterized protein inhibitors of LPL. ANGPTL8 forms a complex with ANGPTL3, and the complex is a potent endogenous inhibitor of LPL. However, the nature of the structural interaction between ANGPTL3/8 and LPL is unknown. To probe the conformational changes in LPL induced by ANGPTL3/8, we found that HDX-MS detected significantly altered deuteration in the lid region, ApoC2 binding site, and furin cleavage region of LPL in the presence of ANGPTL3/8. Supporting this HDX structural evidence, we found that ANGPTL3/8 inhibits LPL enzymatic activities and increases LPL cleavage. ANGPTL3/8-induced effects on LPL activity and LPL cleavage are much stronger than those of ANGPTL3 or ANGPTL8 alone. ANGPTL3/8-mediated LPL cleavage is blocked by both an ANGPTL3 antibody and a furin inhibitor. Knock-down of furin expression by siRNA significantly reduced ANGPT3/8-induced cleavage of LPL. Our data suggest ANGPTL3/8 promotes furin-mediated LPL cleavage.

A Residue-Resolved Bayesian Approach to Quantitative Interpretation of Hydrogen-Deuterium Exchange from Mass Spectrometry: Application to Characterizing Protein-Ligand Interactions.

Characterization of interactions between proteins and other molecules is crucial for understanding the mechanisms of action of biological systems and, thus, drug discovery. An increasingly useful approach to mapping these interactions is measurement of hydrogen/deuterium exchange (HDX) using mass spectrometry (HDX-MS), which measures the time-resolved deuterium incorporation of peptides obtained by enzymatic digestion of the protein. Comparison of exchange rates between apo- and ligand-bound conditions results in a mapping of the differential HDX (ΔHDX) of the ligand. Residue-level analysis of these data, however, must account for experimental error, sparseness, and ambiguity due to overlapping peptides. Here, we propose a Bayesian method consisting of a forward model, noise model, prior probabilities, and a Monte Carlo sampling scheme. This method exploits a residue-resolved exponential rate model of HDX-MS data obtained from all peptides simultaneously, and explicitly models experimental error. The result is the best possible estimate of ΔHDX magnitude and significance for each residue given the data. We demonstrate the method by revealing richer structural interpretation of ΔHDX data on two nuclear receptors: vitamin D-receptor (VDR) and retinoic acid receptor gamma (RORγ). The method is implemented in HDX Workbench and as a standalone module of the open source Integrative Modeling Platform.

The chemical basis of serine palmitoyltransferase inhibition by myriocin.

Sphingolipids (SLs) are essential components of cellular membranes formed from the condensation of L-serine and a long-chain acyl thioester. This first step is catalyzed by the pyridoxal-5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) which is a promising therapeutic target. The fungal natural product myriocin is a potent inhibitor of SPT and is widely used to block SL biosynthesis despite a lack of a detailed understanding of its molecular mechanism. By combining spectroscopy, mass spectrometry, X-ray crystallography, and kinetics, we have characterized the molecular details of SPT inhibition by myriocin. Myriocin initially forms an external aldimine with PLP at the active site, and a structure of the resulting co-complex explains its nanomolar affinity for the enzyme. This co-complex then catalytically degrades via an unexpected 'retro-aldol-like' cleavage mechanism to a C18 aldehyde which in turn acts as a suicide inhibitor of SPT by covalent modification of the essential catalytic lysine. This surprising dual mechanism of inhibition rationalizes the extraordinary potency and longevity of myriocin inhibition.

The synthesis of azadirachtin: a potent insect antifeedant.

We describe in full the first synthesis of the potent insect antifeedant azadirachtin through a highly convergent approach. An O-alkylation reaction is used to unite decalin ketone and propargylic mesylate fragments, after which a Claisen rearrangement constructs the central C8-C14 bond in a stereoselective fashion. The allene which results from this sequence then enables a second critical carbon-carbon bond forming event whereby the [3.2.1] bicyclic system, present in the natural product, is generated via a 5-exo-radical cyclisation process. Finally, using knowledge gained through our early studies into the reactivity of the natural product, a series of carefully designed steps completes the synthesis of this challenging molecule.

Selection of heterocycles for drug design.

A method has been devised to obtain heterocyclic ring systems suitable for use in drug design and library design, with an emphasis on the selection of systems with good absorption, distribution, metabolism, excretion and toxicity (ADMET) properties in man. This has been achieved by extraction of the ring systems found in drugs that have reached Phase II or later stages of drug development and launch. Properties have been calculated for these ring systems to enable them to be rationally selected from the database, including descriptors based on molecular size, shape, hydrogen bonding and orbital properties. In many cases, the properties have been calculated for different attachment points of the same heterocycle. Principal components analysis has been used to enable visualization of the set of heterocycles in a useful "chemical space". Using this space, it is possible to select heterocycles for drug design to explore specific aspects of the properties of the heterocycle, such as size or hydrogen bonding, while maintaining other parameters near constant, or to select heterocycles with extreme values of these properties but which are nonetheless likely to be acceptable in a drug. The differences between the properties calculated for the most- and least-frequently used heterocycles from the late-phase drug set have been analyzed, and may suggest that heterocycles in successful drugs are more likely to have calculated quantities associated with lower chemical reactivity.

Chemistry of epoxysulfones: straightforward synthesis of versatile chiral building blocks.

[reaction: see text] Several chiral building blocks have been obtained easily in large quantities from an epoxysulfone (9) that could be obtained in both enantiomeric forms from accessible starting materials.

Stereocontrolled synthesis of cyclopropanol amino acids from allylic sulfones: conformationally restricted building blocks.

[reaction: see text] A new amino acid methyl ester with a cyclopropanol has been synthesized starting from the allyl sulfone 10. The starting material, 10, could be obtained in both enantiomeric forms. The stereoselectivity of the cyclopropane formation has been studied by molecular modeling.

Identification of a novel, selective GABA(A) alpha5 receptor inverse agonist which enhances cognition.

In pursuit of a GABA(A) alpha5-subtype-selective inverse agonist to enhance cognition, a series of 6,7-dihydro-2-benzothiophen-4(5H)-ones has been identified as a novel class of GABA(A) receptor ligands. These thiophenes have higher binding affinity for the GABA(A) alpha5 receptor subtype compared to the GABA(A) alpha1, alpha2, and alpha3 subtypes, and several analogues exhibit high GABA(A) alpha5 receptor inverse agonism. 6,6-Dimethyl-3-(2-hydroxyethyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one (43) has been identified as a full inverse agonist at the GABA(A) alpha5 receptor and is functionally selective over the other major GABA(A) receptor subtypes. 43 readily penetrates into the CNS to give selective occupancy of GABA(A) alpha5 receptors. In addition, 43 enhances cognitive performance in rats in the delayed 'matching-to-place' Morris water maze test-a hippocampal-dependent memory task-without the convulsant or proconvulsant activity associated with nonselective, GABA(A) receptor inverse agonists.

6,7-Dihydro-2-benzothiophen-4(5H)-ones: a novel class of GABA-A alpha5 receptor inverse agonists.

Nonselective inverse agonists at the benzodiazepine binding site on the GABA-A chloride ion channel enhance cognitive performance in animals but cannot be used in the treatment of cognitive disorders because of anxiogenic and convulsant side effects. We have identified a novel series of GABA-A alpha5 receptor ligands during our search for alpha5 receptor inverse agonists as potential cognition enhancers. In particular, 6,6-dimethyl-3-(2-hydroxyethyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one (26) has been identified as a functionally selective GABA-A alpha5 inverse agonist.

Molecular binding interactions: their estimation and rationalization in QSARs in terms of theoretically derived parameters.

An extensive survey of molecular binding interactions and parameters used in QSARs is reported, which includes consideration of lipophilicity and the derivation of Linear Free Energy Relationships associated with drug-receptor binding, together with an overview of the various contributions to binding energy. The lipophilic parameter, log P, and its relevance to desolvation energy is outlined and explanation of the parameters derived from electronic structure calculation is provided, leading into a summary of molecular dynamics simulations.

Molecular Binding Interactions: Their Estimation and Rationalization in QSARs in Terms of Theoretically Derived Parameters.

An extensive survey of molecular binding interactions and parameters used in QSARs is reported, which includes consideration of lipophilicity and the derivation of Linear Free Energy Relationships associated with drug-receptor binding, together with an overview of the various contributions to binding energy. The lipophilic parameter, log P, and its relevance to desolvation energy is outlined and explanation of the parameters derived from electronic structure calculation is provided, leading into a summary of molecular dynamics simulations.