Methods to assess small molecule allosteric modulators of the STRAD pseudokinase.

With the increased appreciation of the biological relevance of pseudokinase (PSK) allostery, the broadening of small molecule strategies to target PSK function is of particular importance. We and others have pursued the development of small molecule allosteric modulators of the STRAD pseudokinase by targeting its ATP binding pocket. The purpose of this effort is to modulate the function of the LKB1 tumor suppressor kinase, which exists in a trimer with the STRAD PSK and the adaptor protein MO25. Here we provide detailed guidance regarding the different methods we have used for medium throughput screening to identify STRAD ligands and measure their impact on LKB1 kinase activity. Our experience supports preferential use of direct measurements of LKB1 kinase activity, and demonstrates the limitations of indirect assessment methods in the development trans-acting allosteric modulators.

Disubstituted pyrimidines as Lck inhibitors.

We have developed a family of 4-benzimidazolyl-N-piperazinethyl-pyrimidin-2-amines that are subnanomolar inhibitors of Lck. A subset of these Lck inhibitors, with heterocyclic substituents at the benzimidazole C5, are also low-nanomolar inhibitors of cellular IL2 release.

Molecular modeling aided design of nicotinic acid receptor GPR109A agonists.

A homology model of the nicotinic acid receptor GPR109A was constructed based on the X-ray crystal structure of bovine rhodopsin. An HTS hit was docked into the homology model. Characterization of the binding pocket by a grid-based surface calculation of the docking model suggested that a larger hydrophobic body plus a polar tail would improve interaction between the ligand and the receptor. The designed compounds were synthesized, and showed significantly improved binding affinity and activation of GPR109A.

Neuroactive Steroids. 2. 3α-Hydroxy-3ß-methyl-21-(4-cyano-1H-pyrazol-1'-yl)-19-nor-5ß-pregnan-20-one (SAGE-217): A Clinical Next Generation Neuroactive Steroid Positive Allosteric Modulator of the (γ-Aminobutyric Acid)A Receptor.

Certain classes of neuroactive steroids (NASs) are positive allosteric modulators (PAM) of synaptic and extrasynaptic GABAA receptors. Herein, we report new SAR insights in a series of 5ß-nor-19-pregnan-20-one analogues bearing substituted pyrazoles and triazoles at C-21, culminating in the discovery of 3α-hydroxy-3ß-methyl-21-(4-cyano-1H-pyrazol-1'-yl)-19-nor-5ß-pregnan-20-one (SAGE-217, 3), a potent GABAA receptor modulator at both synaptic and extrasynaptic receptor subtypes, with excellent oral DMPK properties. Compound 3 has completed a phase 1 single ascending dose (SAD) and multiple ascending dose (MAD) clinical trial and is currently being studied in parallel phase 2 clinical trials for the treatment of postpartum depression (PPD), major depressive disorder (MDD), and essential tremor (ET).

Neuroactive Steroids. 1. Positive Allosteric Modulators of the (γ-Aminobutyric Acid)A Receptor: Structure-Activity Relationships of Heterocyclic Substitution at C-21.

Neuroactive steroids (NASs) have been shown to impact central nervous system (CNS) function through positive allosteric modulation of the GABA(A) receptor (GABA(A)-R). Herein we report the effects on the activity and pharmacokinetic properties of a series of nor-19 pregnanolone analogues bearing a heterocyclic substituent at C-21. These efforts resulted in the identification of SGE-516, a balanced synaptic/extrasynaptic GABA(A) receptor modulator, and SGE-872, a selective extrasynaptic GABA(A) receptor modulator. Both molecules possess excellent druglike properties, making them advanced leads for oral delivery of GABA(A) receptor modulators.

Vancomycin CD and DE Macrocyclization and Atropisomerism Studies.

Continued studies on the synthesis and atropisomerism of the vancomycin CD and DE ring systems based on aromatic nucleophilic substitution macrocyclization reactions for formation of the biaryl ethers are detailed in efforts that further define substituent effects, explore the impact of protecting groups, and establish the stereochemical integrity of peripheral substituents. These have led to the identification of a previously unrecognized site of epimerization within our original approach to the DE ring system and the introduction of significant improvements in the approach that will find utilization in syntheses of the vancomycin CDE ring system and of the natural product itself. The preparation of a complete set of DE ring system isomers bearing the unnatural stereochemistry at the labile C8, C11, and C14 sites was accomplished for comparison and established that C8 is prone to epimerization to the more stable, unnatural S versus R absolute stereochemistry if it bears an ester, but not a carboxamide, substituent. Additionally, an improved synthesis of the CD ring system, enlisting a C14 carboxamide versus ester substituent, is disclosed and establishes the stereochemical integrity of our prior approach which incorporated a C14 ester. A set of fully functionalized CD and DE ring systems were prepared and include the development of conditions for the final deprotections required for incorporation into efforts on the natural product. The examination of the antimicrobial activity of these key substructures of vancomycin is detailed.