Lee R. Roberts

Design and structure of stapled peptides binding to estrogen receptors

Synthetic peptides that specifically bind nuclear hormone receptors offer an alternative approach to small molecules for the modulation of receptor signaling and subsequent gene expression. Here we describe the design of a series of novel stapled peptides that bind the coactivator peptide site of estrogen receptors. Using a number of biophysical techniques, including crystal structure analysis of receptor-stapled peptide complexes, we describe in detail the molecular interactions and demonstrate that all-hydrocarbon staples modulate molecular recognition events. The findings have implications for the design of stapled peptides in general.

(R)-PFI-2 is a potent and selective inhibitor of SETD7 methyltransferase activity in cells

Significance Protein methyltransferases constitute an emerging but undercharacterized class of therapeutic targets with diverse roles in normal human biology and disease. Small-molecule “chemical probes” can be powerful tools for the functional characterization of such enzymes, and here we report the discovery of (R)-PFI-2—a first-in-class, potent, highly selective, and cell-active inhibitor of the methyltransferase activity of SETD7 [SET domain containing (lysine methyltransferase) 7]—and two related compounds for control and chemoproteomics studies. We used these compounds to characterize the role of SETD7 in signaling, in the Hippo pathway, that controls cell growth and organ size. Our work establishes a chemical biology tool kit for the study of the diverse roles of SETD7 in cells and further validates protein methyltransferases as a druggable target class. SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the discovery of (R)-PFI-2—a first-in-class, potent (Kiapp = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7—and its 500-fold less active enantiomer, (S)-PFI-2. (R)-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S-adenosylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with (R)-PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent MCF7 cells, (R)-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish (R)-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a druggable target class.

The Benzyne Aza‐Claisen Reaction

Adding an aryne to a tertiary allylamine affords o-allylaniline products of an aza-Claisen rearrangement. The aryne simultaneously provides the pi component for the rearrangement and the quaternization event that lowers the activation energy for the sigmatropic shift. The reaction was applied to the synthesis of medium-ring benzannulated amines (see scheme).

Generation of benzyne from benzoic acid using C–H activation

ortho C-H activation of benzoic acids with Pd(II) generates an oxapalladacycle that can decarboxylate to produce a palladium-associated aryne. The arynes then undergo [2+2+2] trimerisation to afford triphenylenes.

Rational Targeting of Active-Site Tyrosine Residues Using Sulfonyl Fluoride Probes

This work describes the first rational targeting of tyrosine residues in a protein binding site by small-molecule covalent probes. Specific tyrosine residues in the active site of the mRNA-decapping scavenger enzyme DcpS were modified using reactive sulfonyl fluoride covalent inhibitors. Structure-based molecular design was used to create an alkyne-tagged probe bearing the sulfonyl fluoride warhead, thus enabling the efficient capture of the protein from a complex proteome. Use of the probe in competition experiments with a diaminoquinazoline DcpS inhibitor permitted the quantification of intracellular target occupancy. As a result, diaminoquinazoline upregulators of survival motor neuron protein that are used for the treatment of spinal muscular atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes designed to react with specific tyrosine residues of a protein and augments the chemical biology toolkit by these probes uses in target validation and molecular pharmacology.

Lead discovery and optimization strategies for peptide macrocycles.

Peptide macrocycles represent a chemical space where the best of biological tools can synergize with the best of chemical approaches in the quest for leads against undruggable targets. Peptide macrocycles offer some key advantages in both lead discovery and lead optimization phases of drug discovery when compared to natural product and synthetic macrocycles. In addition, they are uniquely positioned to capitalize on the therapeutic potential of peptides because cyclization can help drive selectivity, potency and overcome the common limitations of metabolic instability of peptides.

Acidic triazoles as soluble guanylate cyclase stimulators

A series of acidic triazoles with activity as soluble guanylate cyclase stimulators is described. Incorporation of the CF(3) triazole improved the overall physicochemical and drug-like properties of the molecule and is exemplified by compound 25.

7-Sulfonamido-3-benzazepines as potent and selective 5-HT2C receptor agonists: Hit-to-lead optimization

New 7-sulfonamido-3-benzazepines 3 are disclosed as 5-HT(2C) receptor agonists. Appropriate substitution of the amino group (R(1)R(2)N-) gave compounds that were potent 5-HT(2C) agonists with minimal activation of the 5-HT(2A) and 5-HT(2B) receptors. Furthermore, representative examples had excellent in vitro ADME properties and good selectivity over ion channel activity.

Myxovirescin analogues via macrocyclic ring–closing metathesis

A short, efficient route has been developed to analogues of myxovirescin using ring-closing metathesis whereby the antibacterial activity has been retained.

Novel 2-imidazoles as potent, selective and CNS penetrant α1A adrenoceptor partial agonists

A novel series of central nervous system (CNS) penetrant indane 2-imidazoles have been identified as potent, partial agonists of the alpha(1A) adrenergic receptor, having good selectivity over the alpha(1B), alpha(1D) and alpha(2) sub-types. A key structural motif to impart selectivity is a methylene spacer between the indane and a pendant substituent, which includes heterocycles, sulphones and ethers. Introduction of an ortho-halogen to this group led to a lowering of intrinsic efficacy (E(max)).