Discovery and characterization of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists.

PPAR gamma (PPARG) is a ligand activated transcription factor that regulates genes involved in inflammation, bone biology, lipid homeostasis, as well as a master regulator of adipogenesis and a potential lineage driver of luminal bladder cancer. While PPARG agonists lead to transcriptional activation of canonical target genes, inverse agonists have the opposite effect through inducing a transcriptionally repressive complex leading to repression of canonical target gene expression. While many agonists have been described and tested clinically, inverse agonists offer an underexplored avenue to modulate PPARG biology in vivo. Current inverse agonists lack favorable in vivo properties; herein we describe the discovery and characterization of a series of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists, BAY-5516, BAY-5094, and BAY-9683. Structural studies of this series revealed distinct pre- and post-covalent binding positions, which led to the hypothesis that interactions in the pre-covalent conformation are primarily responsible for driving affinity, while interactions in the post-covalent conformation are more responsible for cellular functional effects by enhancing PPARG interactions with its corepressors. The need to simultaneously optimize for two distinct states may partially explain the steep SAR observed. Exquisite selectivity was achieved over related nuclear receptors in the subfamily due in part to a covalent warhead with low reactivity through an SNAr mechanism in addition to the specificity gained through covalent binding to a reactive cysteine uniquely positioned within the PPARG LBD. BAY-5516, BAY-5094, and BAY-9683 lead to pharmacodynamic regulation of PPARG target gene expression in vivo comparable to known inverse agonist SR10221 and represent new tools for future in vivo studies to explore their potential utility for treatment of disorders of hyperactivated PPARG including luminal bladder cancer and other disorders.

Inhibition of Mcl-1 through covalent modification of a noncatalytic lysine side chain.

Targeted covalent inhibition of disease-associated proteins has become a powerful methodology in the field of drug discovery, leading to the approval of new therapeutics. Nevertheless, current approaches are often limited owing to their reliance on a cysteine residue to generate the covalent linkage. Here we used aryl boronic acid carbonyl warheads to covalently target a noncatalytic lysine side chain, and generated to our knowledge the first reversible covalent inhibitors for Mcl-1, a protein-protein interaction (PPI) target that has proven difficult to inhibit via traditional medicinal chemistry strategies. These covalent binders exhibited improved potency in comparison to noncovalent congeners, as demonstrated in biochemical and cell-based assays. We identified Lys234 as the residue involved in covalent modification, via point mutation. The covalent binders discovered in this study will serve as useful starting points for the development of Mcl-1 therapeutics and probes to interrogate Mcl-1-dependent biological phenomena.

Translocation of Non-Canonical Polypeptides into Cells Using Protective Antigen.

A variety of pathogenic bacteria infect host eukaryotic cells using protein toxins, which enter the cytosol and exert their cytotoxic effects. Anthrax lethal toxin, for example, utilizes the membrane-spanning translocase, protective antigen (PA) pore, to deliver the protein toxin lethal factor (LF) from the endosome into the cytosol of cells. Previous work has investigated the delivery of natural peptides and enzymatic domains appended to the C-terminus of the PA-binding domain of lethal factor (LFN) into the cytosol via PA pore. Here, we move beyond natural amino acids and systematically investigate the translocation of polypeptide cargo containing non-canonical amino acids and functionalities through PA pore. Our results indicate translocation is not perturbed with alterations to the peptide backbone or side-chain. Moreover, despite their structural complexity, we found that the small molecule drugs, doxorubicin and monomethyl auristatin F (MMAF) translocated efficiently through PA pore. However, we found cyclic peptides and the small molecule drug docetaxel abrogated translocation due to their large size and structural rigidity. For cargos that reached the cytosol, we demonstrated that each remained intact after translocation. These studies show PA is capable of translocating non-canonical cargo provided it is in a conformational state conducive for passage through the narrow pore.

Total synthesis of trifluorobutyryl-modified, globally protected sialyl Lewis x by a convergent [2+2] approach.

Structural and quantitative changes in the expression of sialic acid residues on the surface of eukaryotic cells profoundly influence a broad range of biological processes including inflammation, antigen recognition, microbial attachment, and tumor metastasis. Uptake and incorporation of sialic acid analogues in mammalian cells enable structure-function studies and perturbation of specific recognition events. Our group has recently shown that a trifluorobutyryl-modified sialic acid metabolite diminishes the adhesion of mammalian cells to E and P-selectin, presumably by leading to the expression of fluorinated sLex epitopes on cell surfaces, and interfering with the sLex-selectin interactions that are well known in mediating tumor cell migration.1 For studies directed towards understanding the molecular basis of this reduced adhesion, chemical synthesis of trifluorobutyrylated sialyl Lewis x (C4F3--sLex) was crucial. We have developed a highly efficient [2+2] approach for the assembly of C4F3-sLex on a preparative scale that contains versatile protective groups allowing the glycan to be surface immobilized or solubilized as needed for biophysical studies to investigate selectin interactions. This strategy can, in principle, be used for preparation of other N-modified sLex analogues.

A New Paradigm for Protein Design and Biological Self-Assembly.

Very few molecules with biological origins contain the element fluorine. Nature's inability to incorporate fluorine into biomolecules is related to the low concentration of free fluoride in sea and surface water. However, judicious introduction of fluorine into proteins, nucleic acids, lipids and carbohydrates has allowed mechanistic scrutiny of enzyme catalysis, control of protein oligomerization in membranes, clustered display of ligands on surfaces of living cells, and in increasing the protease stability of protein and peptide therapeutics.