Darryl Rideout

Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-alpha.

The binding of tumor necrosis factor alpha (TNF-α) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein–protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-α to TNFRc1 (IC50 = 50 nM) and also blocked TNF-stimulated phosphorylation of Iκ-B in Ramos cells (IC50 = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 μM. Detailed evaluation of this and related molecules revealed that compounds in this class are “photochemically enhanced” inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40–100 μM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-α to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-α–TNFRc1 interaction.

Stimulation of Apoptosis by Computationally Derived Small Molecules that Bind to BCL-2

METHODS. The NMR structure of Bcl-xL complexed with a portion of the BH3 region of its ligand Bak (PDB accession number 1BXL) (6) was used to generate a computational model of Bcl-2 complexed with a corresponding portion of its intracellular ligand Bax. The model was used in a 3-D computational search for non-peptide small molecules that disrupt Bcl-2/Bax interaction. Selected compounds were tested for their ability to inhibit binding of Eu 3+ -labelled Bax to Bcl-2 as measured by time-resolved fluorometry. Compounds with Ki values less than 50 µM were tested for their ability to induce apoptosis in Jurkat cells that overexpress Bcl-2 (Jurkat/Bcl-2 cells). The published Bcl-2 binding compound HA14-1 (7), was used as a control in the biochemical and cellular assays.

A Potent Non-Peptide Inhibitor of Anthrax Lethal Factor

Bacillus anthracis, the cause of anthrax, poses a considerable danger because of its potential use as a weapon by military organizations and terrorist groups. Respiratory anthrax infections are almost invariably fatal despite antibiotic treatment. Anthrax lethal factor (LF) is a zinc metalloprotease [1] that cleaves mitogen-activating protein kinase kinase 1 and 2 (MAPKK1 and MAPKK2) followed by a series of biological reactions that leads to death [2]. Existing vaccines provide no protection against certain strains of Bacillus anthracis. The most potent inhibitor currently known is too weak to be useful in vivo [1]. A potent, non-peptide inhibitor of LF could be used to ameliorate and prevent the lethal effects of anthrax. Our approach involves identifying small molecules that mimic the substrate MAPKK1 in the calculated MAPKK/LF complex, which differs from traditional rational drug design methods.