Percy H. Carter

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.

Chemokine receptor antagonism as an approach to anti-inflammatory therapy: 'just right' or plain wrong?

Inflammation plays a pivotal role in exacerbating a wide array of human diseases. The chemokines are a group of proteins that control the movement and activation of the immune cells involved in all aspects of the inflammatory response. Recently, their cognate receptors have attracted considerable interest as therapeutic targets, in part because they are G-protein-coupled receptors, which have been antagonized successfully before by the pharmaceutical industry. Indeed, several companies have now reported the development of selective small-molecule chemokine receptor antagonists, and some of these compounds have even entered human Phase I clinical trials. Preclinical studies of the responsiveness of murine models of inflammation to either pharmacologic or genetic intervention have suggested that antagonism of some chemokine receptors may well prove to be a safe and efficacious approach to anti-inflammatory therapy.

Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists.

CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer. These disease associations have motivated numerous preclinical studies and clinical trials (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2–chemokine axis. To aid drug discovery efforts, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein–protein interactions, receptor–chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.

The Roles of Parathyroid Hormone and Calcitonin in Bone Remodeling: Prospects for Novel Therapeutics

Inappropriate regulation of the bone resorption and bone formation processes that occur as a normal part of bone remodeling can lead to net bone loss, as found in osteoporosis. Parathyroid hormone (PTH) and calcitonin (CT) are two peptide hormones that play important roles in calcium homeostasis through their actions on osteoblasts (bone forming cells) and osteoclasts (bone resorbing cells), respectively. Paradoxically, even though genetic deletion of either PTH or CT produces mice with increased bone mass (presumably through different mechanisms), derivatives of both PTH and CT have now been approved for clinical use in the treatment of bone loss in osteoporosis. In this review, we focus on the biology and pharmacology of these two peptides. Specifically, we sequentially address the following three topics in detail: (1) the biological mechanisms of action of PTH and CT, focusing on data from in vitro studies and animal models; (2) the clinical utility of PTH and CT in treating osteoporosis, examining how their pharmacological efficacy correlates with our understanding of their biological mechanism of action; and (3) future prospects for combination therapy, alternative formulation of PTH and CT into oral and transdermal therapies, and replacement of PTH and CT with modified peptides or small molecules. The past four years have witnessed dramatic advances in each of these three areas, and the review places in context the challenges that lie ahead for this complicated, but clinically-relevant field.

Selective Inhibition of Eosinophil Influx into the Lung by Small Molecule CC Chemokine Receptor 3 Antagonists in Mouse Models of Allergic Inflammation

CC chemokine receptor (CCR) 3 is a chemokine receptor implicated in recruiting cells, particularly eosinophils (EΦ), to the lung in episodes of allergic asthma. To investigate the efficacy of selective, small molecule antagonists of CCR3, we developed a murine model of EΦ recruitment to the lung. Murine eotaxin was delivered intranasally to mice that had previously received i.p. injections of ovalbumin (OVA), and the effects were monitored by bronchoalveolar lavage. A selective eosinophilic influx was produced in animals receiving eotaxin but not saline. Furthermore, the number of EΦ was concentration- and time-dependent. Although anti-CCR3 antibody reduced the number of EΦ, the effect of eotaxin in OVA-sensitized mice was not a direct chemotactic stimulus because mast cell deficiency (in WBB6F1-Kitw/Kitw-v mice) significantly reduced the response. Two representative small molecule CCR3 antagonists from our program were characterized as being active at mouse CCR3. They were administered p.o. to wild-type mice and found to reduce eotaxin-elicited EΦ selectively in a dose-dependent manner. Pump infusion of one of the inhibitors to achieve steady-state levels showed that efficacy was not achieved at plasma concentrations equivalent to the in vitro chemotaxis IC90 but only at much higher concentrations. To extend the results from our recruitment model, we tested one of the inhibitors in an allergenic model of airway inflammation, generated by adoptive transfer of OVA-sensitive murine T helper 2 cells and aerosolized OVA challenge of recipient mice, and found that it inhibited EΦ recruitment. We conclude that small molecule CCR3 antagonists reduce pulmonary eosinophilic inflammation elicited by chemokine or allergenic challenge.

The Discovery of Macrocyclic XIAP Antagonists from a DNA-Programmed Chemistry Library, and Their Optimization To Give Lead Compounds with in Vivo Antitumor Activity.

Affinity selection screening of macrocycle libraries derived from DNA-programmed chemistry identified XIAP BIR2 and BIR3 domain inhibitors that displace bound pro-apoptotic caspases. X-ray cocrystal structures of key compounds with XIAP BIR2 suggested potency-enhancing structural modifications. Optimization of dimeric macrocycles with similar affinity for both domains were potent pro-apoptotic agents in cancer cell lines and efficacious in shrinking tumors in a mouse xenograft model.

Discovery of Disubstituted Cyclohexanes as a New Class of CC Chemokine Receptor 2 Antagonists

We describe the design, synthesis, and evaluation of novel disubstituted cyclohexanes as potent CCR2 antagonists. Exploratory SAR studies led to the cis-disubstituted derivative 22, which displayed excellent binding affinity for CCR2 (binding IC50 = 5.1 nM) and potent functional antagonism (calcium flux IC50 = 18 nM and chemotaxis IC 50 = 1 nM). Site-directed mutagenesis studies with 22 suggest the compound is binding near the key receptor residue Glu291, however, 22 is not reliant on Glu291 for its binding affinity.

Both 5-arylidene-2-thioxodihydropyrimidine-4,6(1H,5H)-diones and 3-thioxo-2,3-dihydro-1H-imidazo[1,5-a]indol-1-ones are light-Dependent tumor necrosis factor-α antagonists

Based on the realization that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones are tumor necrosis factor-alpha antagonists, we discovered two additional classes of antagonists: 3-thioxo-2,3-dihydro-1H-imidazo[1,5-a]indol-1-ones (via rational design) and 5-arylidene-2-thioxodihydropyrimidine-4,6(1H,5H)-diones (via computer-guided screening). Chemical modification of the lead structures showed that the structure-activity relationship profiles for both of these series were dependent on the electronic properties of the molecules. Subsequent studies showed that they were light-dependent inhibitors.

A new synthesis of cytoxazone and its diastereomers provides key initial SAR information.

A short, enantioselective, and diastereoselective synthesis of cytoxazone, a Th2-selective immunomodulator from Streptomyces, is described. The route was readily adapted to the synthesis of the three other stereoisomers of natural cytoxazone. Evaluation of these compounds revealed that the stereochemical configuration of the oxazolidinone ring did not influence their biological activity.

Chapter 14 Advances in the Discovery of CC Chemokine Receptor 2 Antagonists

Publisher Summary CC Chemokine Receptor 2 (CCR2) is a member of the G protein-coupled receptor (GPCR) superfamily that serves as the receptor for monocyte chemoattractant proteins 1–4 (MCP-1 to -4), a group of pro-inflammatory chemotactic cytokines (chemokines). CCR2 is the primary chemokine receptor on inflammatory monocytes, and is also expressed on T-cells, dendritic cells, and endothelial cells. A number of studies have demonstrated that antagonism of CCR2 and/or MCP-1 reduces disease scores in pre-clinical models of arthritis. Recently, the first reports of the actions of small molecule antagonists in both collagen-induced arthritis (CIA) and adjuvant arthritis models have appeared, and they confirm the earlier findings: blockade of CCR2 reduced disease score. The effects of genetic deletion of CCR2 in mouse CIA showed that CCR2–/– mice exhibited exacerbated disease. CCR2–/–mice developed chronic polyarthritis after infection with Mycobacterium avium, whereas wild-type (WT) littermates did not. Recent data have highlighted that CCR2 plays a more central role in immunology than had been previously anticipated, in that it governs the emigration of activated monocytes from the bone marrow in addition to directing their migration toward certain points of inflammation. New pre-clinical validation for CCR2 antagonism in rodent disease models has also been obtained, using both genetic and chemical approaches.