Thomas Gadek

Small molecule antagonists of proteins

The identification of small molecule antagonists of protein function is at the core of the pharmaceutical industry. Successful approaches to this problem, including screening and rational design, have been developed over the years to identify antagonists of enzymes and cellular receptors. These methods have been extended to the search for inhibitors of protein-protein interactions. While the very possibility of designing a small molecule inhibitor for such interactions was once doubted, there are examples of such inhibitors that are currently marketed products and many more inhibitors in various stages of research and development. Here we review the progress in identifying and designing small molecule protein inhibitors, with particular attention to those that block protein-protein interactions. We also discuss the physical character of protein-protein interfaces, and the resulting implications for small molecule lead discovery and design.

A surprising observation about Mitsunobu reactions in solid phase synthesis

Abstract The Mitsunobu reaction of N-Boc-L-Tyrosine methyl ester with hydroxymethyl polystyrene resin proceeds in practically quantitative yield in the presence of a tertiary amine. Coupling yields in the absence of an amine are significantly lower.

Competition between intercellular adhesion molecule-1 and a small-molecule antagonist for a common binding site on the αl subunit of lymphocyte function-associated antigen-1

The lymphocyte function‐associated antigen‐1 (LFA‐1) binding of a unique class of small‐molecule antagonists as represented by compound 3 was analyzed in comparison to that of soluble intercellular adhesion molecule‐1 (sICAM‐1) and A‐286982, which respectively define direct and allosteric competitive binding sites within LFA‐1s inserted (I) domain. All three molecules antagonized LFA‐1 binding to ICAM‐1‐Immunoglobulin G fusion (ICAM‐1‐Ig) in a competition ELISA, but only compound 3 and sICAM‐1 inhibited the binding of a fluorescein‐labeled analog of compound 3 to LFA‐1. Compound 3 and sICAM‐1 displayed classical direct competitive binding behavior with ICAM‐1. Their antagonism of LFA‐1 was surmountable by both ICAM‐1‐Ig and a fluorescein‐labeled compound 3 analog. The competition of both sICAM‐1 and compound 3 with ICAM‐1‐Ig for LFA‐1 resulted in equivalent and linear Schild plots with slopes of 1.24 and 1.26, respectively. Cross‐linking studies with a photoactivated analog of compound 3 localized the high‐affinity small‐molecule binding site to the N‐terminal 507 amino acid segment of the α chain of LFA‐1, a region that includes the I domain. In addition, cells transfected with a variant of LFA‐1 lacking this I domain showed no significant binding of a fluorescein‐labeled analog of compound 3 or ICAM‐1‐Ig. These results demonstrate that compound 3 inhibits the LFA‐1/ICAM‐1 binding interaction in a directly competitive manner by binding to a high‐affinity site on LFA‐1. This binding site overlaps with the ICAM‐1 binding site on the α subunit of LFA‐1, which has previously been localized to the I domain.

Discovery and Development of Potent LFA-1/ICAM-1 Antagonist SAR 1118 as an Ophthalmic Solution for Treating Dry Eye

LFA-1/ICAM-1 interaction is essential in support of inflammatory and specific T-cell regulated immune responses by mediating cell adhesion, leukocyte extravasation, migration, antigen presentation, formation of immunological synapse, and augmentation of T-cell receptor signaling. The increase of ICAM-1 expression levels in conjunctival epithelial cells and acinar cells was observed in animal models and patients diagnosed with dry eye. Therefore, it has been hypothesized that small molecule LFA-1/ICAM-1 antagonists could be an effective topical treatment for dry eye. In this letter, we describe the discovery of a potent tetrahydroisoquinoline (THIQ)-derived LFA-1/ICAM-1 antagonist (SAR 1118) and its development as an ophthalmic solution for treating dry eye.

RGD Mimetics containing a central hydantoin scaffold: αVβ3 vs αIIbβ3 selectivity requirements

Abstract The synthesis of a series of RGD mimetic α V β 3 antagonists containing a hydantoin scaffold is shown. The results demonstrate some of the structural requirements for the design of selective α V β 3 antagonists (vs α IIb β 3 ) in terms of the Arg-mimetic, the distance between N- and C-terminus and the lipophilic side chain.

N-Benzoyl amino acids as LFA-1/ICAM inhibitors 1: amino acid structure-activity relationship.

The association of ICAM-1 with LFA-1 plays a critical role in several autoimmune diseases. N-2-Bromobenzoyl L-tryptophan, compound 1, was identified as an inhibitor to the formation of the LFA-1/ICAM complex. The SAR of the amino acid indicates that the carboxylic acid is required for inhibition and that L-histidine is the most favored amino acid.

Effect of Selective or Combined Inhibition of Integrins αIIbβ3 and αvβ3 on Thrombosis and Neointima After Oversized Porcine Coronary Angioplasty

Background—Thrombosis and neointima formation limit the efficacy of coronary angioplasty (PTCA). Clinical trials have implicated the adhesion molecules integrin αIIbβ3 and integrin αvβ3 in these processes. The roles of these molecules in vascular smooth muscle cell adhesion, platelet aggregation, and the thrombotic and neointimal response to oversize porcine PTCA was investigated by use of a selective αIIbβ3 antagonist (lamifiban), a selective αvβ3 antagonist (VO514), and a combined αIIbβ3/αvβ3 antagonist (G3580). Methods and Results—In vitro, both αvβ3 inhibitors caused dose-dependent inhibition of porcine vascular smooth muscle cell adhesion to vitronectin but not to collagen type IV, fibronectin, or laminin, whereas selective αIIbβ3 inhibition had no effect. Intravenous infusions of either αIIbβ3 inhibitor in swine profoundly inhibited ex vivo platelet aggregation to ADP, whereas selective αvβ3 inhibition had no effect. In a porcine PTCA model, intravenous infusions of the integrin antagonists were adm...

Chapter 9. Glycoprotein IIb IIIa Antagonists

Publisher Summary This chapter describes the work leading to the discovery of antagonists of GPllbllla/fibrinogen interactions as anti-aggretory and anti-thrombotic agents for the treatment of human vascular diseases. Glycoprotein llbllla is a noncovalent heterodimeric protein complex whose tertiary structure is dependent on the association of divalent cations. Each subunit has a single transmembrane domain and a short cytoplasmic tail. For some time, GPllbllla and other integrins were thought to have a purely adhesive function and as such were considered incapable of signal transduction. However, recent work has shown integrins in general and GPllbllla, in particular, are involved in both inside-out and outside-in signaling. In addition to these signaling and adhesive functions, GPllbllla is involved in the trafficking of proteins from serum into the platelet alpha-granules. A number of reports have implicated GPllbllla in the transport of calcium ions into the platelet upon activation. The affinity of GPllbllla for fibrinogen can also be modulated by the lipid composition of the platelet membrane as well as by partial proteolysis of the extracellular domain. Glanzmanns thrombasthenia, a lifelong bleeding disorder, manifests itself in bleeding from gums and mucotaneous tissue and is not typically life threatening even when no functional GPllbllla is expressed. The chapter discusses proteins, peptides, peptide hybrids, and nonpeptides in connection with glycoprotein antagonists. Vascular occlusion by thrombus formation has been implicated as a primary cause of a variety of cerebral and cardiovascular diseases. Animal models of thrombosis have been devised and used to examine the biological response to GPllbllla inhibition.

Two new procedures for the introduction of benzyl-type protecting groups for thiols

Abstract Two new methods for the benzylation of thiols are described: a) direct S—alkylation with para -substituted benzylic cations; and b) reductive S—alkylations of 2-aminothiols. Both methods provide efficient routes for the introduction of benzyl-type protecting groups in high yields.

Safety and Pharmacokinetics of a Novel Lymphocyte Function-associated Antigen-1 Antagonist Ophthalmic Solution (SAR 1118) in Healthy Adults

PURPOSE To investigate the safety, tolerability, and pharmacokinetics (PKs) of topical SAR 1118 Ophthalmic Solution in healthy adults. SAR 1118 is an investigational small molecule lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18; αLβ2) antagonist that inhibits LFA-1 binding to intercellular adhesion molecule-1 (ICAM-1; CD54) targeting T-cell-mediated inflammation. METHODS A randomized, double-masked, placebo-controlled, dose-escalation study of SAR 1118 was performed in 4 cohorts with 7 randomized subjects per cohort (2 placebo: 5 active drug subjects; 0.1%, 0.3%, 1.0%, 5.0%) in 28 healthy adults. Dosing was divided into 3 periods each separated by a 72-h treatment-free observation: once-daily (QD) × 1, twice-daily (BID) × 10, and thrice-daily (TID) × 10 days. Data obtained at the beginning and end of each period included: slit-lamp, best-corrected visual acuity (BCVA), Schirmer tear test (STT) without anesthesia, tear film break-up time (TBUT), intraocular pressure (IOP), and tear/plasma samples for PK analysis. RESULTS All subjects completed the study; there were no tolerability issues or missed treatments (total, 1,428 administered doses). No serious ocular or nonocular adverse events (AEs) occurred over 1,148 subject study days (41 days/subject) and no significant abnormalities were identified on ocular exam. There were 38 ocular AEs (N = 11 subjects) and 21 nonocular AEs (N = 11 subjects). Most AEs were mild in severity and occurred in the 0.3% and placebo groups. No changes were observed in CD3, CD4, and CD8 blood lymphocyte counts. Tear PK profiles support a QD/BID dosing schedule. Plasma levels of SAR 1118 in the 0.1% and 0.3% groups were below level of quantitation (BLQ; <0.50  ng/mL) at all time points and transiently detected within the first 5 min to ∼1  h following administration in the 1.0% and 5.0% groups. CONCLUSION SAR 1118 Ophthalmic Solution appears safe and well-tolerated up to 5.0% TID in healthy adult subjects. PK analysis shows adequate ocular exposure with minimal systemic exposure.