Terence A. Kelly

A simple method for the protection of aryl amines as their t-butylcarbamoyl (Boc) derivatives

It has been found that aryl amines can be directly protected as their Boc derivatives by treatment of the amine with two equivalents of NaHMDS in THF followed by one equivalent of di-t-butyldicarbonate. This procedure works on a wide variety of both electron-rich and electron-deficient aryl amines.

A small-molecule antagonist of LFA-1 blocks a conformational change important for LFA-1 function

Lymphocyte function‐associated antigen(LFA)‐1/intercellular adhesion molecule (ICAM)‐1interactions mediate several important steps in the evolution of animmune response. LFA‐1 is normally expressed in a quiescent state onthe surface of leukocytes and interacts weakly with its ligands ICAM‐1,‐2, and ‐3. LFA‐1 activity may be regulated by receptor clustering andby increasing the affinity of LFA‐1 for its ligands. Affinitymodulation of LFA‐1 has been shown to occur via a conformational changein the LFA‐1 heterodimer that can be detected by using monoclonalantibody 24 (mAb24). We have recently described a small‐moleculeantagonist of LFA‐1, BIRT 377, that demonstrates selective in vitro andin vivo inhibition of LFA‐1/ICAM‐1‐mediated binding events. We nowdemonstrate that BIRT 377 blocks the induction of the mAb24 reporterepitope on LFA‐1 on the surface of SKW‐3 cells treated with variousagonists known to induce high‐affinity LFA‐1. These data imply thatBIRT 377 exerts its inhibitory effects by preventing up‐regulation ofLFA‐1 to its high‐affinity conformation.

The efficient synthesis and simple resolution of a prolineboronate ester suitable for enzyme-inhibition studies

Abstract A method for the preparation and resolution of the pinanediol ester of prolineboronic acid is described. The method takes advantage of the ease of both the lithiation and the reduction of boc-pyrrole to generate the desired compound rapidly and in high yield.

Small molecule LFA-1 antagonists compete with an anti-LFA-1 monoclonal antibody for binding to the CD11a I domain: development of a flow-cytometry-based receptor occupancy assay.

The beta(2) integrin LFA-1 (CD11a/CD18) is a leukocyte-specific adhesion molecule that mediates leukocyte extravasation, antigen presentation, and T-cell-mediated cytolysis through its interaction with its counter-receptors, ICAM-1, ICAM-2, and ICAM-3. We have recently described a small molecule antagonist of LFA-1 (BIRT 377) that inhibits LFA-1/ICAM-1 molecular interactions, LFA-1-dependent adhesion assays, antigen-induced proliferation of T-cells, and superantigen-induced production of IL-2 in vivo in mice. We have also recently described a unique monoclonal antibody, R3.1, which competes with BIRT 377 and its analogs for binding to both purified full-length LFA-1 and the purified recombinant I domain module. In this manuscript, we extend these studies to cell-based systems and utilize this unique reagent for the development of a receptor occupancy assay. Exploiting these observations, we have designed and validated an assay that allows us to measure receptor occupancy in vitro on monkey and human peripheral blood leukocytes and ex vivo in whole blood from monkeys dosed with small molecule LFA-1 antagonists. Further refinement of these reagents has led to the development of a Fab-based assay that allows rapid and reproducible analysis of whole blood samples. These optimized reagents allow for quantification of the number of receptors expressed on the cell surface and a more accurate quantitation of receptor occupancy.

The discovery of thienopyridine analogues as potent IκB kinase β inhibitors. Part II

An SAR study that identified a series of thienopyridine-based potent IkappaB Kinase beta (IKKbeta) inhibitors is described. With focuses on the structural optimization at C4 and C6 of structure 1 (Fig. 1), the study reveals that small alkyl and certain aromatic groups are preferred at C4, whereas polar groups with proper orientation at C6 efficiently enhance compound potency. The most potent analogues inhibit IKKbeta with IC50s as low as 40 nM, suppress LPS-induced TNF-alpha production in vitro and in vivo, display good kinase selectivity profiles, and are active in a HeLa cell NF-kappaB reporter gene assay, demonstrating that they directly interfere with the NF-kappaB signaling pathway.

Characterization of the allosteric inhibition of a protein-protein interaction by mass spectrometry

The allosteric inhibition of the lymphocyte function associated antigen-1/intercellullar adhesion molecule (LFA-1/ICAM-1) interaction, by a class of small molecules, is characterized by a battery of mass spectrometric techniques. Binding of hydantoins to the I domain of LFA-1 is observed by size exclusion chromatography/mass spectrometry (SEC/MS) and by direct electrospray ionization mass spectrometry (ESI/MS). A photoactive hydantoin analog specifically labels an amino acid residue of LFA-1 I domain. Competition with this photoaffinity labeling by a panel of inhibitors is correlated with their Kd’s for inhibition of the LFA-1/ICAM interaction. Alterations to the tertiary structure of LFA-1 I domain, upon compound binding, are inferred from perturbation in the ESI mass spectrum of the polypeptidés charge state distribution and by an altered level of nonspecific multimer formation. The results demonstrate specific, stoichiometric, reversible binding of the hydantoins to LFA-1. They further show correlation of this binding with activity and indicate alterations in the polypeptide’s tertiary structure, on hydantoin binding, consistent with the proposed mechanism for inhibition of the protein—protein interaction.

Chapter 18 . Antagonists of 02 integrin-mediated cell adhesion

Abstract The biology of the β 2 integrins suggests that they could be attractive targets for the intervention in autoimmune and inflammatory diseases. In the short term, results from the clinical trials using biological reagents should provide critical insights into the validity of this hypothesis. The recent progress in the discovery of small molecules that interfere with β 2 integrin activation and function provides new reagents for probing the role of these proteins in vitro and in vivo. Furthermore, the discovery of an allosteric binding site on LFA-1 provides unique insights for understanding how small molecule antagonists might effectively disrupt the interaction of a large cell surface receptor and its ligand.

A third mode of integrin antagonism

Through the biochemical dissection of conformational changes, Shimaoka et al. (this issue of Immunity) have delineated a novel role for the I-like domain in the allosteric regulation of LFA-1 function and signaling. This work advances our understanding of LFA-1 antagonism and reveals new avenues for approaching LFA-1, and potentially other I-domain containing integrins, as drug targets.