Identification and characterization of novel αLβ2 inhibitors and their differentiation from known inhibitors

Mancuso, Riccardo. Identification and characterization of novel αLβ2 inhibitors and their differentiation from known inhibitors. 2016, Doctoral Thesis, University of Basel, Faculty of Science.

Available under License CC BY-NC-ND (Attribution-NonCommercial-NoDerivatives).


Official URL: http://edoc.unibas.ch/diss/DissB_12433

Downloads: Statistics Overview


αLβ2 is expressed on all leukocytes and plays a major role in immune responses by regulating cell adhesion, leukocyte trafficking, T cell costimulation and immunological synapse (IS) formation. This integrin is centrally involved in immune-mediated diseases of high medical need, including chronic plaque psoriasis, multiple sclerosis, rheumatoid arthritis, small vessel vasculitis, dry eye disease and transplantation indications, among other diseases. In several of these diseases, αLβ2 has been validated by biologic therapies as a target of high therapeutic potential.
In the first part (paper) of this thesis we describe the identification and preclinical characterization of a novel class of αLβ2 inhibitors. These novel compounds with an allosteric mechanism of action, (also termed α I allosteric inhibitors), were designed to overcome major unwanted effects of current αLβ2 targeting drugs. Drug candidates derived from this project are foreseen to be assessed clinically in αLβ2-mediated disease of high unmet medical need.
Computer-assisted drug design (CADD) has been used to identify hits that modulate αLβ2 function via binding to the I allosteric site. The CADD approach pursued addressed the task of finding novel hits and leads from two conceptually complementary angles: (1) ligand-based similarity searching from known allosteric αLβ2 modulators but structurally different bioactive compounds, and (2) structure-based similarity searching with the aim to find novel allosteric αLβ2 modulators. A collection of several million commercially available compounds with drug-like properties has been compiled. These virtual screens yielded in ranked lists of compounds.
To determine the activity of these hits, an adhesion assay has been established (V well adhesion assay) to measure the binding of leukocytes (expressing αLβ2) to recombinant intercellular adhesion molecule-1 (ICAM-1), the ligand of αLβ2.
The α I allosteric mode of action of the compounds has been confirmed by quantifying the binding of the anti- αLβ2 mAb R7.1.
The novel and potent αLβ2 silencing compounds identified were chemically derivatized employing also CADD technologies. Based on first SAR data and CADD feedback, the scaffold of the lead compound has been modified.
These newly synthesized αLβ2 inhibitors discovered do not induce “agonistic” effects such as αLβ2 internalization (as observed in same experiments with biologics), induction of αLβ2 activation epitopes (as observed in same experiments with ligand mimetic α/β I allosteric αLβ2 inhibitors), and induction of ZAP70 phosphorylation. Moreover, they do not interfere with the internalization/recycling of engaged T cell receptor/CD3 complexes and they do not show in vitro cytotoxicity.
The lead drug candidate identified, characterized and optimized has been transitioned to preclinical pharmacokinetic (PK) characterization and formulation development in vivo.
In the second part (paper) of this thesis we systematically compared different modes of αLβ2 inhibition for their αLβ2 inhibitory as well as their potential unwanted downstream events, such as paradoxic agonism.
Three major classes of αLβ2 inhibitors with distinct modes of action have been described to date: monoclonal antibodies (mAbs), small molecule α/β I allosteric and small molecule α I allosteric inhibitors. All inhibitors assessed were found to potently block αLβ2-mediated leukocyte adhesion in the low nanomolar to picomolar range. None of the inhibitors induced ZAP70 phosphorylation, indicating absence of agonistic outside-in signalling.
Paradoxically, however, the α/β I allosteric inhibitor XVA143 induced conformational changes within αLβ2 characteristic for an intermediate affinity state, an effect that was not observed with the α I allosteric inhibitor LFA878 or the anti- αLβ2 mAb efalizumab.
On the other hand, efalizumab triggered the unscheduled internalization of αLβ2 while LFA878 and XVA143 did not affect or only mildly reduced αLβ2 surface expression, respectively.
Moreover, anti-αLβ2 mAb efalizumab, in contrast to the small molecule inhibitors, disturbed the fine-tuned internalization/recycling of engaged T cell receptor/CD3 complexes, concomitantly decreasing intracellular ZAP70 expression levels.
In conclusion, different modes of αLβ2 inhibition are associated with fundamentally different biologic effect profiles. The differential established here provides important translational guidance for novel αLβ2 inhibitors.
In the third part (paper) we described a flow cytometry-based technology that simultaneously quantitates αLβ2 conformational change upon inhibitor binding, αLβ2 expression and T cell activation at the single-cell level in human blood. Two classes of allosteric low molecular weight inhibitors, designated α I and α/β I allosteric αLβ2 inhibitors, were investigated.
The multi-parameter whole blood αLβ2 assay described may enable therapeutic monitoring of αLβ2 inhibitors in patients’ blood. The assay dissects differential effect profiles of different classes of αLβ2 inhibitors.
The flow cytometry-based technology described allows, for the first time, to simultaneously assess and correlate, at the single-cell level, inhibitor-specific αLβ2 conformational change, αLβ2 expression and T cell activation in human whole blood.
The format, robustness and sensitivity of the assay indicate that it may be suitable for bedside monitoring of newly developed αLβ2 inhibitors.
Advisors:Krähenbühl, Stephan and Weitz-Schmidt, Gabriele and Huwyler, Jörg
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Pharmakologie (Krähenbühl)
UniBasel Contributors:Krähenbühl, Stephan and Huwyler, Jörg
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12433
Thesis status:Complete
Number of Pages:1 Online-Ressource (119 Seiten)
Identification Number:
edoc DOI:
Last Modified:08 Feb 2020 14:44
Deposited On:01 Feb 2018 10:17

Repository Staff Only: item control page