Michelle Bradley

A common intracellular allosteric binding site for antagonists of the CXCR2 receptor

Background and purpose:  We have previously shown that SB265610 (1‐(2‐bromo‐phenyl)‐3‐(7‐cyano‐3H‐benzotriazol‐4‐yl)‐urea) behaves as an allosteric, inverse agonist at the C‐X‐C chemokine (CXCR)2 receptor. The aim of this study was to determine whether SB265610, in addition to two other known antagonists, bind to either of the two putative, topographically distinct, allosteric binding sites previously reported in the Literature.

SB265610 is an allosteric, inverse agonist at the human CXCR2 receptor

Background and purpose:  In several previous studies, the C‐X‐C chemokine receptor (CXCR)2 antagonist 1‐(2‐bromo‐phenyl)‐3‐(7‐cyano‐3H‐benzotriazol‐4‐yl)‐urea (SB265610) has been described as binding competitively with the endogenous agonist. This is in contrast to many other chemokine receptor antagonists, where the mechanism of antagonism has been described as allosteric.

Investigating the molecular mechanisms through which FTY720-P causes persistent S1P1 receptor internalization.

The molecular mechanism underlying the clinical efficacy of FTY720‐P is thought to involve persistent internalization and enhanced degradation of the S1P1 receptor subtype (S1P1R). We have investigated whether receptor binding kinetics and β‐arrestin recruitment could play a role in the persistent internalization of the S1P1R by FTY720‐P.

Fevipiprant (QAW039), a Slowly Dissociating CRTh2 Antagonist with the Potential for Improved Clinical Efficacy

Here we describe the pharmacologic properties of a series of clinically relevant chemoattractant receptor-homologous molecules expressed on T-helper type 2 (CRTh2) receptor antagonists, including fevipiprant (NVP-QAW039 or QAW039), which is currently in development for the treatment of allergic diseases. [3H]-QAW039 displayed high affinity for the human CRTh2 receptor (1.14 ± 0.44 nM) expressed in Chinese hamster ovary cells, the binding being reversible and competitive with the native agonist prostaglandin D2 (PGD2). The binding kinetics of QAW039 determined directly using [3H]-QAW039 revealed mean kinetic on (kon) and off (koff) values for QAW039 of 4.5 × 107 M-1min−1 and 0.048 minute−1, respectively. Importantly, the koff of QAW039 (half-life = 14.4 minutes) was >7-fold slower than the slowest reference compound tested, AZD-1981. In functional studies, QAW039 behaved as an insurmountable antagonist of PGD2-stimulated [35S]-GTPγS activation, and its effects were not fully reversed by increasing concentrations of PGD2 after an initial 15-minute incubation period. This behavior is consistent with its relatively slow dissociation from the human CRTh2 receptor. In contrast for the other ligands tested this time-dependent effect on maximal stimulation was fully reversed by the 15-minute time point, whereas QAW039’s effects persisted for >180 minutes. All CRTh2 antagonists tested inhibited PGD2-stimulated human eosinophil shape change, but importantly QAW039 retained its potency in the whole-blood shape-change assay relative to the isolated shape change assay, potentially reflective of its relatively slower off rate from the CRTh2 receptor. QAW039 was also a potent inhibitor of PGD2-induced cytokine release in human Th2 cells. Slow CRTh2 antagonist dissociation could provide increased receptor coverage in the face of pathologic PGD2 concentrations, which may be clinically relevant.

A physical properties based approach for the exploration of a 4-hydroxybenzothiazolone series of β2-adrenoceptor agonists as inhaled long-acting bronchodilators

The chiral synthesis of a 4-hydroxybenzothiazolone based series of beta(2)-adrenoceptor agonists is described. Using this methodology a library of N-substituted analogues were prepared for the rapid identification of leads with the potential to be fast onset and long-acting inhaled bronchodilators with improved therapeutic margins. The design of the library to achieve the targeted profile was based upon lipophilicity and metabolism based hypotheses. This approach identified beta-phenethyl, alpha-substituted cyclopentyl and monoterpene N-substituents to be of particular interest for further evaluation, as exemplified by structures 19, 29 and 33, respectively.

Potent and Efficacious Inhibition of CXCR2 Signaling by Biparatopic Nanobodies Combining Two Distinct Modes of Action

Chemokines and chemokine receptors are key modulators in inflammatory diseases and malignancies. Here, we describe the identification and pharmacologic characterization of nanobodies selectively blocking CXCR2, the most promiscuous of all chemokine receptors. Two classes of selective monovalent nanobodies were identified, and detailed epitope mapping showed that these bind to distinct, nonoverlapping epitopes on the CXCR2 receptor. The N-terminal–binding or class 1 monovalent nanobodies possessed potencies in the single-digit nanomolar range but lacked complete efficacy at high agonist concentrations. In contrast, the extracellular loop-binding or class 2 monovalent nanobodies were of lower potency but were more efficacious and competitively inhibited the CXCR2-mediated functional response in both recombinant and neutrophil in vitro assays. In addition to blocking CXCR2 signaling mediated by CXCL1 (growth-related oncogene α) and CXCL8 (interleukin-8), both classes of nanobodies displayed inverse agonist behavior. Bivalent and biparatopic nanobodies were generated, respectively combining nanobodies from the same or different classes via glycine/serine linkers. Interestingly, receptor mutation and competition studies demonstrated that the biparatopic nanobodies were able to avidly bind epitopes within one or across two CXCR2 receptor molecules. Most importantly, the biparatopic nanobodies were superior over their monovalent and bivalent counterparts in terms of potency and efficacy.

The discovery of potent, orally bioavailable pyrimidine-5-carbonitrile-6-alkyl CXCR2 receptor antagonists.

A hit-to-lead optimisation programme was carried out on the Novartis archive screening hit, pyrimidine 2-((2,6-dichlorobenzyl)thio)-5-isocyano-6-phenylpyrimidin-4-ol 4, resulting in the discovery of CXCR2 receptor antagonist 2-((2,3-difluorobenzyl)thio)-6-(2-(hydroxymethyl)cyclopropyl)-5-isocyanopyrimidin-4-ol 24. The SAR was investigated by systematic variation of the aromatic group at c-6, the linker between c-2 and the halogenated ring, and the c-5 nitrile moiety.

The discovery of potent, orally bioavailable pyrazolo and triazolopyrimidine CXCR2 receptor antagonists.

A hit-to-lead optimisation programme was carried out on the Novartis archive screening hit, pyrazolopyrimidine 2-methyl-5-((phenylthio)methyl)pyrazolo[1,5-a]pyrimidin-7-ol 1, resulting in the discovery of CXCR2 receptor antagonist 2-benzyl-5-(((2,3-difluorophenyl)thio)methyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol 14. The SAR was investigated by systematic variation of the pendant thiol, alkyl and pyrimidinol groups. Replacement of the pyrazolopyrimidine core with a triazolo alternative led to a dual series of antagonists with favourable biological and pharmacokinetic properties.

Functional desensitization of the β2 adrenoceptor is not dependent on agonist efficacy

Chronic treatment with β2 adrenoceptor agonists is recommended as a first‐line maintenance therapy for chronic obstructive pulmonary disease (COPD). However, a potential consequence of long‐term treatment may be the loss of functional response (tachyphylaxis) over time. In this study, we have investigated the tendency of such agonists, with a range of efficacies, to develop functional desensitization to cAMP responses in primary human bronchial smooth muscle cells following prolonged agonist exposure. The data show that upon repeat exposure, all agonists produced functional desensitization to the same degree and rate. In addition, β2 adrenoceptor internalization and β‐arrestin‐2 recruitment were monitored using β2·eGFP visualization and the PathHunter™ β‐arrestin‐2 assay, respectively. All agonists were capable of causing robust receptor internalization and β‐arrestin‐2 recruitment, the rate of which was influenced by agonist efficacy, as measured in those assays. In summary, although a relationship exists between agonist efficacy and the rate of both receptor internalization and β‐arrestin‐2 recruitment, there is no correlation between agonist efficacy and the rate or extent of functional desensitization.

Design and synthesis of a library of chemokine antagonists.

A library of chemokine antagonists has been synthesized using a combination of solid and solution-phase chemistry. Structures of known chemokine antagonists were used to produce a pharmacophore which served to guide monomer selection. Several combinations of monomers have resulted in providing novel chemokine antagonists which in some cases display dual chemokine receptor antagonism.