Llama-derived single variable domains (nanobodies) directed against chemokine receptor CXCR7 reduce head and neck cancer cell growth in vivo.

The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the "Nanobody platform" to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced ß-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.

Orthogonal activation of the reengineered A3 adenosine receptor (neoceptor) using tailored nucleoside agonists.

An alternative approach to overcome the inherent lack of specificity of conventional agonist therapy can be the reengineering of the GPCRs and their agonists. A reengineered receptor (neoceptor) could be selectively activated by a modified agonist, but not by the endogenous agonist. Assisted by rhodopsin-based molecular modeling, we pinpointed mutations of the A(3) adenosine receptor (AR) for selective affinity enhancement following complementary modifications of adenosine. Ribose modifications examined included, at 3': amino, aminomethyl, azido, guanidino, ureido; and at 5': uronamido, azidodeoxy. N(6)-Variations included 3-iodobenzyl, 5-chloro-2-methyloxybenzyl, and methyl. An N(6)-3-iodobenzyl-3'-ureido adenosine derivative 10 activated phospholipase C in COS-7 cells (EC(50) = 0.18 microM) or phospholipase D in chick primary cardiomyocytes, both mediated by a mutant (H272E), but not the wild-type, A(3)AR. The affinity enhancements for 10 and the corresponding 3'-acetamidomethyl analogue 6 were >100-fold and >20-fold, respectively. 10 concentration-dependently protected cardiomyocytes transfected with the neoceptor against hypoxia. Unlike 10, adenosine activated the wild-type A(3)AR (EC(50) of 1.0 microM), but had no effect on the H272E mutant A(3)AR (100 microM). Compound 10 was inactive at human A(1), A(2A), and A(2B)ARs. The orthogonal pair comprising an engineered receptor and a modified agonist should be useful for elucidating signaling pathways and could be therapeutically applied to diseases following organ-targeted delivery of the neoceptor gene.

Synthesis of hypermodified adenosine derivatives as selective adenosine A3 receptor ligands.

We investigated the A(3)AR affinity and selectivity of a series of 2-substituted 3'-azido and 3'-amino adenosine derivatives as well as some 5'-uronamide derivatives thereof. All compounds showed high A(3)AR selectivity. While the 3'-azides appeared to be A(3)AR antagonists with moderate A(3)AR affinity, their 3'-amino congeners exhibit significantly improved A(3)AR affinity and behave as partial agonists. For both the 3'-azides and the 3'-amines, the 5'-methylcarbamoyl modification improved the overall affinity. Introduction of a 2-phenylethynyl substituent provided high affinity for the A(3)AR.

Exploring human adenosine A3 receptor complementarity and activity for adenosine analogues modified in the ribose and purine moiety.

In this paper we investigated the influence on affinity, selectivity and intrinsic activity upon modification of the adenosine agonist scaffold at the 3'- and 5'-positions of the ribofuranosyl moiety and the 2- and N6-positions of the purine base. This resulted in the synthesis of various analogues, that is, 3-12 and 24-33, with good hA3AR selectivity and moderate-to-high affinities (as in 32, Ki=27 nM). Interesting was the ability to tune the intrinsic activity depending on the substituent introduced at the 3'-position.

Modulation of adenosine receptor affinity and intrinsic efficacy in adenine nucleosides substituted at the 2-position.

We studied the structural determinants of binding affinity and efficacy of adenosine receptor (AR) agonists. Substituents at the 2-position of adenosine were combined with N(6)-substitutions known to enhance human A(3)AR affinity. Selectivity of binding of the analogues and their functional effects on cAMP production were studied using recombinant human A(1), A(2A), A(2B), and A(3)ARs. Mainly sterically small substituents at the 2-position modulated both the affinity and intrinsic efficacy at all subtypes. The 2-cyano group decreased hA(3)AR affinity and efficacy in the cases of N(6)-(3-iodobenzyl) and N(6)-(trans-2-phenyl-1-cyclopropyl), for which a full A(3)AR agonist was converted into a selective antagonist; the 2-cyano-N(6)-methyl analogue was a full A(3)AR agonist. The combination of N(6)-benzyl and various 2-substitutions (chloro, trifluoromethyl, and cyano) resulted in reduced efficacy at the A(1)AR. The environment surrounding the 2-position within the putative A(3)AR binding site was explored using rhodopsin-based homology modeling and ligand docking.

Modeling the adenosine receptors: comparison of the binding domains of A2A agonists and antagonists.

A three-dimensional model of the human A(2A) adenosine receptor (AR) and its docked ligands was built by homology to rhodopsin and validated with site-directed mutagenesis and the synthesis of chemically complementary agonists. Different binding modes of A(2A)AR antagonists and agonists were compared by using the FlexiDock automated docking procedure, with manual adjustment. Putative binding regions for the 9H-purine ring in agonist NECA 3 and the 1H-[1,2,4]triazolo[1,5-c]quinazoline ring in antagonist CGS15943 1 overlapped, and the exocyclic amino groups of each were H-bonded to the side chain of N(6.55). For bound agonist, H-bonds formed between the ribose 3'- and 5'-substituents and the hydrophilic amino acids T(3.36), S(7.42), and H(7.43), and the terminal methyl group of the 5'-uronamide interacted with the hydrophobic side chain of F(6.44). Formation of the agonist complex destabilized the ground-state structure of the A(2A)AR, which was stabilized through a network of H-bonding and hydrophobic interactions in the transmembrane helical domain (TM) regions, facilitating a conformational change upon activation. Both flexibility of the ribose moiety, required for the movement of TM6, and its H-bonding to the receptor were important for agonism. Two sets of interhelical H-bonds involved residues conserved among ARs but not in rhodopsin: (1) E13(1.39) and H278(7.43) and (2) D52(2.50), with the highly conserved amino acids N280(7.45) and S281(7.46), and N284(7.49) with S91(3.39). Most of the amino acid residues lining the putative binding site(s) were conserved among the four AR subtypes. The A(2A)AR/3 complex showed a preference for an intermediate conformation about the glycosidic bond, unlike in the A(3)AR/3 complex, which featured an anti-conformation. Hydrophilic amino acids of TMs 3 and 7 (ribose-binding region) were replaced with anionic residues for enhanced binding to amine-derivatized agonists. We identified new neoceptor (T88D)-neoligand pairs that were consistent with the model.

Thymidine and thymidine-5'-O-monophosphate analogues as inhibitors of Mycobacterium tuberculosis thymidylate kinase.

The affinity of a series of 2', 3'- and 5-modified thymidine analogues for Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt) was evaluated. The affinities of several non-phosphorylated analogues are in the same order of magnitude as those of their phosphorylated congeners. In view of drug delivery problems associated with phosphorylated compounds, these 'free' nucleosides seem more promising leads in the search of TMPKmt inhibitors as novel anti-tuberculosis agents.

Chemotaxonomic features associated with flavonoids of cannabinoid-free cannabis (Cannabis sativa subsp. sativa L.) in relation to hops (Humulus lupulus L.).

The major flavonoids present in the leaves and flowers of the cannabinoid-free cannabis (Cannabis sativa subsp. sativa L.) cultivars Felina and Futura are orientin (1), vitexin (2), luteolin-7-O-beta-D-glucuronide (3), and apigenin-7-O-beta-D-glucuronide (4), while prenylated flavonoids, to which the potent estrogenicity of hops (Humilus lupulus L.) is associated, are absent. The different composition of flavonoids has chemotaxonomic value.