CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells.

The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.

Search for influence of spatial properties on affinity at α1-adrenoceptor subtypes for phenylpiperazine derivatives of phenytoin.

A series of phenylpiperazine derivatives of phenytoin was evaluated for their affinity at α(1)-adrenoceptor subtypes in functional bioassays (rat tail artery: α(1A) and/or α(1B); guinea pig spleen: α(1B); rat aorta: α(1D)). The most potent compounds at α(1A)-, α(1B)- and α(1D)-adrenoceptors, 11, 18 and 8, showed affinities in the submicromolar range. The role of a hydrogen bond donor group for affinity and selectivity at α(1B)-adrenoceptors, postulated by Bremner's pharmacophore model, was confirmed by functional and molecular modelling studies.

Pharmacological properties of a wide array of ergolines at functional alpha(1)-adrenoceptor subtypes.

Ergot alkaloids act as (partial) agonists or antagonists at serotonergic, dopaminergic and alpha-adrenergic receptors. In contrast to their affinity at serotonergic (5-HT) and dopaminergic receptor subtypes, only limited information is available concerning their interaction with alpha-adrenoceptor subtypes. This especially holds true for native alpha-adrenoceptors. Therefore, we studied the pharmacological profile of 25 ergolines at alpha(1A)-, alpha(1B)- and alpha(1D)-adrenoceptors in vascular rings or strips of rat and guinea pig endowed with these receptors. Contractile responses were studied by measurement of isometric tension changes in rat tail artery (alpha(1A), alpha(1B)), guinea pig spleen (alpha(1B)) and rat thoracic aorta (alpha(1D)). The anti-migraine drugs ergotamine and dihydroergotamine, the anti-parkinsonian drug lisuride and the anti-hyperprolactinemic drug terguride behaved as antagonists or low-efficacy partial agonists at all three alpha(1)-adrenoceptor subtypes with nanomolar receptor affinity. Derivatives of these drugs showed lower affinity at alpha(1)-adrenoceptors than the parent compounds. Each individual ergoline derivative tested showed low discriminatory capability at the subtypes, alpha(1A), alpha(1B), alpha(1D). A low discriminatory power between the subtypes (alpha(1A), alpha(1B), alpha(1D)) seems to be a class effect of the ergolines. The nanomolar affinities of ergotamine, dihydroergotamine and lisuride for alpha(1)-adrenoceptors may affect their effectiveness as anti-migraine and anti-parkinsonian drugs, respectively.

Agonism at 5-HT2B receptors is not a class effect of the ergolines.

Restrictive cardiac valvulopathies observed in Parkinson patients treated with the ergoline dopamine agonist pergolide have recently been associated with the agonist efficacy of the drug at 5-hydroxytryptamine2B (5-HT2B) receptors. To evaluate whether agonism at 5-HT2B receptors is a phenomenon of the class of the ergolines, we studied 5-HT2B receptor-mediated relaxation in porcine pulmonary arteries to five ergolines which are used as antiparkinsonian drugs. Pergolide and cabergoline were potent full agonists in this tissue (pEC50 8.42 and 8.72). Bromocriptine acted as a partial agonist (pEC50 6.86). Lisuride and terguride, however, failed to relax the arteries but potently antagonized 5-HT-induced relaxation (pKB 10.32 and 8.49). Thus, agonism at 5-HT2B receptors seems not to be a class effect of the ergolines.

Evidence that alpha(1B)-adrenoceptors are involved in noradrenaline-induced contractions of rat tail artery.

The present study characterizes the alpha(1)-adrenoceptor subtypes mediating contractions to noradrenaline in isolated ring preparations of rat tail artery. Concentration-response (E/[A]) curves to noradrenaline were apparently monophasic (pEC(50) 6.47) but became biphasic in the presence of the selective alpha(1A)-adrenoceptor antagonist (+/-)-1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin-5-yl)-1-piperazinyl]propyl]amino]-2,4(1H,3H)-pyrimidinedione (B8805-033). Whereas the first phase of contraction to noradrenaline remained nearly unaffected in the presence of B8805-033 (0.03-3 microM), the second phase was concentration-dependently shifted to the right (pK(B) 8.06). In the presence of B8805-033 (3 microM), noradrenaline-induced contractions (pEC(50) 6.55) were antagonized in a competitive manner by prazosin (pK(B) 9.24), tamsulosin (pK(B) 8.55), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101; pK(B) 7.81), spiperone (pK(B) 7.69), 4-amino-2-[4-[1-(benzyloxycarbonyl)-2(S)-[[(1,1-dimethylethyl)amino]carbonyl]-piperazinyl]-6,7-dimethoxyquinazoline (L-765,314; pK(B) 7.31), 5-methylurapidil (pK(B) 6.55), 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY 7378; pK(B) 6.43), and 8-[2-(1,4-benzodioxan-2-ylmethylamino)ethyl]-8-azaspiro[4.5]decane-7,9-dione (MDL 73005EF; pK(B) 5.71), and were also antagonized by 100 microM chloroethylclonidine. N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethanamine (RS-17053) behaved as a noncompetitive antagonist (apparent pA(2) 6.55). Antagonist affinities obtained under these experimental conditions correlated highly with affinities at native and cloned alpha(1B)-adrenoceptors. Pretreatment of arterial rings with B8805-033 (3 microM) followed by receptor inactivation with chloroethylclonidine (100 microM) yielded monophasic E/[A] curves to noradrenaline (pEC(50) 6.14). Noradrenaline-induced contractions were competitively antagonized by tamsulosin (pK(B) 10.32), 5-methylurapidil (pK(B) 8.66), RS-17053 (pK(B) 8.44), B8805-033 (pK(B) 7.87), BMY 7378 (pK(B) 6.54), and L-765,314 (pK(B) 6.41). Antagonist affinities obtained under these experimental conditions correlated highly with affinities at native and cloned alpha(1A)-adrenoceptors. It is concluded that the contraction to noradrenaline in rat tail artery is mediated by both alpha(1B)- and alpha(1A)-adrenoceptors, each component of contraction being separable by use of selective alpha(1A)-adrenoceptor blockade and alpha(1B)-adrenoceptor alkylation, respectively.