Recognition of essential purines by the U1A protein

<p>Abstract</p> <p>Background</p> <p>The RNA recognition motif (RRM) is one of the largest families of RNA binding domains. The RRM is modulated so that individual proteins containing RRMs can specifically recognize RNA targets with diverse sequences and structures. Understanding the principles governing this specificity will be important for the rational modification and design of RRM-RNA complexes.</p> <p>Results</p> <p>In this paper we have investigated the origins of specificity of the N terminal RRM of the U1A protein for stem loop 2 (SL2) of U1 snRNA by substituting modified bases for essential purines in SL2 RNA. In one series of modified bases, hydrogen bond donors and acceptors were replaced by aliphatic groups to probe the importance of these functional groups to binding. In a second series of modified bases, hydrogen bond donors and acceptors were incorrectly placed on the purine bases to analyze the origins of discrimination between cognate and non-cognate RNA. The results of these experiments show that three different approaches are used by the U1A protein to gain specificity for purines. Specificity for the first base in the loop, A1, is based primarily on discrimination against RNA containing the incorrect base, specificity for the fourth base in the loop, G4, is based largely on recognition of the donors and acceptors of G4, while specificity for the sixth base in the loop, A6, results from a combination of direct recognition of the base and discrimination against incorrectly placed functional groups.</p> <p>Conclusion</p> <p>These investigations identify different roles that hydrogen bond donors and acceptors on bases in both cognate and non-cognate RNA play in the specific recognition of RNA by the U1A protein. Taken together with investigations of other RNA-RRM complexes, the results contribute to a general understanding of the origins of RNA-RRM specificity and highlight, in particular, the contribution of steric and electrostatic repulsion to binding specificity.</p

Nicotinic alpha 7 receptor agonists EVP-6124 and BMS-933043, attenuate scopolamine-induced deficits in visuo-spatial paired associates learning - Fig 1

<p>Chemical structures of A) BMS-933043, B) RG3847, C) EVP-6124 and D) donepezil.</p

Comparison of the dose-effect curves to reverse scopolamine-induced impairment of learning in the vsPAL task (% correct eventual).

<p>For each subject, treatment effects on the % correct eventual response were expressed as a difference from the vehicle+scopolamine condition, and group means and SEMs were replotted for side-by-side comparisons. Y axis is percent change from vehicle+scopolamine condition. X axis indicates test condition. Hatched bars indicate a test combination differed significantly from vehicle+scopolamine in post-hoc tests from RM ANOVA dose-response functions (e.g. at <i>p</i><0.05 or <i>p</i><0.01).</p

Effect of treatment with RG3487 on scopolamine-induced impairment of learning in the vsPAL task (N = 8).

<p>Results are presented as the mean ± SEM % eventual correct responses achieved within 6 attempts after presentation of trials with 2-, 3- or 4- stimuli. Results were analyzed by 2 way RM ANOVA followed by Holm-Sidak’s post-hoc analysis; † <i>p<0</i>.<i>05</i> and †† <i>p<0</i>.<i>01</i> compared to vehicle/vehicle treatment.</p

The Novel, Nicotinic Alpha7 Receptor Partial Agonist, BMS-933043, Improves Cognition and Sensory Processing in Preclinical Models of Schizophrenia.

The development of alpha7 nicotinic acetylcholine receptor agonists is considered a promising approach for the treatment of cognitive symptoms in schizophrenia patients. In the present studies we characterized the novel agent, (2R)-N-(6-(1H-imidazol-1-yl)-4-pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'-[1,3]oxazol]-2'-amine (BMS-933043), in vitro and in rodent models of schizophrenia-like deficits in cognition and sensory processing. BMS-933043 showed potent binding affinity to native rat (Ki = 3.3 nM) and recombinant human alpha7 nicotinic acetylcholine receptors (Ki = 8.1 nM) and agonist activity in a calcium fluorescence assay (EC50 = 23.4 nM) and whole cell voltage clamp electrophysiology (EC50 = 0.14 micromolar (rat) and 0.29 micromolar (human)). BMS-933043 exhibited a partial agonist profile relative to acetylcholine; the relative efficacy for net charge crossing the cell membrane was 67% and 78% at rat and human alpha7 nicotinic acetylcholine receptors respectively. BMS-933043 showed no agonist or antagonist activity at other nicotinic acetylcholine receptor subtypes and was at least 300 fold weaker at binding to and antagonizing human 5-HT3A receptors (Ki = 2,451 nM; IC50 = 8,066 nM). BMS-933043 treatment i) improved 24 hour novel object recognition memory in mice (0.1-10 mg/kg, sc), ii) reversed MK-801-induced deficits in Y maze performance in mice (1-10 mg/kg, sc) and set shift performance in rats (1-10 mg/kg, po) and iii) reduced the number of trials required to complete the extradimensional shift discrimination in neonatal PCP treated rats performing the intra-dimensional/extradimensional set shifting task (0.1-3 mg/kg, po). BMS-933043 also improved auditory gating (0.56-3 mg/kg, sc) and mismatch negativity (0.03-3 mg/kg, sc) in rats treated with S(+)ketamine or neonatal phencyclidine respectively. Given this favorable preclinical profile BMS-933043 was selected for further development to support clinical evaluation in humans

BMS-933043 improves 24 h recognition memory in mice.

<p>Subjects were treated 30 min prior to the training session with either A/B) vehicle or low doses of BMS-933043 (n = 11–13/group), C/D) vehicle or high doses of BMS-933043 (n = 10–13/group) or E/F) vehicle or NS-6740 (10 mg/kg, sc) 10 min prior to BMS-933043 (0.3 mg/kg, sc; n = 15–16/group) and then tested for memory retention 24 h later. Results are presented as the mean ± SEM % discrimination index for the training (A, C, E) and test sessions (B, D, F) and were analyzed by ANOVA followed by Dunnett’s test; ** p<0.01, *** p<0.001 versus vehicle or vehicle/BMS-933043 treated mice.</p