Selectivity For Different Subtypes Research Articles

The effect of differences in the third domain of the glycoprotein E of tick-borne encephalitis virus of the Far Eastern, Siberian and European subtypes on the binding of recombinant D3 proteins with a chimeric antibody

Currently, a therapeutic drug based on recombinant antibodies for the prevention and treatment of tick-borne encephalitis virus (TBEV) is developed in ICBFM SB RAS, and the chimeric antibody ch14D5 is considered as one of the key components of this drug. It was previously shown that this antibody is directed to the domain D3 of the glycoprotein E of TBEV. It was previously shown that this antibody is able to protect mice from the European subtype of TBEV, strain “Absettarov”, and the presence of virus-neutralizing activity against the Far Eastern subtype of TBEV, strain 205 was also shown for this antibody. However, it remains unclear whether this antibody exhibits selectivity for different subtypes of TBEV. The aim of this study was to investigate the effect of amino acid sequence differences of recombinant D3 domains derived from the glycoprotein E of TBEV of the Far Eastern, Siberian and European subtypes on the binding of the protective antibody ch14D5 to these proteins. Using Western blot analysis and surface plasmon resonance, it was shown that ch14D5 antibody has the highest affinity (KD= 1.7±0.5 nM) for the D3 domain of the TBEV of the “Sofjin-Ru” strain belonging to the Far Eastern subtype of the virus. At the same time, the affinity of ch14D5 antibody for similar D3 proteins derived from “Zausaev”, “1528-99” and “Absettarov” strains of the Siberian and European subtypes of TBEV was noticeably lower (KD= 25±4, 300±50, 250±50 nM, respectively). In addition, information about the spatial arrangement of amino acid residues that are different for the studied recombinant proteins indicates that the epitope recognized by the ch14D5 antibody is in close proximity to the lateral ridge of D3 domain of E glycoprotein.

The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential.

Voltage-gated calcium channels are required for many key functions in the body. In this review, the different subtypes of voltage-gated calcium channels are described and their physiologic roles and pharmacology are outlined. We describe the current uses of drugs interacting with the different calcium channel subtypes and subunits, as well as specific areas in which there is strong potential for future drug development. Current therapeutic agents include drugs targeting L-type Ca(V)1.2 calcium channels, particularly 1,4-dihydropyridines, which are widely used in the treatment of hypertension. T-type (Ca(V)3) channels are a target of ethosuximide, widely used in absence epilepsy. The auxiliary subunit α2δ-1 is the therapeutic target of the gabapentinoid drugs, which are of value in certain epilepsies and chronic neuropathic pain. The limited use of intrathecal ziconotide, a peptide blocker of N-type (Ca(V)2.2) calcium channels, as a treatment of intractable pain, gives an indication that these channels represent excellent drug targets for various pain conditions. We describe how selectivity for different subtypes of calcium channels (e.g., Ca(V)1.2 and Ca(V)1.3 L-type channels) may be achieved in the future by exploiting differences between channel isoforms in terms of sequence and biophysical properties, variation in splicing in different target tissues, and differences in the properties of the target tissues themselves in terms of membrane potential or firing frequency. Thus, use-dependent blockers of the different isoforms could selectively block calcium channels in particular pathologies, such as nociceptive neurons in pain states or in epileptic brain circuits. Of important future potential are selective Ca(V)1.3 blockers for neuropsychiatric diseases, neuroprotection in Parkinson's disease, and resistant hypertension. In addition, selective or nonselective T-type channel blockers are considered potential therapeutic targets in epilepsy, pain, obesity, sleep, and anxiety. Use-dependent N-type calcium channel blockers are likely to be of therapeutic use in chronic pain conditions. Thus, more selective calcium channel blockers hold promise for therapeutic intervention.

A Synthetic Combinatorial Strategy for Developing α-Conotoxin Analogs as Potent α7 Nicotinic Acetylcholine Receptor Antagonists

alpha-Conotoxins are peptide neurotoxins isolated from venomous cone snails that display exquisite selectivity for different subtypes of nicotinic acetylcholine receptors (nAChR). They are valuable research tools that have profound implications in the discovery of new drugs for a myriad of neuropharmacological conditions. They are characterized by a conserved two-disulfide bond framework, which gives rise to two intervening loops of extensively mutated amino acids that determine their selectivity for different nAChR subtypes. We have used a multistep synthetic combinatorial approach using alpha-conotoxin ImI to develop potent and selective alpha(7) nAChR antagonists. A positional scan synthetic combinatorial library was constructed based on the three residues of the n-loop of alpha-conotoxin ImI to give a total of 10,648 possible combinations that were screened for functional activity in an alpha(7) nAChR Fluo-4/Ca2+ assay, allowing amino acids that confer antagonistic activity for this receptor to be identified. A second series of individual alpha-conotoxin analogs based on the combinations of defined active amino acid residues from positional scan synthetic combinatorial library screening data were synthesized. Several analogs exhibited significantly improved antagonist activity for the alpha(7) nAChR compared with WT-ImI. Binding interactions between the analogs and the alpha(7) nAChR were explored using a homology model of the amino-terminal domain based on a crystal structure of an acetylcholine-binding protein. Finally, a third series of refined analogs was synthesized based on modeling studies, which led to several analogs with refined pharmacological properties. Of the 96 individual alpha-conotoxin analogs synthesized, three displayed > or =10-fold increases in antagonist potency compared with WT-ImI.

Establishing regiocontrol of disulfide bond isomers of α‐conotoxin ImI via the synthesis of N‐to‐C cyclic analogs

alpha-Conotoxins are multiple disulfide bond containing peptides that are isolated from venomous marine cone snails. They display remarkable selectivity for different subtypes of nicotinic acetylcholine receptors (nAChRs). While alpha-conotoxins display poor resistance to in vivo degradation by proteases, which limits their use as drug leads, N-to-C cyclization via an oligopeptide spacer unit has been previously shown to improve stability. However, the effect of N-to-C cyclization on the formation of the disulfide bond framework is not fully understood. Four N-to-C cyclic analogs of alpha-conotoxin ImI; cImI-A, cImI-betaA, cImI-AG, and cImI-AGG were synthesized to evaluate the effect of oligopeptide spacer length on disulfide bond selectivity and stability to proteolysis. Different ratios of disulfide bond isomers were obtained for each analog using a nonselective random disulfide bond forming strategy, which was dependent on the length of the spacer. To identify each isomer obtained using the random strategy, and to gain access to disulfide bond isomers otherwise unattainable using the random strategy, both the native (globular) and ribbon isomers were synthesized in good yield and purity using a selective orthogonal cysteine protecting group strategy. As such, a random oxidation strategy showed a clear preference for the ribbon isomer in cImI-A. The cyclic globular isomers showed a high resistance to enzymatic degradation compared to the ribbon isomers, with the cImI-A and cImI-AG globular isomers demonstrating the highest stability. These results suggest that cyclization can improve the biochemical stability of conotoxins with potential applications in the development of drugs.

New drugs for the treatment of schizophrenic patients.

Conventional neuroleptics are widely accepted as being effective against the positive symptoms of schizophrenia, but do not benefit all patients. Furthermore, they are relatively ineffective against negative symptoms and cognitive disorders, and most have unpleasant side effect profiles. New strategies for treating schizophrenia include the development of dopamine antagonists with high selectivity for different subtypes of dopamine receptors, dopamine partial agonists, antagonists at different serotonin (5-hydroxytryptamine; 5-HT) receptor subtypes, drugs with mixed pharmacological profiles and drugs which modify transmission via amino acids or peptides in the brain. The prospect is that some of these strategies will lead to the introduction of new drugs and that some of these will become the standards against which future drugs will be compared. The search for new drugs and their use in clinical practice will also lead to developments in our knowledge and understanding of schizophrenia.

Actions of two new antagonists showing selectivity for different sub-types of metabotropic glutamate receptor in the neonatal rat spinal cord.

1. The presynaptic depressant action of L-2-amino-4-phosphonobutyrate (L-AP4) on the monosynaptic excitation of neonatal rat motoneurones has been differentiated from the similar effects produced by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate ((1S,3R)-ACPD), (1S,3S)-ACPD and (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I), and from the postsynaptic motoneuronal depolarization produced by (1S,3R)-ACPD, by the actions of two new antagonists, alpha-methyl-L-AP4 (MAP4) and alpha-methyl-L-CCG-I (MCCG). Such selectivity was not seen with a previously reported antagonist, (+)-alpha-methyl-4-carboxyphenylglycine (MCPG). 2. MAP4 selectively and competitively antagonized the depression of monosynaptic excitation produced by L-AP4 (KD 22 microM). At ten fold higher concentrations, MAP4 also antagonized synaptic depression produced by L-CCG-I but in an apparently non-competitive manner. MAP4 was virtually without effect on depression produced by (1S,3R)- or (1S,3S)-ACPD. 3. MCCG differentially antagonized the presynaptic depression produced by the range of agonists used. This antagonist had minimal effect on L-AP4-induced depression. The antagonism of the synaptic depression effected by (1S,3S)-ACPD and L-CCG-I was apparently competitive in each case but of varying effectiveness, with apparent KD values for the interaction between MCCG and the receptors activated by the two depressants calculated as 103 and 259 microM, respectively. MCCG also antagonized the presynaptic depression produced by (1S,3R)-ACPD. 4. Neither MAP4 nor MCCG (200-500 microM) significantly affected motoneuronal depolarizations produced by (1S,3R)-ACPD. At the same concentrations the two antagonists produced only very weak and variable effects (slight antagonism or potentiation) on depolarizations produced by (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA).5. It is concluded that MAP4 is a potent and selective antagonist for those excitatory amino acid(EAA) receptors on neonatal rat primary afferent terminals that are preferentially activated by L-AP4,and that MCCG is a relatively selective antagonist for different presynaptic EAA receptors that are preferentially activated by (1S,3S)-ACPD and (perhaps less selectively) by L-CCG-I. These receptors probably comprise two sub-types of metabotropic glutamate receptors negatively linked to adenylyl cyclase activity.

Differential antagonism of transmitter release by subtypes of omega-agatoxins

1. The omega-agatoxins from Agelenopsis aperta spider venom are a diverse group of voltage-sensitive calcium channel antagonists. Subtypes of omega-agatoxins are distinguished as type I (omega-Aga-IA, omega-Aga-IB, and omega-Aga-IC), type II (omega-Aga-IIA and omega-Aga-IIB), and type III (omega-Aga-IIIA and omega-Aga-IIIB). All except type III toxins block calcium channels in insect motor nerve terminals and in neuronal cell bodies at nanomolar concentrations. 2. The potency and maximum level of block of the excitatory junctional potential (EJP) by omega-agatoxins are dependent on the extracellular calcium concentration ([Ca]o). Saturating concentrations of type I or II omega-agatoxins block 97-99% of the evoked EJP in low [Ca]o (0.75 mM) saline. A remnant of the EJP (1-3%) that persists after toxin exposure suggests that a small amount of voltage-dependent calcium entry is toxin resistant. When [Ca]o is elevated to 5 mM, this resistant component increases dramatically, revealing differences between type I and type II block. Under these conditions, 60-70% of the EJP is resistant to type I toxins and approximately 20% is resistant to type II toxins. 3. Sequential application of type I and II toxins in high [Ca]o leads to enhanced block of the EJP, suggesting that type I and II omega-agatoxins may block calcium channels by different mechanisms. 4. Type I and type II omega-agatoxins also block calcium channels in the somata of locust dorsal unpaired median (DUM) neurons. In agreement with studies on neuromuscular transmission, block of barium action potentials is incomplete after either type I or type II toxin exposure and combined application of the toxins results in enhanced block. 5. Partial calcium channel antagonism by type I and type II toxins could be explained either by altered kinetics of toxin-modified channels or by selectivity for different subtypes of presynaptic calcium channels.

Antidepressant-like activity of 5-HT1A agonists measured with the forced swim test

This study examined the abilities of 5-hydroxytryptamine (5-HT) agonists with varying selectivity for different subtypes of 5-HT receptors to produce antidepressant-like behavioral effects in the forced swim test in rats. The 5-HT1A agonists 8-OH-DPAT (0.125-1.0 mg/kg, SC) and tandospirone (SM-3997) (5-20 mg/kg, SC) both produced dose-related decreases in immobility time following subchronic treatment in rats. These effects were similar to those of the tricyclic antidepressants imipramine (5-15 mg/kg) and desipramine (5-15 mg/kg). In addition, the 5-HT1A agonists, buspirone (20 mg/kg), gepirone (20 mg/kg) and ipsapirone (10 and 20 mg/kg) demonstrated antidepressant-like effects. Other groups of rats treated subchronically with each of the 5-HT1A agonists or antidepressants showed no increase in locomotor activity, so that general changes in activity could not account for the reduction of immobility time in the forced swim test. 5-HT agonists selective for other receptor subtypes, such as the 5-HT1B/1C agonist m-CPP (5 mg/kg) and the 5-HT2/1C agonist DOB (1 mg/kg), were not effective in this behavioral test. The benzodiazepine diazepam (5 mg/kg) also failed to reduce immobility time, suggesting that anxiolytic properties of 5-HT1A agonists did not mediate this behavioral effect. A common metabolite of some of the 5-HT1A agonists, 1-PP, was ineffective in reducing immobility time. The stimulant d-amphetamine (2 mg/kg) significantly reduced immobility time but also significantly increased locomotor activity.(ABSTRACT TRUNCATED AT 250 WORDS)