Thomas Wein

The Glutamic Acid-Rich Protein Is a Gating Inhibitor of Cyclic Nucleotide-Gated Channels

The cyclic nucleotide-gated (CNG) cation channel of rod photoreceptors is a heterotetramer consisting of homologous CNGA1 and CNGB1a subunits. While CNGA1 is indispensable for channel activation, the specific role of CNGB1a in this process has remained elusive. Here, we show that the N-terminal glutamic acid-rich protein (GARP) domain of CNGB1a and soluble GARP2, which corresponds to the proximal portion of the GARP domain, act as autoinhibitory domains that decrease the opening probability of the CNG channel. In the presence of mutations that structurally impair the cyclic nucleotide-binding domain (CNBD) of CNGB1a, the GARP domain completely abolishes channel activity. In agreement with an inhibitory function of GARP, the activity of mutant CNG channels could be fully restored by deletion of the GARP domain. We identified two sequences within the GARP domain that confer most of the inhibitory effect and demonstrate that the profound inhibition imposed by the GARP domain is caused by direct and autonomous protein–protein interaction with the CNG channel complex. In wild-type rod CNG channels, this inhibitory effect can be relieved by binding of cGMP to the CNBD of CNGB1a. In conclusion, we propose that the N terminus of CNGB1a and soluble GARPs act as molecular gate keepers that control the activation of heteromeric rod CNG channels. Our results suggest that the GARP domain has evolved in rod photoreceptors to reduce current noise resulting from openings of CNG channels in the absence of cGMP.

Generation of a 3D model for human GABA transporter hGAT-1 using molecular modeling and investigation of the binding of GABA

A three-dimensional model of the human Na+/Cl−-dependent γ-aminobutyric acid (GABA) transporter hGAT-1 was developed by homology modeling and refined by subsequent molecular modeling using the crystal structure of a bacterial homologue leucine transporter from Aquifex aeolicus (LeuTAa) as the template. Protein structure quality checks show that the resulting structure is particularly suited for the analysis of the substrate binding pocket and virtual screening experiments. Interactions of GABA and the substrate binding pocket were investigated using docking studies. The difference of 6 out of 13 substrate interacting side chains between hGAT-1 and LeuTAa lead to the different substrate preference which can be explained using our three-dimensional model of hGAT-1. In particular the replacement of serine 256 and isoleucine 359 in LeuTAa with glycine and threonine in hGAT-1 seems to facilitate the selection of GABA as the main substrate by changing the hydrogen bonding pattern in the active site to the amino group of the substrate. For a set of 12 compounds flexible docking experiments were performed using LigandFit in combination with the Jain scoring function. With few exceptions the obtained rank order of potency was in line with experimental data. Thus, the method can be assumed to give at least a rough estimate of the potency of the potential of GABA uptake inhibitors.

Different Binding Modes of Small and Large Binders of GAT1.

Well‐known inhibitors of the γ‐aminobutyric acid (GABA) transporter GAT1 share a common scaffold of a small cyclic amino acid linked by an alkyl chain to a moiety with two aromatic rings. Tiagabine, the only FDA‐approved GAT1 inhibitor, is a typical example. Some small amino acids such as (R)‐nipecotic acid are medium‐to‐strong binders of GAT1, but similar compounds, such as proline, are very weak binders. When substituted with 4,4‐diphenylbut‐3‐en‐1‐yl (DPB) or 4,4‐bis(3‐methylthiophen‐2‐yl)but‐3‐en‐1‐yl (BTB) groups, the resulting compounds have similar pKi and pIC50 values, even though the pure amino acids have very different values. To investigate if small amino acids and their substituted counterparts share a similar binding mode, we synthesized butyl‐, DPB‐, and BTB‐substituted derivatives of small amino acids. Supported by the results of docking studies, we propose different binding modes not only for unsubstituted und substituted, but also for strong‐ and weak‐binding amino acids. These data lead to the conclusion that following a fragment‐based approach, not pure but N‐butyl‐substituted amino acids should be used as starting points, giving a better estimate of the activity when a BTB or DPB substituent is added.

Development of Highly Potent GAT1 Inhibitors: Synthesis of Nipecotic Acid Derivatives by Suzuki–Miyaura Cross‐Coupling Reactions

A new series of potent and selective mGAT1 inhibitors has been identified, featuring a nipecotic acid residue and an N‐butenyl linker with a 2‐biphenyl residue at the ω‐position. Docking, combined with MD calculations, revealed a binding mode for the new compounds similar to that of tiagabine, the only mGAT1 inhibitor currently approved as antiepileptic drug. For the synthesis, a Suzuki–Miyaura cross‐coupling reaction was used as a key step by which variously substituted biaryl subunits were assembled. Biological evaluation revealed several compounds that possess binding affinities and inhibitory potencies toward mGAT1, together with subtype selectivities against mGAT2–mGAT4 that were similar to or even higher than those for tiagabine. A derivative carrying the 2′,4′‐dichloro‐2‐biphenyl moiety attached to N‐but‐3‐enylnipecotic acid at the terminal position of the linker chain was found to be the most potent binder, with the racemic form of the compound displaying a binding affinity of 8.05±0.13 (pKi), while the R enantiomer exhibited an affinity value of 8.33±0.06 (pKi).

Synthesis of 4-substituted nipecotic acid derivatives and their evaluation as potential GABA uptake inhibitors.

In this study, we disclose the design and synthesis of novel 4-susbtituted nipecotic acid derivatives as inhibitors of the GABA transporter mGAT1. Based on molecular modeling studies the compounds are assumed to adopt a binding pose similar to that of the potent mGAT1 inhibitor nipecotic acid. As substitution in 4-position should not cause an energetically unfavorable orientation of nipecotic acid as it is the case for N-substituted derivatives this is expected to lead to highly potent binders. For the synthesis of novel 4-substituted nipecotic acid derivatives a linear synthetic strategy was employed. As a key step, palladium catalyzed cross coupling reactions were used to attach the required biaryl moieties to the ω-position of the alkenyl- or alkynyl spacers of varying length in the 4-position of the nipecotic acid scaffold. The resulting amino acids were characterized with respect to their binding affinities and inhibitory potencies at mGAT1. Though the biological activities found were generally insignificant to poor, two compounds, one of which possesses a reasonable binding affinity for mGAT1, rac-57, the other a notable inhibitory potency at mGAT4, rac-84, both displaying a slight subtype selectivity for the individual transporters, could be identified.

Searching for putative binding sites of the bispyridinium compound MB327 in the nicotinic acetylcholine receptor

Irreversible inhibition of the acetylcholine esterase upon intoxication with organophosphorus compounds leads to an accumulation of acetylcholine in the synaptic cleft and a subsequent desensitization of nicotinic acetylcholine receptors which may ultimately result in respiratory failure. The bispyridinium compound MB327 has been found to restore functional activity of nAChR thus representing a promising starting point for the development of new drugs for the treatment of organophosphate poisoning. In order to optimize the resensitizing effect of MB327 on nAChR, it would be very helpful to know the MB327 specific binding site to apply structure based molecular modeling. The binding site for MB327 at the nAChR is not known and so far goal of speculations, but it has been shown that MB327 does not bind to the orthosteric acetylcholine binding site. We have used docking calculations to screen the surface of nAChR for possible binding sites of MB327. The results indicate that at least two potential binding sites for MB327 at nAChR are present inside the channel pore. In these binding sites, MB327 intercalates between the γ-α and β-δ subunits of nAChR, respectively. Both putative MB327 binding sites show an unsymmetrical distribution of surrounding hydrophilic and lipophilic amino acids. This suggests that substitution of MB327-related bispyridinium compounds on one of the two pyridinium rings with polar substituents should have a favorable effect on the pharmacological function.

Development of New Photoswitchable Azobenzene Based γ-Aminobutyric Acid (GABA) Uptake Inhibitors with Distinctly Enhanced Potency upon Photoactivation

A series of nipecotic acid derivatives with new azo benzene based photoswitchable N-substituents was synthesized and characterized in their ( E)- and ( Z)-form for their functional inhibitory activity at γ-aminobutyric acid transporters subtype 1 (GAT1), the most common γ-aminobutyric acid (GABA) transporter subtype in the central nervous system (CNS). This led to the identification of the first photoswitchable ligands exhibiting a moderate uptake inhibition of GABA in their ( E)- but distinctive higher inhibitory potency in their ( Z)-form resulting from photoirradiation. For the most efficient photoactivatable nipecotic acid derivative displaying an N-but-3-yn-1-yl linker with a terminal diphenyldiazene unit, an inhibitory potency of 4.65 ± 0.05 (pIC50) was found for its ( E)-form. which increased by almost two log units up to 6.38 ± 0.04 when irradiated. The effect of this photoswitchable mGAT1 inhibitor has also been evaluated and confirmed in patch-clamp recordings in acute hippocampal slices from mice.

Synthesis of a Series of Non-Symmetric Bispyridinium and Related Compounds and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor

The current standard therapy to counteract organophosphate intoxication is not effective in equal measure against all types of organophosphorus compounds (OPCs), as the outcome of oxime‐induced reactivation of inactivated acetylcholinesterase (AChE) strongly depends on the particular OPC. In case the reactivation is insufficient, acetylcholine concentrations that rise to pathophysiological levels force the nicotinic acetylcholine receptor (nAChR) into a desensitized state and hence a functionally inactive state. As a consequence, neurotransmission is irreversibly disrupted at the neuromuscular junction. Previous electrophysiological studies identified the symmetric bispyridinium compound 1,1′‐(propane‐1,3‐diyl)bis[4‐(tert‐butyl)pyridin‐1‐ium] diiodide (MB327) as a re‐sensitizer of the desensitized nAChR. MB327 is thereby capable of restoring the functional activity. Very recently, in silico modeling studies suggested non‐symmetric derivatives of MB327 as potential re‐sensitizers with enhanced binding affinity and thus possible enhanced efficacy. In this study, 26 novel non‐symmetric bispyridinium compounds and related derivatives were synthesized. For the synthesis of the highly polar target compounds in sufficient quantities, newly developed and highly efficient two‐step procedures were used. Compounds were characterized in terms of their binding affinity toward the MB327 binding site at the nAChR using recently developed mass spectrometry (MS) Binding Assays. Regarding structure–affinity relationships at the MB327 binding site, the presence of two quaternary aromatic nitrogen centers as well as pyridinium systems with a tert‐butyl group at the 4‐position or a NMe2 group at the 3‐ or 4‐positions appeared to be beneficial for high binding affinities.

Synthesis of a Series of Structurally Diverse MB327 Derivatives and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor

A novel series of 30 symmetric bispyridinium and related N‐heteroaromatic bisquaternary salts with a propane‐1,3‐diyl linker was synthesized and characterized for their binding affinity at the MB327 binding site of nicotinic acetylcholine receptor (nAChR) from Torpedo californica. Compounds targeting this binding site are of particular interest for research into new antidotes against organophosphate poisoning, as therapeutically active 4‐tert‐butyl‐substituted bispyridinium salt MB327 was previously identified as a nAChR re‐sensitizer. Efficient access to the target compounds was provided by newly developed methods enabling N‐alkylation of sterically hindered or electronically deactivated heterocycles exhibiting a wide variety of functional groups. Determination of binding affinities toward the MB327 binding site at the nAChR, using a recently developed mass spectrometry (MS)‐based Binding Assay, revealed that several compounds reached affinities similar to that of MB327 (pKi=4.73±0.03). Notably, the newly prepared lipophilic 4‐tert‐butyl‐3‐phenyl‐substituted bispyridinium salt PTM0022 (3 h) was found to have significantly higher binding affinity, with a pKi value of 5.16±0.07, thus representing considerable progress toward the development of more potent nAChR re‐sensitizers.

Development of MS Binding Assays targeting the binding site of MB327 at the nicotinic acetylcholine receptor

The bispyridinium compound MB327 has been shown previously to have a positive pharmacological effect against poisoning with organophosphorous compounds (OPCs). The mechanism by which it exerts its therapeutic effect seems to be directly mediated by the nicotinic acetylcholine receptor (nAChR). In the present study, the development of mass spectrometry based binding assays (MS Binding Assays) for characterization of the binding site of MB327 at the nAChR from Torpedo californica is described. MS Binding Assays follow the principle of radioligand binding assays, but do not, in contrast to the latter, require a radiolabeled reporter ligand, as the readout is in this case based on mass spectrometric detection. For [2H6]MB327, a deuterated MB327 analogue employed as reporter ligand in the MS Binding Assays, an LC-ESI-MS/MS method was established allowing for its fast and reliable quantification in samples resulting from binding experiments. Using centrifugation for separation of non-bound [2H6]MB327 from target-bound [2H6]MB327 in saturation and autocompetition experiments (employing native MB327 as competitor) enabled reliable determination of specific binding. In this way, the affinities for [2H6]MB327 (Kd=15.5±0.9μmolL-1) and for MB327 (Ki=18.3±2.6μmolL-1) towards the nAChR could be determined for the first time. The almost exactly matching affinities for MB327 and [2H6]MB327 obtained in the MS Binding Assays are in agreement with potencies previously found in functional studies. In summary, our results demonstrate that the established MS Binding Assays represent a promising tool for affinity determination of test compounds towards the binding site of MB327 at the nAChR.