MS Binding Assays for Glycine Transporter 2 (GlyT2) Employing Org25543 as Reporter Ligand

This study describes the first binding assay for glycine transporter 2 (GlyT2) following the concept of MS Binding Assays. The selective GlyT2 inhibitor Org25543 was employed as a reporter ligand and it was quantified with a highly sensitive and rapid LC-ESI-MS/MS method. Binding of Org25543 at GlyT2 was characterized in kinetic and saturation experiments with an off-rate of 7.07×10⁻³ s⁻¹, an on-rate of 1.01×10⁶ M⁻¹ s⁻¹, and an equilibrium dissociation constant of 7.45 nM. Furthermore, the inhibitory constants of 19 GlyT ligands were determined in competition experiments. The validity of the GlyT2 affinities determined with the binding assay was examined by a comparison with published inhibitory potencies from various functional assays. With the capability for affinity determination towards GlyT2 the developed MS Binding Assays provide the first tool for affinity profiling of potential ligands and it represents a valuable new alternative to functional assays addressing GlyT2.     

MS-Based Screening of 5-Substituted Nipecotic Acid Derived Hydrazone Libraries as Ligands of the GABA Transporter 1

A screening of compound libraries based on nipecotic acid derivatives with lipophilic residues attached to the scarcely explored 5-position of the core structure was used for the search of new inhibitors of the γ-aminobutyric acid (GABA) transporter 1 (mGAT1). The generated compound libraries, which were based on hydrazone chemistry commonly used in dynamic combinatorial chemistry but rendered pseudostatic, were screened for their binding affinities toward mGAT1 by means of MS Binding Assays. With nipecotic acid derived hydrazone rac- 16 h [rac-(3R,5S)-{5-[(E)-2-{[5-(2-phenylethynyl)thiophen-2-yl]methylidene}hydrazin-1-yl]piperidine-3-carboxylic acid}-sodium chloride (1/2)], one hit was found and evaluated displaying sub-micromolar potency (pK_i=6.62±0.04) and a noncompetitive interaction mode at mGAT1. By bearing a 5-(2-phenylethynyl)thiophen-2-yl residue attached to the 5-position of nipecotic acid via a three-atom spacer, compound rac- 16 h contains a structural moiety so far unprecedented for these kinds of bioactive molecules, and complements novel 5-substituted nipecotic acid derived ligands of mGAT1 revealed in a recently published screening campaign. This new class of ligands, with an inhibition mode distinct from that of benchmark mGAT1 inhibitors, could serve as research tools for investigations of mGAT1-mediated GABA transport.     

Simultaneous Multiple MS Binding Assays for the Dopamine,Norepinephrine, and Serotonin Transporters

In this work, we present label‐free, mass‐spectrometry‐based binding assays (MS Binding Assays), targeting the human dopamine, norepinephrine, and serotonin transporters (hDAT, hNET, and hSERT) in simultaneous binding experiments. Using a validated LC–ESI‐MS/MS method for quantification of the selective dopamine transporter inhibitor (R,R)‐4‐(2‐benzhydryloxyethyl)‐1‐(4‐fluorobenzyl)piperidin‐3‐ol ((R,R)‐D‐84), the selective norepinephrine transporter inhibitor (S,S)‐reboxetine, and the selective serotonin reuptake inhibitor (S)‐citalopram, binding affinities at the three monoamine transporters could be characterized simultaneously in a single binding experiment. The performed simultaneous saturation and competition experiments yielded results that are in good accordance with those determined in MS Binding Assays addressing the monoamine transporters individually. The results obtained from this study underscore the potential of MS Binding Assays for simultaneous affinity determination at different targets, which is difficult to accomplish with conventional radioligand binding assays.     

De Novo Design,Synthesis and Evaluation of Benzylpiperazine Derivatives as Highly Selective Binders of Mcl‐1

Considerable efforts have been made to the development of small‐molecule inhibitors of antiapoptotic B‐cell lymphoma 2 (Bcl‐2) family proteins (such as Bcl‐2, Bcl‐x_L, and Mcl‐1) as a new class of anticancer therapies. Unlike general inhibitors of the entire family, selective inhibitors of each member protein can hopefully reduce the adverse side effects in chemotherapy treatments of cancers overexpressing different Bcl‐2 family proteins. In this study, we designed four series of benzylpiperazine derivatives as plausible Bcl‐2 inhibitors based on the outcomes of a computational algorithm. A total of 81 compounds were synthesized, and their binding affinities to Bcl‐2, Bcl‐x_L, and Mcl‐1 measured. Encouragingly, 22 compounds exhibited binding affinities in the micromolar range (K_i<20 μM ) to at least one target protein. Moreover, some compounds were observed to be highly selective binders to Mcl‐1 with no detectable binding to Bcl‐2 or Bcl‐x_L, among which the most potent one has a K_i value of 0.18 μM for Mcl‐1. Binding modes of four selected compounds to Mcl‐1 and Bcl‐x_L were derived through molecular docking and molecular dynamics simulations. It seems that the binding affinity and selectivity of these compounds can be reasonably interpreted with these models. Our study demonstrated the possibility for obtaining selective Mcl‐1 inhibitors with relatively simple chemical scaffolds. The active compounds identified by us could be used as lead compounds for developing even more potent selective Mcl‐1 inhibitors with potential pharmaceutical applications.     

MS Binding Assays for D1 and D5 Dopamine Receptors

MS Binding Assays are a label‐free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but an unlabeled reporter ligand is used instead of a radioligand. The study presented herein describes the development of MS Binding Assays that address D₁ and D₅ dopamine receptors. A highly sensitive, rapid and robust LC–ESI‐MS/MS quantification method for the selective D₁ dopamine receptor antagonist SCH23390 ((5R)‐8‐chloro‐3‐methyl‐5‐phenyl‐1,2,4,5‐tetrahydro‐3‐benzazepin‐7‐ol) was established and validated, using its 8‐bromo analogue SKF83566 as an internal standard. This quantification method proved to be suitable for the characterization of SCH23390 binding to human D₁ and D₅ receptors. Following the concept of MS Binding Assays, saturation experiments for D₁ and D₅ receptors were performed, as well as competition experiments for D₁ receptors. The results obtained are in good agreement with results from radioligand binding assays and therefore indicate that the established MS Binding Assays addressing D₁ and D₅ receptors are well‐suited substitutes for radioligand binding assays, the technique that has so far dominated affinity determinations toward these targets.     

Generation and Screening of Oxime Libraries Addressing the Neuronal GABA Transporter GAT1

The objective of the present study was to transfer the concept of library screening by MS binding assays, so far applied to pseudostatic hydrazine libraries, to static oxime libraries to screen for new potent inhibitors of mGAT1, the most abundant GABA transporter in the central nervous system that represents a validated drug target for the treatment of epilepsy. Library generation was performed by reaction of guvacine derivatives possessing a hydroxylamine functionality with various sets of four aldehydes. After dilution, the libraries were screened by competitive MS binding assays. Deconvolution experiments allowed hits in the most active libraries to be identified, and they were resynthesized for biological evaluation. That way a series of compounds was identified that displayed binding affinities ≥8.00 (pK_i) at mGAT1, one of which was found to be the most potent mGAT1 inhibitor known to date in a functional GABA uptake assay with a pIC₅₀ value of 8.27±0.03.     

Library Screening by Means of Mass Spectrometry (MS) Binding Assays-Exemplarily Demonstrated for a Pseudostatic Library Addressing γ-Aminobutyric Acid (GABA) Transporter 1 (GAT1)

In the present study, the application of mass spectrometry (MS) binding assays as a tool for library screening is reported. For library generation, dynamic combinatorial chemistry (DCC) was used. These libraries can be screened by means of MS binding assays when appropriate measures are taken to render the libraries pseudostatic. That way, the efficiency of MS binding assays to determine ligand binding in compound screening with the ease of library generation by DCC is combined. The feasibility of this approach is shown for γ‐aminobutyric acid (GABA) transporter 1 (GAT1) as a target, representing the most important subtype of the GABA transporters. For the screening, hydrazone libraries were employed that were generated in the presence of the target by reacting various sets of aldehydes with a hydrazine derivative that is delineated from piperidine‐3‐carboxylic acid (nipecotic acid), a common fragment of known GAT1 inhibitors. To ensure that the library generated is pseudostatic, a large excess of the nipecotic acid derivative is employed. As the library is generated in a buffer system suitable for binding and the target is already present, the mixtures can be directly analyzed by MS binding assays—the process of library generation and screening thus becoming simple to perform. The binding affinities of the hits identified by deconvolution were confirmed in conventional competitive MS binding assays performed with single compounds obtained by separate synthesis. In this way, two nipecotic acid derivatives exhibiting a biaryl moiety, 1‐{2‐[2′‐(1,1’‐biphenyl‐2‐ylmethylidene)hydrazine]ethyl}piperidine‐3‐carboxylic acid and 1‐(2‐{2′‐[1‐(2‐thiophenylphenyl)methylidene]hydrazine}ethyl)piperidine‐3‐carboxylic acid, were found to be potent GAT1 ligands exhibiting pK_i values of 6.186±0.028 and 6.229±0.039, respectively. This method enables screening of libraries, whether generated by conventional chemistry or DCC, and is applicable to all kinds of targets including membrane‐bound targets such as G protein coupled receptors (GPCRs), ion channels and transporters. As such, this strategy displays high potential in the drug discovery process.     

MS Binding Assays for the Three Monoamine Transporters Using the Triple Reuptake Inhibitor (1R,3S)‐Indatraline as Native Marker

We herein present label‐free, mass‐spectrometry‐based binding assays (MS Binding Assays) for the human dopamine, norepinephrine, and serotonin transporters (hDAT, hNET, and hSERT). Using this approach both enantiomers of the triple reuptake inhibitor indatraline as well as its cis‐configured diastereomer were investigated toward hDAT, hNET, and hSERT in saturation experiments. The dissociation rate constants for (1R,3S)‐indatraline binding at hDAT, hNET, and hSERT were determined in kinetic studies. These experiments revealed an allosteric effect of clomipramine on the dissociation of (1R,3S)‐indatraline from hSERT. Finally, a comprehensive set of known monoamine transport inhibitors and substrates was studied in competition experiments at hDAT, hNET, and hSERT, using (1R,3S)‐indatraline as nonlabeled marker. The results are in excellent agreement with those reported for radioligand binding assays. Therefore, the established MS Binding Assays are a promising alternative to the latter for the characterization of new monoamine reuptake inhibitors at DAT, NET, and SERT.     

Discovery and development of thiazolo[3,2-a]pyrimidinone derivatives as general inhibitors of Bcl-2 family proteins

A class of compounds with a common thiazolo[3,2‐a]pyrimidinone motif has been developed as general inhibitors of Bcl‐2 family proteins. The lead compound was originally identified in a random screening of a small compound library using a fluorescence polarization‐based competitive binding assay. Its binding to the Bcl‐x_L protein was further confirmed by ¹⁵N‐HSQC NMR experiments. Structural modifications on the lead compound were guided by the outcomes of molecular modeling studies. Among the 42 compounds obtained, a number of them exhibited much improved binding affinities to Bcl‐2 family proteins as compared to the lead compound. The most potent compound, BCL‐LZH‐ 40 , inhibited the binding of BH3 peptides to Bcl‐x_L, Bcl‐2, and Mcl‐1 with inhibition constants (K_i) of 17, 534, and 200 nM , respectively.     

A Novel Approach to the Discovery of Small-Molecule Ligands of CDK2

In an attempt to identify novel small-molecule ligands of cyclin-dependent kinase 2 (CDK2) with potential as allosteric inhibitors, we have devised a robust and cost-effective fluorescence-based high-throughput screening assay. The assay is based on the specific interaction of CDK2 with the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS), which binds to a large allosteric pocket adjacent to the ATP site. Hit compounds that displace ANS directly or indirectly from CDK2 are readily classified as ATP site binders or allosteric ligands through the use of staurosporine, which blocks the ATP site without displacing ANS. Pilot screening of 1453 compounds led to the discovery of 12 compounds with displacement activities (EC(50) values) ranging from 6 to 44 μM, all of which were classified as ATP-site-directed ligands. Four new type I inhibitor scaffolds were confirmed by X-ray crystallography. Although this small compound library contained only ATP-site-directed ligands, the application of this assay to large compound libraries has the potential to reveal previously unrecognized chemical scaffolds suitable for structure-based design of CDK2 inhibitors with new mechanisms of action.     

A Virtual Screening Study of the 18 kDa Translocator Protein using Pharmacophore Models Combined with 3D‐QSAR Studies

In the present study, we considered various pharmacophore hypotheses for TSPO ligands and an optimal one was selected on the basis of 3D‐QSAR studies. This hypothesis was used in a ligand‐based virtual screening study on the Maybridge database with the aim of identifying new TSPO ligands. Binding assays revealed that all selected compounds displayed TSPO affinity at 10 μM , and among them two compounds exhibited sub‐micromolar K_i values. These results validated our applied methodologies, and the two compounds with sub‐micromolar affinity could be used as interesting leads for the development of new active TSPO ligands.     

Characterization of the Stereochemical Structures of 2H‐Thiazolo[3,2‐a]pyrimidine Compounds and Their Binding Affinities for Anti‐apoptotic Bcl‐2 Family Proteins

In a previous study we reported a class of compounds with a 2H‐thiazolo[3,2‐a]pyrimidine core structure as general inhibitors of anti‐apoptotic Bcl‐2 family proteins. However, the absolute stereochemical configuration of one carbon atom on the core structure remained unsolved, and its potential impact on the binding affinities of compounds in this class was unknown. In this study, we obtained pure R and S enantiomers of four selected compounds by HPLC separation and chiral synthesis. The absolute configurations of these enantiomers were determined by comparing their circular dichroism spectra to that of an appropriate reference compound. In addition, a crystal structure of one selected compound revealed the exocyclic double bond in these compounds to be in the Z configuration. The binding affinities of all four pairs of enantiomers to Bcl‐x_L, Bcl‐2, and Mcl‐1 proteins were measured in a fluorescence‐polarization‐based binding assay, yielding inhibition constants (K_i values) ranging from 0.24 to 2.20 μM . Interestingly, our results indicate that most R and S enantiomers exhibit similar binding affinities for the three tested proteins. A binding mode for this compound class was derived by molecular docking and molecular dynamics simulations to provide a reasonable interpretation of this observation.     

Development of 3‐Phenyl‐N‐(2‐(3‐phenylureido)ethyl)‐thiophene‐2‐sulfonamide Compounds as Inhibitors of Antiapoptotic Bcl‐2 Family Proteins

Antiapoptotic Bcl‐2 family proteins, such as Bcl‐x_L, Bcl‐2, and Mcl‐1, are often overexpressed in tumor cells, which contributes to tumor cell resistance to chemotherapies and radiotherapies. Inhibitors of these proteins thus have potential applications in cancer treatment. We discovered, through structure‐based virtual screening, a lead compound with micromolar binding affinity to Mcl‐1 (inhibition constant (K_i)=3 μM ). It contains a phenyltetrazole and a hydrazinecarbothioamide moiety, and it represents a structural scaffold not observed among known Bcl‐2 inhibitors. This work presents the structural optimization of this lead compound. By following the scaffold‐hopping strategy, we have designed and synthesized a total of 82 compounds in three sets. All of the compounds were evaluated in a fluorescence‐polarization binding assay to measure their binding affinities to Bcl‐x_L, Bcl‐2, and Mcl‐1. Some of the compounds with a 3‐phenylthiophene‐2‐sulfonamide core moiety showed sub‐micromolar binding affinities to Mcl‐1 (K_i=0.3–0.4 μM ) or Bcl‐2 (K_i≈1 μM ). They also showed obvious cytotoxicity on tumor cells (IC₅₀<10 μM ). Two‐dimensional heteronuclear single quantum coherence NMR spectra of three selected compounds, that is, YCW‐E5, YCW‐E10, and YCW‐E11, indicated that they bind to the BH3‐binding groove on Bcl‐x_L in a similar mode to ABT‐737. Several apoptotic assays conducted on HL‐60 cells demonstrated that these compounds are able to induce cell apoptosis through the mitochondrial pathway. We propose that the compounds with the 3‐phenylthiophene‐2‐sulfonamide core moiety are worth further optimization as effective apoptosis inducers with an interesting selectivity towards Mcl‐1 and Bcl‐2.     

Synthesis and Biological Evaluation of Nipecotic Acid and Guvacine Derived 1,3-Disubstituted Allenes as Inhibitors of Murine GABA Transporter mGAT1

A new class of nipecotic acid and guvacine derivatives has been synthesized and characterized for their inhibitory potency at mGAT1–4 and binding affinity for mGAT1. Compounds of the described class are defined by a four-carbon-atom allenyl spacer connecting the nitrogen atom of the nipecotic acid or guvacine head with an aromatic residue. Among the compounds investigated, the mixture of nipecotic acid derivatives rac-{(R_a)-1-[4-([1,1′:2′,1′′-terphenyl]-2-yl)buta-2,3-dien-1-yl](3R)-piperidine-3-carboxylic acid} and rac-{(S_a)-1-[4-([1,1′:2′,1′′-terphenyl]-2-yl)buta-2,3-dien-1-yl](3R)-piperidine-3-carboxylic acid} ( 21 p ), possessing an o-terphenyl residue, was identified as highly selective and the most potent mGAT1 inhibitor in this study. For the (R)-nipecotic acid derived form of 21 p , the inhibitory potency in [³H]GABA uptake assays was determined as pIC₅₀=6.78±0.08, and the binding affinity in MS Binding Assays as pK_i=7.10±0.12. The synthesis of the designed compounds was carried out by a two-step procedure, generating the allene moiety via allenylation of terminal alkynes which allows broad variation of the terminal phenyl and biphenyl subunit.     

Synthesis and Pharmacological Evaluation of σ2 Receptor Ligands Based on a 3-Alkoxyisoxazole Scaffold: Potential Antitumor Effects against Osteosarcoma

Since its initial discovery as the basis for nicotinic acetylcholine receptor ligands, the 3-alkoxyisoxazole scaffold has been shown to be a versatile platform for the development of potent σ1 and σ2 receptor ligands. Herein we report a further SAR exploration of the 3-alkoxyisoxazole scaffold with the aim of obtaining potent σ2 receptor ligands. Various substitutions on the benzene ring and at the basic amino regions resulted in a total of 21 compounds that were tested for their binding affinities for the σ2 receptor. In particular, compound 51 [(2S)-1-(4-ammoniobutyl)-2-(((5-((3,4-dichlorophenoxy)methyl)isoxazol-3-yl)oxy)methyl)pyrrolidin-1-ium chloride] was identified as one of the most potent σ2 ligands within the series, with a K_i value of 7.9 nM. It demonstrated potent antiproliferative effects on both osteosarcoma cell lines 143B and MOS−J (IC₅₀ values of 0.89 and 0.71 μM, respectively), relative to siramesine (IC₅₀ values of 1.81 and 2.01 μM). Moreover, compound 51 inhibited clonal formation of osteosarcoma 143B cells at 1 μM, corresponding to half the dose required of siramesine for similar effects. The general cytotoxicity profile of compound 51 was assessed in a number of normal cell lines, including HaCaT, HAF, and LO2 cells. Furthermore, FACS analysis showed that compound 51 likely inhibits osteosarcoma cell growth by disruption of the cell cycle and promotion of apoptosis.     

Discovery of Non‐ATP‐Competitive Inhibitors of Polo‐like Kinase 1

Polo‐like kinase 1 (Plk1) is an evolutionarily conserved serine/threonine kinase, and its N‐terminal kinase domain (KD) controls cell signaling through phosphorylation. Inhibitors of Plk1 are potential anticancer drugs. Most known Plk1 KD inhibitors are ATP‐competitive compounds, which may suffer from low selectivity. In this study we discovered novel non‐ATP‐competitive Plk1 KD inhibitors by virtual screening and experimental studies. Potential binding sites in Plk1 KD were identified by using the protein binding site detection program Cavity. The identified site was subjected to molecular‐docking‐based virtual screening. The activities of top‐ranking compounds were evaluated by in vitro enzyme assay with full‐length Plk1 and direct binding assay with Plk1 KD. Several compounds showed inhibitory activity, and the most potent was found to be 3‐((2‐oxo‐2‐(thiophen‐2‐yl)ethyl)thio)‐6‐(pyridin‐3‐ylmethyl)‐1,2,4‐triazin‐5(4H)‐one (compound 4 ) with an IC₅₀ value of 13.1±1.7 μm . Our work provides new insight into the design of kinase inhibitors that target non‐ATP binding sites.     

High-Throughput Screening Identifies Ascorbyl Palmitate as a SIRT2 Deacetylase and Defatty-Acylase Inhibitor

Small-molecule inhibitors of the human sirtuin SIRT2 are being developed because of their therapeutic potential in a variety of diseases. Here, we developed a high-throughput screen to identify novel SIRT2 inhibitors using a fluorescent SIRT2 probe, 1-aminoanthracene (AMA). AMA has high fluorescence when bound to SIRT2, and its fluorescence reduces >10-fold when it is displaced from SIRT2 by other ligands. We used this property of AMA to screen a library of known bioactive compounds for SIRT2 binding and discovered two known pharmaceutical compounds that bind SIRT2 with K_d values in the low μM range, ascorbyl palmitate and pictilisib. Both compounds inhibit the deacetylase and defatty-acylase activities of SIRT2. While pictilisib has selectivity for SIRT2, ascorbyl palmitate also inhibits the enzymatic activities of SIRT1 and SIRT6. Finally, we show that ascorbyl palmitate inhibits SIRT2 deacetylase and defatty-acylase activities in cells, and SIRT2 inhibition by ascorbyl palmitate contributes to the cytotoxicity of the compound. Our work discovered novel SIRT2 deacylase inhibitors and presents a screening approach that can be applied on a larger scale.     

Structure‐Based Virtual Screening for Dopamine D2 Receptor Ligands as Potential Antipsychotics

Structure‐based virtual screening using a D₂ receptor homology model was performed to identify dopamine D₂ receptor ligands as potential antipsychotics. From screening a library of 6.5 million compounds, 21 were selected and were subjected to experimental validation. From these 21 compounds tested, ten D₂ ligands were identified (47.6 % success rate, among them D₂ receptor antagonists, as expected) that have additional affinity for other receptors tested, in particular 5‐HT_2A receptors. The affinity (K_i values) of the compounds ranged from 58 nm to about 24 μm . Similarity and fragment analysis indicated a significant degree of structural novelty among the identified compounds. We found one D₂ receptor antagonist that did not have a protonatable nitrogen atom, which is a key structural element of the classical D₂ pharmacophore model necessary for interaction with the conserved Asp(3.32) residue. This compound exhibited greater than 20‐fold binding selectivity for the D₂ receptor over the D₃ receptor. We provide additional evidence that the amide hydrogen atom of this compound forms a hydrogen bond with Asp(3.32), as determined by tests of its derivatives that cannot maintain this interaction.     

Synthesis and Pharmacological Evaluation of SNC80 Analogues with a Bridged Piperazine Ring

To discover novel δ‐opioid receptor ligands derived from SNC80 ( 1 ), a series of 6,8‐diazabicyclo[3.2.2]nonane derivatives bearing two aromatic moieties was prepared, and the affinity toward δ, μ, and κ receptors, as well as σ receptors, was investigated. After removal of the 4‐methoxybenzyl and 2,4‐dimethoxybenzyl protecting groups, the pharmacophoric N,N‐diethylcarbamoylbenzyl residue was attached to the 6,8‐diazabicyclo[3.2.2]nonane framework to yield the designed δ receptor ligands. In a first series of compounds the benzhydryl moiety of SNC80 was dissected, and one phenyl ring was attached to the bicyclic framework. In a second series of δ ligands the complete benzhydryl moiety was introduced into the bicyclic scaffold. The determined δ receptor affinities show that compounds based on an (R)‐glutamate‐derived bicyclic scaffold possess higher δ receptor affinity than their (S)‐glutamate‐derived counterparts. Furthermore, an intact benzhydryl moiety leads to δ receptor ligands that are more potent than compounds with two separated aromatic moieties. Compound 24 , with the same spatial arrangement of substituents around the benzhydryl stereocenter as SNC80, shows the highest δ receptor affinity of this series: K_i=24 nM . Whereas the highly potent δ ligands reveal good selectivity against μ and κ receptors, the σ₁ and/or σ₂ affinities of some compounds are almost in the same range as their δ receptor affinities, such as compound 25 (σ₂: K_i=83 nM ; δ: K_i=75 nM ). In [³⁵S]GTPγS assays the most potent δ ligands 24 and 25 showed almost the same intrinsic activity as the full agonist SNC80, proving the agonistic activity of 24 and 25 . The enantiomeric 4‐benzylidene derivatives 15 and ent‐ 15 showed selective cytotoxicity toward the 5637 (bladder) and A‐427 (small‐cell lung) human tumor cell lines.     

Pharmacological Evidence on Augmented Antiallodynia Following Systemic Co-Treatment with GlyT-1 and GlyT-2 Inhibitors in Rat Neuropathic Pain Model

The limited effect of current medications on neuropathic pain (NP) has initiated large efforts to develop effective treatments. Animal studies showed that glycine transporter (GlyT) inhibitors are promising analgesics in NP, though concerns regarding adverse effects were raised. We aimed to study NFPS and Org-25543, GlyT-1 and GlyT-2 inhibitors, respectively and their combination in rat mononeuropathic pain evoked by partial sciatic nerve ligation. Cerebrospinal fluid (CSF) glycine content was also determined by capillary electrophoresis. Subcutaneous (s.c.) 4 mg/kg NFPS or Org-25543 showed analgesia following acute administration (30–60 min). Small doses of each compound failed to produce antiallodynia up to 180 min after the acute administration. However, NFPS (1 mg/kg) produced antiallodynia after four days of treatment. Co-treatment with subanalgesic doses of NFPS (1 mg/kg) and Org-25543 (2 mg/kg) produced analgesia at 60 min and thereafter meanwhile increased significantly the CSF glycine content. This combination alleviated NP without affecting motor function. Test compounds failed to activate G-proteins in spinal cord. To the best of our knowledge for the first time we demonstrated augmented analgesia by combining GlyT-1 and 2 inhibitors. Increased CSF glycine content supports involvement of glycinergic system. Combining selective GlyT inhibitors or developing non-selective GlyT inhibitors might have therapeutic value in NP.