Fenamates and ibuprofen as foundational components in the synthesis of innovative, targeted COX-2 anti-inflammatory drugs, undergoing thorough biopharmacological assessments and in-silico computational studies.

Cyclooxygenase-2 plays a vital role in inflammation by catalyzing arachidonic acid conversion toward prostaglandins, making it a prime therapeutic objective. Selective COX-2 inhibitors represent significant progress in anti-inflammatory therapy, offering improved efficacy and fewer side effects. This study describes the synthesis of novel anti-inflammatory compounds from established pharmaceutically marketed agents like fenamates III-V and ibuprofen VI. Through rigorous in vitro testing, compounds 7b-c, and 12a-b demonstrated substantial in vitro selective inhibition, with IC50 values of 0.07 to 0.09 µM, indicating potent pharmacological activity. In vivo assessment, particularly focusing on compound 7c, revealed significant anti-inflammatory effects. Markedly, it demonstrated the highest inhibition of paw thickness (58.62 %) at the 5-hr mark compared to the carrageenan group, indicating its potency in mitigating inflammation. Furthermore, it exhibited a rapid onset of action, with a 54.88 % inhibition observed at the 1-hr mark. Subsequent comprehensive evaluations encompassing analgesic efficacy, histological characteristics, and toxicological properties indicated that compound 7c did not induce gastric ulcers, in contrast to the ulcerogenic tendency associated with mefenamic acid. Moreover, compound 7c underwent additional investigations through in silico methodologies such as molecular modelling, field alignment, and density functional theory. These analyses underscored the therapeutic potential and safety profile of this novel compound, warranting further exploration and development in the realm of pharmaceutical research.

New Anthranilic Acid Hydrazones as Fenamate Isosteres: Synthesis, Characterization, Molecular Docking, Dynamics & in Silico ADME, in Vitro Anti-Inflammatory and Anticancer Activity Studies.

In this study, twenty new anthranilic acid hydrazones 6-9 (a-e) were synthesized and their structures were characterized by Fourier-transform Infrared (FT-IR), Nuclear Magnetic Resonance (1 H-NMR - 13 C-NMR), and High-resolution Mass Spectroscopy (HR-MS). The inhibitory effects of the compounds against COX-II were evaluated. IC50 values of the compounds were found in the range of >200-0.32 µM and compounds 6e, 8d, 8e, 9b, 9c, and 9e were determined to be the most effective inhibitors. Cytotoxic effects of the most potent compounds were investigated against human hepatoblastoma (Hep-G2) and human healthy embryonic kidney (Hek-293) cell lines. Doxorubicin (IC50 : 8.68±0.16 µM for Hep-G2, 55.29±0.56 µM for Hek-293) was used as standard. 8e is the most active compound, with low IC50 against Hep-G2 (4.80±0.04 µM), high against Hek-293 (159.30±3.12), and high selectivity (33.15). Finally, molecular docking and dynamics studies were performed to understand ligand-protein interactions between the most potent compounds and COX II, Epidermal Growth Factor Receptor (EGFR), and Transforming Growth Factor beta II (TGF-ßII). The docking scores were calculated in the range of -10.609--6.705 kcal/mol for COX-II, -8.652--7.743 kcal/mol for EGFR, and -10.708--8.596 kcal/mol for TGF-ßII.

The MEK1/2-inhibitor ATR-002 efficiently blocks SARS-CoV-2 propagation and alleviates pro-inflammatory cytokine/chemokine responses.

Coronavirus disease 2019 (COVID-19), the illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 260 million confirmed infections and 5 million deaths to date. While vaccination is a powerful tool to control pandemic spread, medication to relieve COVID-19-associated symptoms and alleviate disease progression especially in high-risk patients is still lacking. In this study, we explore the suitability of the rapid accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway as a druggable target in the treatment of SARS-CoV-2 infections. We find that SARS-CoV-2 transiently activates Raf/MEK/ERK signaling in the very early infection phase and that ERK1/2 knockdown limits virus replication in cell culture models. We demonstrate that ATR-002, a specific inhibitor of the upstream MEK1/2 kinases which is currently evaluated in clinical trials as an anti-influenza drug, displays strong anti-SARS-CoV-2 activity in cell lines as well as in primary air-liquid-interphase epithelial cell (ALI) cultures, with a safe and selective treatment window. We also observe that ATR-002 treatment impairs the SARS-CoV-2-induced expression of pro-inflammatory cytokines, and thus might prevent COVID-19-associated hyperinflammation, a key player in COVID-19 progression. Thus, our data suggest that the Raf/MEK/ERK signaling cascade may represent a target for therapeutic intervention strategies against SARS-CoV-2 infections and that ATR-002 is a promising candidate for further drug evaluation.

Antiviral efficacy against influenza virus and pharmacokinetic analysis of a novel MEK-inhibitor, ATR-002, in cell culture and in the mouse model.

Antiviral therapies against influenza are required, especially for high-risk patients, severe influenza and in case of highly pathogenic influenza virus (IV) strains. However, currently, licensed drugs that target the virus directly are not very effective and often lead to the development of resistant IV variants. This may be overcome by targeting host cell factors that are required for IV propagation. IV induces a variety of host cell signaling cascades, such as the Raf/MEK/ERK kinase pathway. The activation of this pathway is necessary for IV propagation. MEK-inhibitors block the activation of the pathway on the bottleneck of the signaling cascade leading to impaired virus propagation. In the present study, we aimed to compare the antiviral potency and bioavailability of the MEK-inhibitor CI-1040 versus its major active metabolite ATR-002, in vitro as well as in the mouse model. In cell culture assays, an approximately 10-fold higher concentration of ATR-002 is required to generate the same antiviral activity as for CI-1040. Interestingly, we observed that considerably lower concentrations of ATR-002 were required to achieve a reduction of the viral load in vivo. Pharmacokinetic studies with ATR-002 and CI-1040 in mice have found the Cmax and AUC to be far higher for ATR-002 than for CI-1040. Our results thereby demonstrate the in vivo superiority of the active metabolite ATR-002 over CI-1040 as an antiviral agent despite its weaker cell membrane permeability. Therefore, ATR-002 is an attractive candidate for development as an efficient antiviral agent, especially given the fact that a treatment based on cellular pathway inhibition would be far less likely to lead to viral drug resistance.

Fenamates Inhibit Human Sodium Channel Nav1.2 and Protect Glutamate-Induced Injury in SH-SY5Y Cells.

Voltage-gated sodium channels are crucial mediators of neuronal damage in ischemic and excitotoxicity disease models. Fenamates have been reported to have anti-inflammatory properties following a decrease in prostaglandin synthesis. Several researches showed that fenamates appear to be ion channel modulators and potential neuroprotectants. In this study, the neuroprotective effects of tolfenamic acid, flufenamic acid, and mefenamic acid were tested by glutamate-induced injury in SH-SY5Y cells. Following this, fenamates' effects were examined on both the expression level and the function of hNav1.1 and hNav1.2, which were closely associated with neuroprotection, using Western blot and patch clamp. Finally, the effect of fenamates on the expression of apoptosis-related proteins in SH-SY5Y cells was examined. The results showed that both flufenamic acid and mefenamic acid exhibited neuroprotective effects against glutamate-induced injury in SH-SY5Y cells. They inhibited peak currents of both hNav1.1 and hNav1.2. However, fenamates exhibited decreased inhibitory effects on hNav1.1 when compared to hNav1.2. Correspondingly, the inhibitory effect of fenamates was found to be consistent with the level of neuroprotective effects in vitro. Fenamates inhibited glutamate-induced apoptosis through the modulation of the Bcl-2/Bax-dependent cell death pathways. Taken together, Nav1.2 might play a part in fenamates' neuroprotection mechanism. Nav1.2 and NMDAR might take part in the neuroprotection mechanism of the fenamates. The fenamates inhibited glutamate-induced apoptosis through modulation of the Bcl-2/Bax-dependent cell death pathways.

Multiple actions of fenamates and other nonsteroidal anti-inflammatory drugs on GABAA receptors.

The nonsteroidal anti-inflammatory drug (NSAID) niflumic acid, a fenamate in structure, has many molecular targets, one of them being specific subtypes of the main inhibitory ligand-gated anion channel, the GABAA receptor. Here, we report on the effects of other fenamates and other classes of NSAIDs on brain picrotoxinin-sensitive GABAA receptors, using an autoradiographic assay with [35S]TBPS as a ligand on mouse brain sections. We found that the other fenamates studied (flufenamic acid, meclofenamic acid, mefenamic acid and tolfenamic acid) affected the autoradiographic signal at low micromolar concentrations in a facilitatory-like allosteric fashion, i.e., without having affinity to the [35S]TBPS binding site. Unlike niflumic acid that shows clear preference for inhibiting cerebellar granule cell layer GABAA receptors, the other fenamates showed little brain regional selectivity, indicating that their actions are not receptor-subtype selective. Of the non-fenamate NSAIDs studied at 100 µM concentration, diclofenac induced the greatest inhibition of the binding, which is not surprising as it has close structural similarity with the potent fenamate meclofenamic acid. Using two-electrode voltage-clamp assays on Xenopus oocytes, the effect of niflumic acid was found to be dependent on the ß subunit variant and the presence of γ2 subunit in rat recombinant α1ß and α1ßγ2 GABAA receptors, with the ß1 allowing the niflumic acid inhibition and ß3 the stimulation of the receptor-mediated currents. In summary, the fenamate NSAIDs constitute an interesting class of compounds that could be used for development of potent GABAA receptor allosteric agonists with other targets to moderate inflammation, pain and associated anxiety/depression.

Redox-cycling and intercalating properties of novel mixed copper(II) complexes with non-steroidal anti-inflammatory drugs tolfenamic, mefenamic and flufenamic acids and phenanthroline functionality: Structure, SOD-mimetic activity, interaction with albumin, DNA damage study and anticancer activity.

Copper(II) complexes containing non-steroidal anti-inflammatory drugs (NSAIDs) have been the subject of many research papers and reviews. Here we report the synthesis, spectroscopic study and biological activity of novel mixed copper(II) complexes with NSAIDs: tolfenamic (tolf), mefenamic (mef) and flufenamic (fluf) acids and phenanthroline (phen): [Cu(tolf-O,O')2(phen)] (1), [Cu(mef-O,O')2(phen)] (2), [Cu(fluf-O,O')2(phen)] (3). Complexes were characterized by X-ray analysis and EPR spectroscopy. Complexes 1-3 are monomeric, six-coordinate and crystallize in a monoclinic space group. Interaction of Cu(II) complexes with DNA was studied by means of absorption titrations, viscosity measurements and gel electrophoresis. The relative ability of the complexes to cleave DNA even in the absence of hydrogen peroxide is in the order 3 > 2 > 1. Application of the reactive oxygen species (ROS) scavengers, L-histidine, DMSO and SOD confirmed that singlet oxygen, hydroxyl radicals (Fenton reaction) and superoxide radical were formed, respectively. Thus, in addition to mechanism of intercalation, redox-cycling mechanism which in turn lead to the formation of ROS contribute to DNA damage. Cu(II) complexes exhibit excellent SOD-mimetic activity in the order 3~1 > 2. The fluorescence spectroscopy revealed that albumin may act as a targeted drug delivery vehicle for Cu(II) complexes (K~106). The anticancer activities of complexes 1-3 were investigated using an MTS assay (reduction of the tetrazolium compound) against three cancer cell lines (HT-29 human colon adenocarcinoma, HeLa and T-47D breast cancer cells) and mesenchymal stromal cells (MSC). The most promising compound, from the viewpoint of its NSAID biological activity is 3, due to the presence of the three fluorine atoms participating in the formation of weak hydrogen-bonds at the DNA surface.

Identification of Novel Inhibitors of Auxin-Induced Ca2+ Signaling via a Plant-Based Chemical Screen.

Many signal perception mechanisms are connected to Ca2+-based second messenger signaling to modulate specific cellular responses. The well-characterized plant hormone auxin elicits a very rapid Ca2+ signal. However, the cellular targets of auxin-induced Ca2+ are largely unknown. Here, we screened a biologically annotated chemical library for inhibitors of auxin-induced Ca2+ entry in plant cell suspensions to better understand the molecular mechanism of auxin-induced Ca2+ and to explore the physiological relevance of Ca2+ in auxin signal transduction. Using this approach, we defined a set of diverse, small molecules that interfere with auxin-induced Ca2+ entry. Based on annotated biological activities of the hit molecules, we found that auxin-induced Ca2+ signaling is, among others, highly sensitive to disruption of membrane proton gradients and the mammalian Ca2+ channel inhibitor bepridil. Whereas protonophores nonselectively inhibited auxin-induced and osmotic stress-induced Ca2+ signals, bepridil specifically inhibited auxin-induced Ca2+ We found evidence that bepridil severely alters vacuolar morphology and antagonized auxin-induced vacuolar remodeling. Further exploration of this plant-tailored collection of inhibitors will lead to a better understanding of auxin-induced Ca2+ entry and its relevance for auxin responses.

Fenamates inhibit human sodium channel Nav1.7 and Nav1.8.

Fenamates are N-substituted anthranilic acid derivatives, clinically used as nonsteroidal anti-inflammatory drugs (NSAIDs) in fever, pain and inflammation treatments. Previous studies have shown that they are also modulators of diverse ion channels, exhibiting either activation or inhibitory effects. However, the effects of fenamates on sodium channel subtypes are still unknown. In this study, fenamates, including mefenamic acid, flufenamic acid and tolfenamic acid, were examined by whole-cell patch clamp techniques on the sodium channels hNav1.7 and hNav1.8, which are closely associated with pain. The results showed that the mefenamic acid, flufenamic acid, and tolfenamic acid inhibited the peak currents of hNav1.7 and hNav1.8 in CHO cells stably expressing hNav1.7 and hNav1.8. However, much lighter inhibition effects of hNav1.8 were registered in the experimental system. Furthermore, the mefenamic acid, flufenamic acid and tolfenamic acid significantly affected the inactivation processes of hNav1.7 and hNav1.8 with I-V curves left-shifted to hyperpolarized direction. These data indicate that the inhibition effects of Nav1.7 and Nav1.8 by mefenamic acid, flufenamic acid and tolfenamic acid might contribute to their analgesic activity in addition to their inhibition of cyclooxygenase. These findings provide a basis for further studies in the discovery of other potential targets for NSAIDs.

Inhibition of the mechano-enzymatic amyloidogenesis of transthyretin: role of ligand affinity, binding cooperativity and occupancy of the inner channel.

Dissociation of the native transthyretin (TTR) tetramer is widely accepted as the critical step in TTR amyloid fibrillogenesis. It is modelled by exposure of the protein to non-physiological low pH in vitro and is inhibited by small molecule compounds, such as the drug tafamidis. We have recently identified a new mechano-enzymatic pathway of TTR fibrillogenesis in vitro, catalysed by selective proteolytic cleavage, which produces a high yield of genuine amyloid fibrils. This pathway is efficiently inhibited only by ligands that occupy both binding sites in TTR. Tolcapone, which is bound with similar high affinity in both TTR binding sites without the usual negative cooperativity, is therefore of interest. Here we show that TTR fibrillogenesis by the mechano-enzymatic pathway is indeed more potently inhibited by tolcapone than by tafamidis but neither, even in large molar excess, completely prevents amyloid fibril formation. In contrast, mds84, the prototype of our previously reported bivalent ligand TTR 'superstabiliser' family, is notably more potent than the monovalent ligands and we show here that this apparently reflects the critical additional interactions of its linker within the TTR central channel. Our findings have major implications for therapeutic approaches in TTR amyloidosis.

Substrate-selective Inhibition of Cyclooxygeanse-2 by Fenamic Acid Derivatives Is Dependent on Peroxide Tone.

Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid (AA) and endocannabinoid substrates, placing the enzyme at a unique junction between the eicosanoid and endocannabinoid signaling pathways. COX-2 is a sequence homodimer, but the enzyme displays half-of-site reactivity, such that only one monomer of the dimer is active at a given time. Certain rapid reversible, competitive nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit COX-2 in a substrate-selective manner, with the binding of inhibitor to a single monomer sufficient to inhibit the oxygenation of endocannabinoids but not arachidonic acid. The underlying mechanism responsible for substrate-selective inhibition has remained elusive. We utilized structural and biophysical methods to evaluate flufenamic acid, meclofenamic acid, mefenamic acid, and tolfenamic acid for their ability to act as substrate-selective inhibitors. Crystal structures of each drug in complex with human COX-2 revealed that the inhibitor binds within the cyclooxygenase channel in an inverted orientation, with the carboxylate group interacting with Tyr-385 and Ser-530 at the top of the channel. Tryptophan fluorescence quenching, continuous-wave electron spin resonance, and UV-visible spectroscopy demonstrate that flufenamic acid, mefenamic acid, and tolfenamic acid are substrate-selective inhibitors that bind rapidly to COX-2, quench tyrosyl radicals, and reduce higher oxidation states of the heme moiety. Substrate-selective inhibition was attenuated by the addition of the lipid peroxide 15-hydroperoxyeicosatertaenoic acid. Collectively, these studies implicate peroxide tone as an important mechanistic component of substrate-selective inhibition by flufenamic acid, mefenamic acid, and tolfenamic acid.

Influence of the N terminus on the biophysical properties and pharmacology of TREK1 potassium channels.

TWIK-related K(+) 1 (TREK1) potassium channels are members of the two-pore domain potassium channel family and contribute to background potassium conductances in many cell types, where their activity can be regulated by a variety of physiologic and pharmacologic mediators. Fenamates such as FFA (flufenamic acid; 2-{[3-(trifluoromethyl)phenyl]amino}benzoic acid), MFA [mefenamic acid; 2-(2,3-dimethylphenyl)aminobenzoic acid], NFA [niflumic acid; 2-{[3-(trifluoromethyl)phenyl]amino}nicotinic acid], and diclofenac [2-(2-(2,6-dichlorophenylamino)phenyl)acetic acid] and the related experimental drug BL-1249 [(5,6,7,8-tetrahydro-naphthalen-1-yl)-[2-(1H-tetrazol-5-yl)-phenyl]-amine] enhance the activity of TREK1 currents, and we show that BL-1249 is the most potent of these compounds. Alternative translation initiation produces a shorter, N terminus truncated form of TREK1 with a much reduced open probability and a proposed increased permeability to sodium compared with the longer form. We show that both forms of TREK1 can be activated by fenamates and that a number of mutations that affect TREK1 channel gating occlude the action of fenamates but only in the longer form of TREK1. Furthermore, fenamates produce a marked enhancement of current through the shorter, truncated form of TREK1 and reveal a K(+)-selective channel, like the long form. These results provide insight into the mechanism of TREK1 channel activation by fenamates, and, given the role of TREK1 channels in pain, they suggest a novel analgesic mechanism for these compounds.

The potential and promise of mefenamic acid.

Clinical use of mefenamic acid has generally declined in an era where other NSAID use has flourished. While having modes of action and general toxicities similar to other NSAIDs, mefenamic acid, as a member of the fenamates, nevertheless possesses some unique in vitro effects that have the potential to distinguish this agent from others. Use of this drug remains relevant for pain syndromes and some gynecological disorders, albeit with considerable competition from other NSAIDs. New basic science has considerably improved the understanding of the biochemistry of mefenamic acid. As well as maintaining its use in traditional settings, there is a tremendous potential for expanding the application of mefenamic acid to niche roles.

Structure-activity relationship of fenamates as Slo2.1 channel activators.

Niflumic acid, 2-{[3-(trifluoromethyl)phenyl]amino}pyridine-3-carboxylic acid (NFA), a nonsteroidal anti-inflammatory drug that blocks cyclooxygenase (COX), was shown previously to activate [Na(+)](i)-regulated Slo2.1 channels. In this study, we report that other fenamates, including flufenamic acid, mefenamic acid, tolfenamic acid, meclofenamic acid, and a phenyl acetic acid derivative, diclofenac, also are low-potency (EC(50) = 80 µM to 2.1 mM), partial agonists of human Slo2.1 channels heterologously expressed in Xenopus oocytes. Substituent analysis determined that N-phenylanthranilic acid was the minimal pharmacophore for fenamate activation of Slo2.1 channels. The effects of fenamates were biphasic, with an initial rapid activation phase followed by a slow phase of current inhibition. Ibuprofen, a structurally dissimilar COX inhibitor, did not activate Slo2.1. Preincubation of oocytes with ibuprofen did not significantly alter the effects of NFA, suggesting that neither channel activation nor inhibition is associated with COX activity. A point mutation (A278R) in the pore-lining S6 segment of Slo2.1 increased the sensitivity to activation and reduced the inhibition induced by NFA. Together, our results suggest that fenamates bind to two sites on Slo2.1 channels: an extracellular accessible site to activate and a cytoplasmic accessible site in the pore to inhibit currents.

Pharmacological comparison of novel synthetic fenamate analogues with econazole and 2-APB on the inhibition of TRPM2 channels.

BACKGROUND AND PURPOSE: Fenamate analogues, econazole and 2-aminoethoxydiphenyl borate (2-APB) are inhibitors of transient receptor potential melastatin 2 (TRPM2) channels and are used as research tools. However, these compounds have different chemical structures and therapeutic applications. Here we have investigated the pharmacological profile of TRPM2 channels by application of newly synthesized fenamate analogues and the existing channel blockers. EXPERIMENTAL APPROACH: Human TRPM2 channels in tetracycline-regulated pcDNA4/TO vectors were transfected into HEK293 T-REx cells and the expression was induced by tetracycline. Whole cell currents were recorded by patch-clamp techniques. Ca(2+) influx or release was monitored by fluorometry. KEY RESULTS: Flufenamic acid (FFA), mefenamic acid (MFA) and niflumic acid (NFA) concentration-dependently inhibited TRPM2 current with potency order FFA > MFA = NFA. Modification of the 2-phenylamino ring by substitution of the trifluoromethyl group in FFA with -CH(3), -F, -CF(3), -OCH(3), -OCH(2)CH(3), -COOH, and -NO(2) at various positions, reduced channel blocking potency. The conservative substitution of 3-CF(3) in FFA by -CH(3) (3-MFA), however, gave the most potent fenamate analogue with an IC(50) of 76 µM, comparable to that of FFA, but unlike FFA, had no effect on Ca(2+) release. 3-MFA and FFA inhibited the channel intracellularly. Econazole and 2-APB showed non-selectivity by altering cytosolic Ca(2+) movement. Econazole also evoked a non-selective current. CONCLUSION AND IMPLICATIONS: The fenamate analogue 3-MFA was more selective than other TRPM2 channel blockers. FFA, 2-APB and econazole should be used with caution as TRPM2 channel blockers, as these compounds can interfere with intracellular Ca(2+) movement.

Effect of non-steroidal anti-inflammatory drugs and new fenamate analogues on TRPC4 and TRPC5 channels.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used anti-inflammatory therapeutic agents, among which the fenamate analogues play important roles in regulating intracellular Ca²âº transient and ion channels. However, the effect of NSAIDs on TRPC4 and TRPC5 is still unknown. To understand the structure-activity of fenamate analogues on TRPC channels, we have synthesized a series of fenamate analogues and investigated their effects on TRPC4 and TRPC5 channels. Human TRPC4 and TRPC5 cDNAs in tetracycline-regulated vectors were transfected into HEK293 T-REx cells. The whole cell current and Ca²âº movement were recorded by patch clamp and calcium imaging, respectively. Flufenamic acid (FFA), mefenamic acid (MFA), niflumic acid (NFA) and diclofenac sodium (DFS) showed inhibition on TRPC4 and TRPC5 channels in a concentration-dependent manner. The potency was FFA>MFA>NFA>DFS. Modification of 2-phenylamino ring by substitution of the trifluoromethyl group in FFA with F, CH3, OCH3, OCH2CH3, COOH, and NO2 led to the changes in their channel blocking activity. However, 2-(2'-methoxy-5'-methylphenyl)aminobenzoic acid stimulated TRPC4 and TRPC5 channels. Selective COX1-3 inhibitors (aspirin, celecoxib, acetaminophen, and indomethacin) had no effect on the channels. Longer perfusion (> 5 min) with FFA (100 µM) and MFA (100 µM) caused a potentiation of TRPC4 and TRPC5 currents after their initial blocking effects that appeared to be partially mediated by the mitochondrial Ca²âº release. Our results suggest that fenamate analogues are direct modulators of TRPC4 and TRPC5 channels. The substitution pattern and conformation of the 2-phenylamino ring could alter their blocking activity, which is important for understanding fenamate pharmacology and new drug development targeting the TRPC channels.

Development of potent and selective inhibitors of aldo-keto reductase 1C3 (type 5 17ß-hydroxysteroid dehydrogenase) based on N-phenyl-aminobenzoates and their structure-activity relationships.

Aldo-keto reductase 1C3 (AKR1C3; type 5 17ß-hydroxysteroid dehydrogenase) is overexpressed in castration resistant prostate cancer (CRPC) and is implicated in the intratumoral biosynthesis of testosterone and 5α-dihydrotestosterone. Selective AKR1C3 inhibitors are required because compounds should not inhibit the highly related AKR1C1 and AKR1C2 isoforms which are involved in the inactivation of 5α-dihydrotestosterone. NSAIDs, N-phenylanthranilates in particular, are potent but nonselective AKR1C3 inhibitors. Using flufenamic acid, 2-{[3-(trifluoromethyl)phenyl]amino}benzoic acid, as lead compound, five classes of structural analogues were synthesized and evaluated for AKR1C3 inhibitory potency and selectivity. Structure-activity relationship (SAR) studies revealed that a meta-carboxylic acid group relative to the amine conferred pronounced AKR1C3 selectivity without loss of potency, while electron withdrawing groups on the phenylamino B-ring were optimal for AKR1C3 inhibition. Lead compounds did not inhibit COX-1 or COX-2 but blocked the AKR1C3 mediated production of testosterone in LNCaP-AKR1C3 cells. These compounds offer promising leads toward new therapeutics for CRPC.

The pharmacological profile of brain liver intestine Na+ channel: inhibition by diarylamidines and activation by fenamates.

The brain liver intestine Na(+) channel (BLINaC) is a member of the degenerin/epithelial Na(+) channel gene family of unknown function. Elucidation of the physiological function of BLINaC would benefit greatly from pharmacological tools that specifically affect BLINaC activity. Guided by the close molecular relation of BLINaC to acid-sensing ion channels, we discovered in this study that rat BLINaC (rBLINaC) and mouse BLINaC are inhibited by micromolar concentrations of diarylamidines and nafamostat, similar to acid-sensing ion channels. Inhibition was voltage-dependent, suggesting pore block as the mechanism of inhibition. Furthermore, we identified the fenamate flufenamic acid and related compounds as agonists of rBLINaC. Application of millimolar concentrations of flufenamic acid to rBLINaC induced a robust, Na(+)-selective current, which was blocked partially by amiloride. The identification of an artificial agonist of rBLINaC supports the hypothesis that rBLINaC is opened by an unknown physiological ligand. Inhibition by diarylamidines and activation by fenamates define a unique pharmacological profile for BLINaC, which will be useful to unravel the physiological function of this ion channel.

Fenamates as TRP channel blockers: mefenamic acid selectively blocks TRPM3.

BACKGROUND AND PURPOSE: Fenamates are N-phenyl-substituted anthranilic acid derivatives clinically used as non-steroid anti-inflammatory drugs in pain treatment. Reports describing fenamates as tools to interfere with cellular volume regulation attracted our attention based on our interest in the role of the volume-modulated transient receptor potential (TRP) channels TRPM3 and TRPV4. EXPERIMENTAL APPROACH: Firstly, we measured the blocking potencies and selectivities of fenamates on TRPM3 and TRPV4 as well as TRPC6 and TRPM2 by Ca(2+) imaging in the heterologous HEK293 cell system. Secondly, we further investigated the effects of mefenamic acid on cytosolic Ca(2+) and on the membrane voltage in single HEK293 cells that exogenously express TRPM3. Thirdly, in insulin-secreting INS-1E cells, which endogenously express TRPM3, we validated the effect of mefenamic acid on cytosolic Ca(2+) and insulin secretion. KEY RESULTS: We identified and characterized mefenamic acid as a selective and potent TRPM3 blocker, whereas other fenamate structures non-selectively blocked TRPM3, TRPV4, TRPC6 and TRPM2. CONCLUSIONS AND IMPLICATIONS: This study reveals that mefenamic acid selectively inhibits TRPM3-mediated calcium entry. This selectivity was further confirmed using insulin-secreting cells. K(ATP) channel-dependent increases in cytosolic Ca(2+) and insulin secretion were not blocked by mefenamic acid, but the selective stimulation of TRPM3-dependent Ca(2+) entry and insulin secretion induced by pregnenolone sulphate were inhibited. However, the physiological regulator of TRPM3 in insulin-secreting cells remains to be elucidated, as well as the conditions under which the inhibition of TRPM3 can impair pancreatic ß-cell function. Our results strongly suggest mefenamic acid is the most selective fenamate to interfere with TRPM3 function.

Ligand-based virtual screening procedure for the prediction and the identification of novel ß-amyloid aggregation inhibitors using Kohonen maps and Counterpropagation Artificial Neural Networks.

In this work we have developed an in silico model to predict the inhibition of ß-amyloid aggregation by small organic molecules. In particular we have explored the inhibitory activity of a series of 62 N-phenylanthranilic acids using Kohonen maps and Counterpropagation Artificial Neural Networks. The effects of various structural modifications on biological activity are investigated and novel structures are designed using the developed in silico model. More specifically a search for optimized pharmacophore patterns by insertions, substitutions, and ring fusions of pharmacophoric substituents of the main building block scaffolds is described. The detection of the domain of applicability defines compounds whose estimations can be accepted with confidence.