Pavel Savechenkov - Academia.edu (original) (raw)
Papers by Pavel Savechenkov
Journal of Biological Chemistry, Sep 1, 2015
Background: For some chiral barbiturates, one isomer potentiates and the other inhibits GABA resp... more Background: For some chiral barbiturates, one isomer potentiates and the other inhibits GABA responses by binding to unknown sites. Results: A photoreactive convulsant barbiturate identifies a transmembrane intersubunit-binding site between the ␥ and  subunits. Conclusion: Positive and negative allosteric modulators can bind to a common intersubunit site. Significance: This study defines a novel mode of regulation of GABA A R responses.
ChemInform, May 31, 2005
Nitration of phenylpropiolic acid derivatives ArCºCX (X=CN, CO 2 Me) in HSO 3 F at-75...50°C affo... more Nitration of phenylpropiolic acid derivatives ArCºCX (X=CN, CO 2 Me) in HSO 3 F at-75...50°C afforded mononitro compounds, for instance, m-O 2 NC 6 H 4 CºCX. Vinyl type cations generated in HSO 3 F from methyl 3-arylpropiolates ArC + =CHCO 2 Me react along two pathways. The first among them results in formation of fluorosulfonates ArC(OSO 2 F)=CHCO 2 Me, and the second one after the attack of vinyl cation on the aryl moiety of the substrate affords a dimer that on nitration is converted into a nitro product with conserved triple bond.
Chemistry: A European Journal, Mar 6, 2018
Expanded calixpyrrole-type macrocycles, calix[2]benzo[4]pyrroles, bearing fluorescent moieties at... more Expanded calixpyrrole-type macrocycles, calix[2]benzo[4]pyrroles, bearing fluorescent moieties attached via conjugated vinyl spacer were synthesized from the corresponding formyl-derivatives using Knoevenagel condensation. The anion-binding properties of the resulting fluorescent macrocycles were studied using NMR, UVvis and fluorescence spectroscopy. The main interest was devoted to dicarboxylates matching the size of the binding cavity of the calix[2]benzo[4]pyrrole skeleton. The observed anion binding properties were compared with the regular calix[4]pyrroles bearing identical fluorophores. Surprisingly, the parent calix[4]pyrroles appear to be equally if not more efficient sensors for anions, including dicarboxylates. The recorded affinity constants for various anions and dianions show the sensors S1-S5 to be highly crossreactive. The cross-reactivity of the sensors was utilized in the microchip based array sensor, which showed perfect (100%) classification of 18 analytes utilizing only 5 sensors. Finally, the same array was used to perform quantitative analysis of dicarboxylates such as oxalate and malonate whereas the data from the array were subjected to linear regression to distinguish varying concentrations of dianions with low error (<2%).
![Research paper thumbnail of Anion Binding Modes in <i>meso</i>-Substituted Hexapyrrolic Calix[4]pyrrole Isomers](https://attachments.academia-assets.com/111532951/thumbnails/1.jpg)
](Journal of the American Chemical Society, Jan 17, 2014
We report on the synthesis of a new receptor for anions, mesosubstituted hexapyrrolic calix[4]pyr... more We report on the synthesis of a new receptor for anions, mesosubstituted hexapyrrolic calix[4]pyrrole 1. The calix[4]pyrrole's core features two additional pyrrole side-arms suspended above or below the calix[4]pyrrole core. This hexapyrrolic calix[4]pyrrole 1 is formed as cis-and trans-configurational isomers, the structures of which have been determined by single crystal X-ray diffraction. The anion binding experiments revealed interesting difference in the binding mode: The cis-1 isomer binds anions in a mixed binding mode featuring a combination of hydrogen bonding and anion−π interactions resulting in an unexpected strong binding. On the other hand, the trans-1 isomer displays only hydrogen bonding and lower affinity for anions. This is unexpected as one would assume both isomers to display the same binding modes. Overall, the titrations of 1 using UV spectrophotometry and NMR titrations by anions reveal that cisisomer 1 displays higher affinity (10 5 −10 6 M −1) and cross-reactivity for anions, while the trans-isomer 1 shows a more selective response to anions. Such differences in binding mode in configurational isomers are so far unexplored and a feature deserving further study.
ChemInform, Jul 4, 2006
Polyphenylalkene derivatives Q 0740 Reactions of Vinyl Type Carbocations Generated in Fluorosulfo... more Polyphenylalkene derivatives Q 0740 Reactions of Vinyl Type Carbocations Generated in Fluorosulfonic Acid with Benzene Derivatives. New Synthesis of Alkyl 3,3-Diarylpropenoates.-A new procedure for the synthesis of alkyl 3,3-diarylpropenoates carrying various substituents in the aryl moieties is presented.-(SAVECHENKOV,
European journal of medicinal chemistry, Aug 1, 2017
Neuroactive steroids are potent positive allosteric modulators of GABAA receptors (GABAAR), but t... more Neuroactive steroids are potent positive allosteric modulators of GABAA receptors (GABAAR), but the locations of their GABAAR binding sites remain poorly defined. To discover these sites, we synthesized two photoreactive analogs of alphaxalone, an anesthetic neurosteroid targeting GABAAR, 11β-(4-azido-2,3,5,6-tetrafluorobenzoyloxy)allopregnanolone, (F4N3Bzoxy-AP) and 11-aziallopregnanolone (11-AziAP). Both photoprobes acted with equal or higher potency than alphaxalone as general anesthetics and potentiators of GABAAR responses, left-shifting the GABA concentration - response curve for human α1β3γ2 GABAARs expressed in Xenopus oocytes, and enhancing [3H]muscimol binding to α1β3γ2 GABAARs expressed in HEK293 cells. With EC50 of 110 nM, 11-AziAP is one the most potent general anesthetics reported. [3H]F4N3Bzoxy-AP and [3H]11-AziAP, at anesthetic concentrations, photoincorporated into α- and β-subunits of purified α1β3γ2 GABAARs, but labeling at the subunit level was not inhibited by alphaxalone (30 μM). The enhancement of photolabeling by 3H-azietomidate and 3H-mTFD-MPAB in the presence of either of the two steroid photoprobes indicates the neurosteroid binding site is different from, but allosterically related to, the etomidate and barbiturate sites. Our observations are consistent with two hypotheses. First, F4N3Bzoxy-AP and 11-aziAP bind to a high affinity site in such a pose that the 11-photoactivatable moiety, that is rigidly attached to the steroid backbone, points away from the protein. Second, F4N3Bzoxy-AP, 11-aziAP and other steroid anesthetics, which are present at very high concentration at the lipid-protein interface due to their high lipophilicity, act via low affinity sites, as proposed by Akk et al. (Psychoneuroendocrinology2009, 34S1, S59-S66).
An entry from the Cambridge Structural Database, the world's repository for small molecule cr... more An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
An entry from the Cambridge Structural Database, the world's repository for small molecule cr... more An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Chemistry (Weinheim an der Bergstrasse, Germany), Jan 3, 2018
Expanded calixpyrrole-type macrocycles, calix[2]benzo[4]pyrroles, bearing fluorescent moieties at... more Expanded calixpyrrole-type macrocycles, calix[2]benzo[4]pyrroles, bearing fluorescent moieties attached via conjugated vinyl spacers, have been synthesized from the corresponding formyl derivatives through Knoevenagel condensation. The anion-binding properties of the resulting fluorescent macrocycles have been studied by means of NMR, UV/Vis, and fluorescence spectroscopies. Our main focus has been on dicarboxylates matching the size of the binding cavity of the calix[2]benzo[4]pyrrole skeleton. The observed anion-binding properties were compared with those of the regular calix[4]pyrroles bearing identical fluorophores. Surprisingly, the parent calix[4]pyrroles appear to be equally efficient, if not more so, for sensing anions such as dicarboxylates. Affinity constants determined for various anions and dianions show the sensors S1-S5 to be highly cross-reactive. The cross-reactivity of the sensors was utilized in a microchip-based array, which showed perfect (100 %) classification o...
![Research paper thumbnail of Anion Binding Modes in meso-Substituted Hexapyrrolic Calix[4]pyrrole Isomers](https://attachments.academia-assets.com/111532975/thumbnails/1.jpg)
](Journal of the American Chemical Society, 2014
We report on the synthesis of a new receptor for anions, mesosubstituted hexapyrrolic calix[4]pyr... more We report on the synthesis of a new receptor for anions, mesosubstituted hexapyrrolic calix[4]pyrrole 1. The calix[4]pyrrole's core features two additional pyrrole side-arms suspended above or below the calix[4]pyrrole core. This hexapyrrolic calix[4]pyrrole 1 is formed as cis-and trans-configurational isomers, the structures of which have been determined by single crystal X-ray diffraction. The anion binding experiments revealed interesting difference in the binding mode: The cis-1 isomer binds anions in a mixed binding mode featuring a combination of hydrogen bonding and anion−π interactions resulting in an unexpected strong binding. On the other hand, the trans-1 isomer displays only hydrogen bonding and lower affinity for anions. This is unexpected as one would assume both isomers to display the same binding modes. Overall, the titrations of 1 using UV spectrophotometry and NMR titrations by anions reveal that cisisomer 1 displays higher affinity (10 5 −10 6 M −1) and cross-reactivity for anions, while the trans-isomer 1 shows a more selective response to anions. Such differences in binding mode in configurational isomers are so far unexplored and a feature deserving further study.
Capping off an era marred by drug development failures and punctuated by waning interest and pres... more Capping off an era marred by drug development failures and punctuated by waning interest and presumed intractability toward direct targeting of KRAS, new technologies and strategies are aiding in the target's resurgence. As previously reported, the tetrahydropyridopyrimidines were identified as irreversible covalent inhibitors of KRAS G12C that bind in the switch-II pocket of KRAS and make a covalent bond to cysteine 12. Using structure-based drug design in conjunction with a focused in vitro absorption, distribution, metabolism and excretion screening approach, analogues were synthesized to increase the potency and reduce metabolic liabilities of this series. The discovery of the clinical development candidate MRTX849 as a potent, selective covalent inhibitor of KRAS G12C is described.
British Journal of Pharmacology, 2019
Background and PurposeGeneral anaesthetics can act on synaptic GABAA receptors by binding to one ... more Background and PurposeGeneral anaesthetics can act on synaptic GABAA receptors by binding to one of three classes of general anaesthetic sites. Canonical drugs that bind selectively to only one class of site are etomidate, alphaxalone, and the mephobarbital derivative, R‐mTFD‐MPAB. We tested the hypothesis that the general anaesthetic potencies of mixtures of such site‐selective agents binding to the same or to different sites would combine additively or synergistically respectively.Experimental ApproachThe potency of general anaesthetics individually or in combinations to cause loss of righting reflexes in tadpoles was determined, and the results were analysed using isobolographic methods.Key ResultsThe potencies of combinations of two or three site‐selective anaesthetics that all acted on a single class of site were strictly additive, regardless of which single site was involved. Combinations of two or three site‐selective anaesthetics that all bound selectively to different sites...
Molecular Pharmacology, 2019
GABA A receptors (GABA A Rs) are targets for important classes of clinical agents (e.g., anxiolyt... more GABA A receptors (GABA A Rs) are targets for important classes of clinical agents (e.g., anxiolytics, anticonvulsants, and general anesthetics) that act as positive allosteric modulators (PAMs). Previously, using photoreactive analogs of etomidate ([ 3 H]azietomidate) and mephobarbital [[ 3 H]1-methyl-5-allyl-5-(mtrifluoromethyl-diazirynylphenyl)barbituric acid ([ 3 H]R-mTFD-MPAB)], we identified two homologous but pharmacologically distinct classes of general anesthetic binding sites in the a1b3g2 GABA A R transmembrane domain at b 1-a 2 (b 1 sites) and a 1-b 2 /g 1-b 2 (b 2 sites) subunit interfaces. We now use competition photolabeling with [ 3 H]azietomidate and [ 3 H]R-mTFD-MPAB to identify para-substituted propofol analogs and other drugs that bind selectively to intersubunit anesthetic sites. Propofol and 4-chloro-propofol bind with 5-fold selectivity to b 1 , while derivatives with bulkier lipophilic substitutions [4-(tert-butyl)-propofol and 4-(hydroxyl(phenyl)methyl)-propofol] bind with ∼10-fold higher affinity to b 2 sites. Similar to R-mTFD-MPAB and propofol, these drugs bind in the presence of GABA with similar affinity to the a 1-b 2 and g 1-b 2 sites. However, we discovered four compounds that bind with different affinities to the two b 2 interface sites. Two of these bind with higher affinity to one of the b 2 sites than to the b 1 sites. We deduce that 4-benzoyl-propofol binds with .100-fold higher affinity to the g 1-b 2 site than to the a 1-b 2 or b 1-a 2 sites, whereas loreclezole, an anticonvulsant, binds with 5-and 100-fold higher affinity to the a 1-b 2 site than to the b 1 and g 1-b 2 sites. These studies provide a first identification of PAMs that bind selectively to a single intersubunit site in the GABA A R transmembrane domain, a property that may facilitate the development of subtype selective GABA A R PAMs.
Journal of Biological Chemistry, 2019
Edited by Henrik G. Dohlman Many neuroactive steroids potently and allosterically modulate pentam... more Edited by Henrik G. Dohlman Many neuroactive steroids potently and allosterically modulate pentameric ligand-gated ion channels, including GABA A receptors (GABA A R) and nicotinic acetylcholine receptors (nAChRs). Allopregnanolone and its synthetic analog alphaxalone are GABA A R-positive allosteric modulators (PAMs), whereas alphaxalone and most neuroactive steroids are nAChR inhibitors. In this report, we used 11-(p-azidotetrafluorobenzoyloxy)allopregnanolone (F 4 N 3 Bzoxy-AP), a general anesthetic and photoreactive allopregnanolone analog that is a potent GABA A R PAM, to characterize steroid-binding sites in the Torpedo ␣ 2 ␥␦ nAChR in its native membrane environment. We found that F 4 N 3 Bzoxy-AP (IC 50 ؍ 31 M) is 7-fold more potent than alphaxalone in inhibiting binding of the channel blocker [ 3 H]tenocyclidine to nAChRs in the desensitized state. At 300 M, neither steroid inhibited binding of [ 3 H]tetracaine, a closed-state selective channel blocker, or of [ 3 H]acetylcholine. Photolabeling identified three distinct [ 3 H]F 4 N 3 Bzoxy-AP-binding sites in the nAChR transmembrane domain: 1) in the ion channel, identified by photolabeling in the M2 helices of Val-261 and ␦Val-269 (position M2-13); 2) at the interface between the ␣M1 and ␣M4 helices, identified by photolabeling in ␣M1 (␣Cys-222/␣Leu-223); and 3) at the lipid-protein interface involving ␥Trp-453 (M4), a residue photolabeled by small lipophilic probes and promegestone, a steroid nAChR antagonist. Photolabeling in the ion channel and ␣M1 was higher in the nAChR-desensitized state than in the resting state and inhibitable by promegestone. These results directly indicate a steroidbinding site in the nAChR ion channel and identify additional steroid-binding sites also occupied by other lipophilic nAChR antagonists. Many steroids, including endogenous 3␣-hydroxy metabolites of progesterone and deoxycorticosterone and synthetic analogs, act as potent general anesthetics, sedatives, anxiolytics, or anticonvulsants (1, 2). They are positive allosteric modula
Biophysical Journal, 2019
European Journal of Medicinal Chemistry, 2018
Pregnanolone and allopregnanolone-type ligands exert general anesthetic, anticonvulsant and anxio... more Pregnanolone and allopregnanolone-type ligands exert general anesthetic, anticonvulsant and anxiolytic effects due to their positive modulatory interactions with the GABA A receptors in the brain. Binding sites for these neurosteroids have been recently identified at subunit interfaces in the transmembrane domain (TMD) of homomeric 3 GABA A receptors using photoaffinity labeling techniques, and in homomeric chimeric receptors containing GABA A receptor subunit TMDs by crystallography. Steroid binding sites have yet to be determined in human, heteromeric, functionally reconstituted, full-length, glycosylated GABA A receptors. Here, we report on the synthesis and pharmacological characterization of several photoaffinity analogs of pregnanolone and allopregnanolone, of which 21-[4-(3-(trifluoromethyl)-3H-diazirin-3yl)benzoxy]allopregnanolone (21-pTFDBzox-AP) was the most potent ligand. It is a partial positive modulator of the human 13 and 132L GABA A receptors at sub-micromolar concentrations. [ 3 H]21-pTFDBzox-AP photoincorporated in a pharmacologically specific manner into the and β subunits of those receptors, with the β3 subunit photolabeled most efficiently. Importantly, photolabeling by [ 3 H]21-pTFDBzox-AP was inhibited by the positive steroid modulators alphaxalone, pregnanolone and allopregnanolone, but not by inhibitory neurosteroid pregnenolone sulfate or by two potent general anesthetics and GABA A R positive allosteric modulators, etomidate and an anesthetic barbiturate. The latter two ligands bind to sites at subunit interfaces in the GABA A R that are different from those interacting with neurosteroids. 21-pTFDBzox-AP's potency and pharmacological specificity of photolabeling indicate its suitability for characterizing neurosteroid binding sites in native GABA A receptors.
Journal of Biological Chemistry, Sep 1, 2015
Background: For some chiral barbiturates, one isomer potentiates and the other inhibits GABA resp... more Background: For some chiral barbiturates, one isomer potentiates and the other inhibits GABA responses by binding to unknown sites. Results: A photoreactive convulsant barbiturate identifies a transmembrane intersubunit-binding site between the ␥ and  subunits. Conclusion: Positive and negative allosteric modulators can bind to a common intersubunit site. Significance: This study defines a novel mode of regulation of GABA A R responses.
ChemInform, May 31, 2005
Nitration of phenylpropiolic acid derivatives ArCºCX (X=CN, CO 2 Me) in HSO 3 F at-75...50°C affo... more Nitration of phenylpropiolic acid derivatives ArCºCX (X=CN, CO 2 Me) in HSO 3 F at-75...50°C afforded mononitro compounds, for instance, m-O 2 NC 6 H 4 CºCX. Vinyl type cations generated in HSO 3 F from methyl 3-arylpropiolates ArC + =CHCO 2 Me react along two pathways. The first among them results in formation of fluorosulfonates ArC(OSO 2 F)=CHCO 2 Me, and the second one after the attack of vinyl cation on the aryl moiety of the substrate affords a dimer that on nitration is converted into a nitro product with conserved triple bond.
Chemistry: A European Journal, Mar 6, 2018
Expanded calixpyrrole-type macrocycles, calix[2]benzo[4]pyrroles, bearing fluorescent moieties at... more Expanded calixpyrrole-type macrocycles, calix[2]benzo[4]pyrroles, bearing fluorescent moieties attached via conjugated vinyl spacer were synthesized from the corresponding formyl-derivatives using Knoevenagel condensation. The anion-binding properties of the resulting fluorescent macrocycles were studied using NMR, UVvis and fluorescence spectroscopy. The main interest was devoted to dicarboxylates matching the size of the binding cavity of the calix[2]benzo[4]pyrrole skeleton. The observed anion binding properties were compared with the regular calix[4]pyrroles bearing identical fluorophores. Surprisingly, the parent calix[4]pyrroles appear to be equally if not more efficient sensors for anions, including dicarboxylates. The recorded affinity constants for various anions and dianions show the sensors S1-S5 to be highly crossreactive. The cross-reactivity of the sensors was utilized in the microchip based array sensor, which showed perfect (100%) classification of 18 analytes utilizing only 5 sensors. Finally, the same array was used to perform quantitative analysis of dicarboxylates such as oxalate and malonate whereas the data from the array were subjected to linear regression to distinguish varying concentrations of dianions with low error (<2%).
Journal of the American Chemical Society, Jan 17, 2014
We report on the synthesis of a new receptor for anions, mesosubstituted hexapyrrolic calix[4]pyr... more We report on the synthesis of a new receptor for anions, mesosubstituted hexapyrrolic calix[4]pyrrole 1. The calix[4]pyrrole's core features two additional pyrrole side-arms suspended above or below the calix[4]pyrrole core. This hexapyrrolic calix[4]pyrrole 1 is formed as cis-and trans-configurational isomers, the structures of which have been determined by single crystal X-ray diffraction. The anion binding experiments revealed interesting difference in the binding mode: The cis-1 isomer binds anions in a mixed binding mode featuring a combination of hydrogen bonding and anion−π interactions resulting in an unexpected strong binding. On the other hand, the trans-1 isomer displays only hydrogen bonding and lower affinity for anions. This is unexpected as one would assume both isomers to display the same binding modes. Overall, the titrations of 1 using UV spectrophotometry and NMR titrations by anions reveal that cisisomer 1 displays higher affinity (10 5 −10 6 M −1) and cross-reactivity for anions, while the trans-isomer 1 shows a more selective response to anions. Such differences in binding mode in configurational isomers are so far unexplored and a feature deserving further study.
ChemInform, Jul 4, 2006
Polyphenylalkene derivatives Q 0740 Reactions of Vinyl Type Carbocations Generated in Fluorosulfo... more Polyphenylalkene derivatives Q 0740 Reactions of Vinyl Type Carbocations Generated in Fluorosulfonic Acid with Benzene Derivatives. New Synthesis of Alkyl 3,3-Diarylpropenoates.-A new procedure for the synthesis of alkyl 3,3-diarylpropenoates carrying various substituents in the aryl moieties is presented.-(SAVECHENKOV,
European journal of medicinal chemistry, Aug 1, 2017
Neuroactive steroids are potent positive allosteric modulators of GABAA receptors (GABAAR), but t... more Neuroactive steroids are potent positive allosteric modulators of GABAA receptors (GABAAR), but the locations of their GABAAR binding sites remain poorly defined. To discover these sites, we synthesized two photoreactive analogs of alphaxalone, an anesthetic neurosteroid targeting GABAAR, 11β-(4-azido-2,3,5,6-tetrafluorobenzoyloxy)allopregnanolone, (F4N3Bzoxy-AP) and 11-aziallopregnanolone (11-AziAP). Both photoprobes acted with equal or higher potency than alphaxalone as general anesthetics and potentiators of GABAAR responses, left-shifting the GABA concentration - response curve for human α1β3γ2 GABAARs expressed in Xenopus oocytes, and enhancing [3H]muscimol binding to α1β3γ2 GABAARs expressed in HEK293 cells. With EC50 of 110 nM, 11-AziAP is one the most potent general anesthetics reported. [3H]F4N3Bzoxy-AP and [3H]11-AziAP, at anesthetic concentrations, photoincorporated into α- and β-subunits of purified α1β3γ2 GABAARs, but labeling at the subunit level was not inhibited by alphaxalone (30 μM). The enhancement of photolabeling by 3H-azietomidate and 3H-mTFD-MPAB in the presence of either of the two steroid photoprobes indicates the neurosteroid binding site is different from, but allosterically related to, the etomidate and barbiturate sites. Our observations are consistent with two hypotheses. First, F4N3Bzoxy-AP and 11-aziAP bind to a high affinity site in such a pose that the 11-photoactivatable moiety, that is rigidly attached to the steroid backbone, points away from the protein. Second, F4N3Bzoxy-AP, 11-aziAP and other steroid anesthetics, which are present at very high concentration at the lipid-protein interface due to their high lipophilicity, act via low affinity sites, as proposed by Akk et al. (Psychoneuroendocrinology2009, 34S1, S59-S66).
An entry from the Cambridge Structural Database, the world's repository for small molecule cr... more An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
An entry from the Cambridge Structural Database, the world's repository for small molecule cr... more An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Chemistry (Weinheim an der Bergstrasse, Germany), Jan 3, 2018
Expanded calixpyrrole-type macrocycles, calix[2]benzo[4]pyrroles, bearing fluorescent moieties at... more Expanded calixpyrrole-type macrocycles, calix[2]benzo[4]pyrroles, bearing fluorescent moieties attached via conjugated vinyl spacers, have been synthesized from the corresponding formyl derivatives through Knoevenagel condensation. The anion-binding properties of the resulting fluorescent macrocycles have been studied by means of NMR, UV/Vis, and fluorescence spectroscopies. Our main focus has been on dicarboxylates matching the size of the binding cavity of the calix[2]benzo[4]pyrrole skeleton. The observed anion-binding properties were compared with those of the regular calix[4]pyrroles bearing identical fluorophores. Surprisingly, the parent calix[4]pyrroles appear to be equally efficient, if not more so, for sensing anions such as dicarboxylates. Affinity constants determined for various anions and dianions show the sensors S1-S5 to be highly cross-reactive. The cross-reactivity of the sensors was utilized in a microchip-based array, which showed perfect (100 %) classification o...
Journal of the American Chemical Society, 2014
We report on the synthesis of a new receptor for anions, mesosubstituted hexapyrrolic calix[4]pyr... more We report on the synthesis of a new receptor for anions, mesosubstituted hexapyrrolic calix[4]pyrrole 1. The calix[4]pyrrole's core features two additional pyrrole side-arms suspended above or below the calix[4]pyrrole core. This hexapyrrolic calix[4]pyrrole 1 is formed as cis-and trans-configurational isomers, the structures of which have been determined by single crystal X-ray diffraction. The anion binding experiments revealed interesting difference in the binding mode: The cis-1 isomer binds anions in a mixed binding mode featuring a combination of hydrogen bonding and anion−π interactions resulting in an unexpected strong binding. On the other hand, the trans-1 isomer displays only hydrogen bonding and lower affinity for anions. This is unexpected as one would assume both isomers to display the same binding modes. Overall, the titrations of 1 using UV spectrophotometry and NMR titrations by anions reveal that cisisomer 1 displays higher affinity (10 5 −10 6 M −1) and cross-reactivity for anions, while the trans-isomer 1 shows a more selective response to anions. Such differences in binding mode in configurational isomers are so far unexplored and a feature deserving further study.
Capping off an era marred by drug development failures and punctuated by waning interest and pres... more Capping off an era marred by drug development failures and punctuated by waning interest and presumed intractability toward direct targeting of KRAS, new technologies and strategies are aiding in the target's resurgence. As previously reported, the tetrahydropyridopyrimidines were identified as irreversible covalent inhibitors of KRAS G12C that bind in the switch-II pocket of KRAS and make a covalent bond to cysteine 12. Using structure-based drug design in conjunction with a focused in vitro absorption, distribution, metabolism and excretion screening approach, analogues were synthesized to increase the potency and reduce metabolic liabilities of this series. The discovery of the clinical development candidate MRTX849 as a potent, selective covalent inhibitor of KRAS G12C is described.
British Journal of Pharmacology, 2019
Background and PurposeGeneral anaesthetics can act on synaptic GABAA receptors by binding to one ... more Background and PurposeGeneral anaesthetics can act on synaptic GABAA receptors by binding to one of three classes of general anaesthetic sites. Canonical drugs that bind selectively to only one class of site are etomidate, alphaxalone, and the mephobarbital derivative, R‐mTFD‐MPAB. We tested the hypothesis that the general anaesthetic potencies of mixtures of such site‐selective agents binding to the same or to different sites would combine additively or synergistically respectively.Experimental ApproachThe potency of general anaesthetics individually or in combinations to cause loss of righting reflexes in tadpoles was determined, and the results were analysed using isobolographic methods.Key ResultsThe potencies of combinations of two or three site‐selective anaesthetics that all acted on a single class of site were strictly additive, regardless of which single site was involved. Combinations of two or three site‐selective anaesthetics that all bound selectively to different sites...
Molecular Pharmacology, 2019
GABA A receptors (GABA A Rs) are targets for important classes of clinical agents (e.g., anxiolyt... more GABA A receptors (GABA A Rs) are targets for important classes of clinical agents (e.g., anxiolytics, anticonvulsants, and general anesthetics) that act as positive allosteric modulators (PAMs). Previously, using photoreactive analogs of etomidate ([ 3 H]azietomidate) and mephobarbital [[ 3 H]1-methyl-5-allyl-5-(mtrifluoromethyl-diazirynylphenyl)barbituric acid ([ 3 H]R-mTFD-MPAB)], we identified two homologous but pharmacologically distinct classes of general anesthetic binding sites in the a1b3g2 GABA A R transmembrane domain at b 1-a 2 (b 1 sites) and a 1-b 2 /g 1-b 2 (b 2 sites) subunit interfaces. We now use competition photolabeling with [ 3 H]azietomidate and [ 3 H]R-mTFD-MPAB to identify para-substituted propofol analogs and other drugs that bind selectively to intersubunit anesthetic sites. Propofol and 4-chloro-propofol bind with 5-fold selectivity to b 1 , while derivatives with bulkier lipophilic substitutions [4-(tert-butyl)-propofol and 4-(hydroxyl(phenyl)methyl)-propofol] bind with ∼10-fold higher affinity to b 2 sites. Similar to R-mTFD-MPAB and propofol, these drugs bind in the presence of GABA with similar affinity to the a 1-b 2 and g 1-b 2 sites. However, we discovered four compounds that bind with different affinities to the two b 2 interface sites. Two of these bind with higher affinity to one of the b 2 sites than to the b 1 sites. We deduce that 4-benzoyl-propofol binds with .100-fold higher affinity to the g 1-b 2 site than to the a 1-b 2 or b 1-a 2 sites, whereas loreclezole, an anticonvulsant, binds with 5-and 100-fold higher affinity to the a 1-b 2 site than to the b 1 and g 1-b 2 sites. These studies provide a first identification of PAMs that bind selectively to a single intersubunit site in the GABA A R transmembrane domain, a property that may facilitate the development of subtype selective GABA A R PAMs.
Journal of Biological Chemistry, 2019
Edited by Henrik G. Dohlman Many neuroactive steroids potently and allosterically modulate pentam... more Edited by Henrik G. Dohlman Many neuroactive steroids potently and allosterically modulate pentameric ligand-gated ion channels, including GABA A receptors (GABA A R) and nicotinic acetylcholine receptors (nAChRs). Allopregnanolone and its synthetic analog alphaxalone are GABA A R-positive allosteric modulators (PAMs), whereas alphaxalone and most neuroactive steroids are nAChR inhibitors. In this report, we used 11-(p-azidotetrafluorobenzoyloxy)allopregnanolone (F 4 N 3 Bzoxy-AP), a general anesthetic and photoreactive allopregnanolone analog that is a potent GABA A R PAM, to characterize steroid-binding sites in the Torpedo ␣ 2 ␥␦ nAChR in its native membrane environment. We found that F 4 N 3 Bzoxy-AP (IC 50 ؍ 31 M) is 7-fold more potent than alphaxalone in inhibiting binding of the channel blocker [ 3 H]tenocyclidine to nAChRs in the desensitized state. At 300 M, neither steroid inhibited binding of [ 3 H]tetracaine, a closed-state selective channel blocker, or of [ 3 H]acetylcholine. Photolabeling identified three distinct [ 3 H]F 4 N 3 Bzoxy-AP-binding sites in the nAChR transmembrane domain: 1) in the ion channel, identified by photolabeling in the M2 helices of Val-261 and ␦Val-269 (position M2-13); 2) at the interface between the ␣M1 and ␣M4 helices, identified by photolabeling in ␣M1 (␣Cys-222/␣Leu-223); and 3) at the lipid-protein interface involving ␥Trp-453 (M4), a residue photolabeled by small lipophilic probes and promegestone, a steroid nAChR antagonist. Photolabeling in the ion channel and ␣M1 was higher in the nAChR-desensitized state than in the resting state and inhibitable by promegestone. These results directly indicate a steroidbinding site in the nAChR ion channel and identify additional steroid-binding sites also occupied by other lipophilic nAChR antagonists. Many steroids, including endogenous 3␣-hydroxy metabolites of progesterone and deoxycorticosterone and synthetic analogs, act as potent general anesthetics, sedatives, anxiolytics, or anticonvulsants (1, 2). They are positive allosteric modula
Biophysical Journal, 2019
European Journal of Medicinal Chemistry, 2018
Pregnanolone and allopregnanolone-type ligands exert general anesthetic, anticonvulsant and anxio... more Pregnanolone and allopregnanolone-type ligands exert general anesthetic, anticonvulsant and anxiolytic effects due to their positive modulatory interactions with the GABA A receptors in the brain. Binding sites for these neurosteroids have been recently identified at subunit interfaces in the transmembrane domain (TMD) of homomeric 3 GABA A receptors using photoaffinity labeling techniques, and in homomeric chimeric receptors containing GABA A receptor subunit TMDs by crystallography. Steroid binding sites have yet to be determined in human, heteromeric, functionally reconstituted, full-length, glycosylated GABA A receptors. Here, we report on the synthesis and pharmacological characterization of several photoaffinity analogs of pregnanolone and allopregnanolone, of which 21-[4-(3-(trifluoromethyl)-3H-diazirin-3yl)benzoxy]allopregnanolone (21-pTFDBzox-AP) was the most potent ligand. It is a partial positive modulator of the human 13 and 132L GABA A receptors at sub-micromolar concentrations. [ 3 H]21-pTFDBzox-AP photoincorporated in a pharmacologically specific manner into the and β subunits of those receptors, with the β3 subunit photolabeled most efficiently. Importantly, photolabeling by [ 3 H]21-pTFDBzox-AP was inhibited by the positive steroid modulators alphaxalone, pregnanolone and allopregnanolone, but not by inhibitory neurosteroid pregnenolone sulfate or by two potent general anesthetics and GABA A R positive allosteric modulators, etomidate and an anesthetic barbiturate. The latter two ligands bind to sites at subunit interfaces in the GABA A R that are different from those interacting with neurosteroids. 21-pTFDBzox-AP's potency and pharmacological specificity of photolabeling indicate its suitability for characterizing neurosteroid binding sites in native GABA A receptors.