Potent in vitro anticancer activities of ring-expanded ("fat") nucleosides containing the imidazo[4,5-e][1,3]diazepine ring system

The ring-expanded ("fat") nucleoside, 4,8-diamino-6-imino-6H-1-beta-D-ribofuranosylimidazo[4,5-e][1,3]diazepine (1) and its 2',3',5'-tri-O-benzoyl derivative (2) exhibited potent broad spectrum anticancer activities in vitro against a wide variety of human tumor cell lines. The tribenzoyl derivative 2 was found to be considerably more active than the parent nucleoside 1. Further studies using human prostate cancer cells PC-3 and DU-145 suggest that the treatment of exponentially growing culture cells with 1 and 2 leads to marked loss of cell viability in a dose-dependent manner.     

On the ring transformation of hydrazine derivatives of l-ascorbic acid into nitrogen heterocyclic derivatives

l-hreo-2,3-hexodiulosono-1,4-lactone 2-(p-methoxyphenylhydrazone) (1) was condensed with arylhydrazines to give mixed bishydrazones, whose acetylation gave the corresponding di-O-acetyl derivatives. The hydrazone 1 undergoes elimination of one molecule of water per molecule during, the acetylation, and gives 4-(2-acetoxy- ethylidene)-4-hydroxy-2,3-dioxobutano-1,4-lactone 2-(p-methoxyphenylhydrazone), which reacts with methylhydrazine, via a ring transformation process, to give 1-methyl-3-(L-methylpyrazolin-3-yl)-4,5-pyrazoledione 4-(p-methoxyphenylhydrazone). Alkali rearranged the mixed bishydrazones to 1-aryl-3-(l-threo-glycerol-1-yl)-4,5- pyrazoledione 4-(p-methoxyphenylhydrazones), which gave triacetyl and tribenzoyl derivatives, and, upon periodate oxidation, afforded 1-aryl-3-formyl-4,5- pyrazolediones 4-(p-methoxyphenylhydrazones) that gave the corresponding phenylhydrazones. The n.m.r. and mass spectra of some of these derivatives have been investigated.     

Effects of defined synthetic phospholipids on glycosylation processes. Modifications of membrane-bound N-acetylglucosaminyl-transferase and solubilized sialyl-transferase activities

In cultures of hamster embryo cells, benzo[a]pyrene (B[a]P) is metabolized primarily in the bay region. In contrast, little or no bay region metabolism of the noncarcinogenic isomer benzo[e]pyrene (B[e]P) could be detected during 12–96-h incubations of hamster embryo cells with 4 μM [³H]B[e]P. The upper limit to 9,10-dihydro-9,10-dihydroxy-B[e]P formation is about 0.2% of the ethyl acetate-soluble metabolites ( <0.1% of the total metabolites). The major identified metabolites of B[e]P were 4,5-dihydro-4,5-dihydroxy B[e]P and the glucuronide conjugates of 3-OH-B[e]P and 4,5-dihydro-4,5-dihydroxy B[e]P. Simultaneous treatment of cells with either B[a]P or 7,8-benzoflavone (BF) did not induce bay region metabolism of [³H]B[e]P.     

Comparative potency and pharmacology of isomers of leukotriene D4 on guinea-pig trachea: Requirement for a 5(S)6(R) configuration

The relative contractile activity of C₅ and C₆ diastereomers of Leukotriene D₄ (LTD₄), as well as 11-trans stereoisomers were evaluated in guinea-pig tracheal smooth muscle. 5(S)6(R) LTD₄ was 1000 times more potent than histamine as a contractile agent. While a change of the 11-ethylenic bond from to resulted in a four fold decrease in potency, a change in configuration of the 5-hydroxyl group and/or the 6-peptide adduct resulted in a decrease in potency of at least 2 to 3 orders of magnitude. The contractile activity of all LTD₄ isomers was inhibited by FPL 55712, whereas indomethacin markedly enhanced the contractile activity of 5(S)6(R) LTD₄, but appeared to have less of an effect on the other diastereomers. The results demonstrate the critical nature of configuration of the 5-hydroxyl and the 6-peptide adduct of eicosatetraenoic acid for maintenance of high affinity for receptors.     

Phenylalanyl-aminocyclophosphamides as model prodrugs for proteolytic activation: synthesis, stability, and stereochemical requirements for enzymatic cleavage

4-Aminocyclophosphamide (4-NH2-CPA, 7) was proposed as a prodrug moiety of phosphoramide mustard. Four diastereomers of phenylalanine-conjugates of 4-NH2-CPA were synthesized and their stereochemistry was assigned based on chromatographic and spectroscopic data. All diastereomers were stable in phosphate buffer but only the cis-(4R)-isomer of 15 was efficiently cleaved by alpha-chymotrypsin with a half-life of 20 min, which is much shorter than the 8.9h to >12h half-lives found for the other diastereomers. LC-MS analysis of the proteolytic products of cis-(4R)-15 indicated that 4-NH2-CPA was released upon proteolysis and further disintegrated to phosphoramide mustard. These results suggest the feasibility of using peptide-conjugated cis-(4R)-4-NH2-CPA as potential prodrugs for proteolytic activation in tumor tissues.     

Facile synthesis and rearrangement of L-threo-2,3-hexodiulosono-1,4-lactone 2-(2-arylhydrazones)

Controlled reaction of L-threo-2,3-hexodiulosono-1,4-lactone with substituted phenylhydrazines gave the 2-(monoarylhydrazones) (2), which underwent dehydrative acetylation to 4-(2-acetoxyethylidene)-4-hydroxy-2,3-dioxohutyro-1,4-lactone 2-(2-arylhydrazones) (3). The latter reacted with methylhydrazine to give 1-methyl-3-(1-methylpyrazolin-3-yl)-4,5-pyrazoledione 4-(2-arylhydrazones) (4). Reaction of the monoarythydrazones (2) with phenylhydrazine gave the mixed bishydrazones (5), which were rearranged by alkali and acidification to the pyrazolediones (6). Compounds 6 gave triacetyl (7) and tribenzoyl derivatives (8), and, on periodite oxidation, the aldehydes (9), which afforded the monohydrazones (10). The i.r.. n.m.r.. and mass-spectral data of some of the compounds were investigated.     

Preparation of (6R)-tetrahydrofolic acid and (6R)-5-formyltetrahydrofolic acid of high stereochemical purity

Commercially available 5-formyltetrahydrofolate (5-CHO-H4PteGlu) is chemically prepared in a reaction that introduces an asymmetric center at the 6 carbon, and hence is the mixture of diastereomers differing in chirality about this position. (6R)-5-CHO-H4PteGlu, the diastereomer that is not normally found in vivo, was prepared from folic acid. Folic acid was chemically reduced and (6R)-tetrahydrofolate (H4PteGlu) was obtained from the resultant (6R,S)-H4PteGlu by enzymatic consumption of the natural diastereomer of (6R,S)-5,10-CH2-H4PteGlu (reversibly formed from (6R,S)-H4PteGlu in the presence of formaldehyde) with Lactobacillus casei thymidylate synthase. The 5 position of purified (6R)-H4PteGlu was directly formylated in a carbodiimide-catalyzed reaction. The level of contamination of these preparations with the corresponding 6S diastereomers was estimated using the binding of fluorodeoxyuridylate to thymidylate synthase promoted by folate cofactor (for H4PteGlu) and by the growth of folate requiring bacteria (for 5-CHO-H4PteGlu). Purified preparations of (6R)-H4PteGlu promoted the binding of fluorodeoxyuridylate to L. casei thymidylate synthase (in the presence of formaldehyde) only at concentrations greater than 1000-fold higher than equiactive levels of (6S)-H4PteGlu. Likewise, the (6R)-5-CHO-H4PteGlu made by this method was 600 times less active as a growth factor for Pediococcus cerevisiae than was authentic (6S)-5-CHO-H4PteGlu. Hence, the minimum stereochemical purity of these preparations was 99.9% for (6R)-H4PteGlu and 99.8% for (6R)-5-CHO-H4PteGlu.     

Efficient synthesis and biological evaluation of all A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3 and its 20-epimer

An improved synthesis of the diastereomers of 1alpha,25-dihydroxyvitamin D3 (1) was accomplished utilizing our practical route to the A-ring synthon. We applied this procedure to synthesize for the first time all possible A-ring diastereomers of 20-epi-1alpha,25-dihydroxyvitamin D3 (2). Ten-step conversion of 1-(4-methoxyphenoxy)but-3-ene (6), including enantiomeric introduction of the C-3 hydroxyl group to the olefin by the Sharpless asymmetric dihydroxylation, provided all four possible stereoisomers of A-ring enynes (3). i.e., (3R,5R)-, (3R,5S)-, (3S,5R)- and (3S,5S)-bis[(tert-butyldimethylsilyl)oxy]oct-1-en-7-yne, in good overall yield. Palladium-catalyzed cross-coupling of the A-ring synthon with the 20-epi CD-ring portion (5), (E)-(20S)-de-A,B-8-(bromomethylene)cholestan-25-ol, followed by deprotection, afforded the requisite diastereomers of 20-epi-1alpha,25-dihydroxyvitamin D3 (2). The biological profiles of the synthesized stereoisomers were assessed in terms of affinities for vitamin D receptor (VDR) and vitamin D binding protein (DBP). HL-60 cell differentiation-inducing activity and in vivo calcium-regulating potency in comparison with the natural hormone.     

Syntheses of ( + )-Phrymarolin II and Its Stereoisomers

Phrymarolin II, a unique naturally-occurring lignan having a 1,2-dioxygenated 3,7-dioxabicyclo[3.3.0]octane skeleton, was synthesized by the reaction of sesamol, in the presence of cadmium carbonate, with 1-acetoxy-2-chloro-6-(2′-methoxy-4′,5′-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octane. The latter was prepared through 13 steps starting from an aldol condensation of β-vinyl-γ-butyrolactone with 2-methoxy-4,5-methylenedioxybenzaldehyde. Three diastereomers of phrymarolin II were also obtained.     

A practical post-modification synthesis of oligodeoxynucleotides containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside

We describe herein the practical post-modification synthesis of oligodeoxynucleotides (ODNs) containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside (ImN^N). Since the ImN^N nucleoside unit possessing tribenzoyl groups on its exocyclic amino groups as the protecting group was quite unstable under acidic conditions, cleavage of its glycosidic linkage in ODN has been suggested throughout the conditions of solid-phase synthesis. As an alternative approach, we investigated a post-modification synthesis of the desired ODNs containing the ImN^N unit. Starting with protected 4-amino-7-chloro-1-(2-deoxy-β-d-ribofuranosyl)imidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine derivative 1, conversion into the corresponding phosphoramidite unit was examined. The p-bromobenzoyl group (p-BrBz) was the best protecting group of 4-amino group of 1 to give the phosphoramidite unit 9 for the post-modification synthesis. After carrying out the ODN synthesis linked to the controlled pore glass (CPG) support, the support was treated with ammonium hydroxide at 55 °C to remove the protecting groups, detach the ODN form the CPG support, and convert the 7-chloro group into a desired amino group. As a result, the desired ODNs containing ImN^N were obtained in good yield.     

Time course of metabolism of benzo[e]pyrene by hamster embryo cells and the effect of chemical modifiers

In cultures of hamster embryo cells, benzo[a]pyrene (B[a]P) is metabolized primarily in the bay region. In contrast, little or no bay region metabolism of the noncarcinogenic isomer benzo[e]pyrene (B[e]P) could be detected during 12–96-h incubations of hamster embryo cells with 4 μM [³H]B[e]P. The upper limit to 9,10-dihydro-9,10-dihydroxy-B[e]P formation is about 0.2% of the ethyl acetate-soluble metabolites ( <0.1% of the total metabolites). The major identified metabolites of B[e]P were 4,5-dihydro-4,5-dihydroxy B[e]P and the glucuronide conjugates of 3-OH-B[e]P and 4,5-dihydro-4,5-dihydroxy B[e]P. Simultaneous treatment of cells with either B[a]P or 7,8-benzoflavone (BF) did not induce bay region metabolism of [³H]B[e]P.     

Stereoselectivity and regioselectivity of uridine-5'-diphosphoglucuronosyltransferase toward vicinal dihydrodiols of polycyclic aromatic hydrocarbons

The ability of a purified rat liver microsomal uridine-5'-diphosphoglucuronosyltransferase to catalyze the glucuronidation of stereoisomeric trans- and cis-9, 10-dihydroxy-9, 10-dihydrophenanthrenes and 4, 5-dihydroxy-4,5-dihydrobenzo[alpha]pyrenes is examined. The enzyme shows the ability to discriminate kinetically between the antipodes of trans-9, 10-dihydroxy-9, 10-dihydrophenanthrene with turnover numbers of 0.070 and 1.4 s-1 and kc/Kmapp values of 4.4 X 10(3) and 1.1 X 10(3) M-1 s-1 for the 9R, 10R and 9S, 10S stereoisomers. Glucuronidation of the nondissymmetric cis-9, 10-dihydroxy-9, 10-dihydrophenanthrene proceeds with a turnover number of 0.037 s-1 and kc/Kmapp of 18 X 10(3) M-1 s-1 to give a 60/40 mixture of the two possible diastereomeric products. Three of the four stereoisomers of 4,5-dihydroxy-4,5-dihydrobenzo[alpha] pyrene are regioselectively glucuronidated by the enzyme with a high degree of kinetic discrimination. Turnover numbers for the 4S,5S, 4R,5R, and 4S,5R stereoisomers are 4.1, 0.37, and 0.23 s-1 with kc/Kmapp values of 23.8 X 10(3), 0.23 X 10(3), and 3.15 X 10(3) M-1 s-1, respectively. The 4R,5S cis isomer is not a substrate. Enzyme-catalyzed reactions of the 4S,5S and 4S,5R isomers give exclusively (greater than or equal to 95%) the 4-glucuronide with the 4R,5R isomer giving the 5-glucuronide. The kinetic and regiochemical results indicate that the enzyme recognizes hydroxyl groups on the beta-face or bottom face of the 4,5-dihydroxy-4,5-dihydrobenzo[alpha]pyrenes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of 2-hydroxylaminoimidazoles and their implications for the biological effects of 2-nitroimidazoles

In aqueous solution, in the presence of ammonium chloride, N1-substituted 2-nitroimidazoles are readily reduced to the corresponding hydroxylamines. In air, under neutral conditions, analogous to the reactions of aromatic hydroxylamines, 2-hydroxylaminoimidazoles are converted to the azoxy derivatives via a base-catalyzed condensation reaction between the hydroxylamine and its oxidation product, the nitroso derivative. In nitrogen, rearrangement to form the 2-amino-4(5)hydroxyimidazole derivative followed by addition of water across the C4-C5 double bond to yield isomers of a 4,5-dihydro-4,5-dihydroxy derivative appears to be a major reaction. 2-hydroxylaminoimidazoles undergo a complex series of reactions with glutathione. The initial reaction is the formation of a labile conjugate involving an N-S-linkage. Subsequently in the presence of excess GSH, under neutral conditions, two stable conjugates identified as 2-amino-4-S-glutathionyl- and 2-amino-5-S-glutathionyl imidazoles are formed. Nucleophilic attack by GSH on the imidazole ring of a nitrenium ion is postulated as the initial step in the formation of the stable GSH conjugates as well as the 2-amino-4,5-dihydro dihydroxy derivative. The results provide a molecular mechanism for many of the biological effects of N1-substituted 2-nitroimidazoles in hypoxic mammalian cells.     

New bisabolane sesquiterpenes and coumarin from Leontopodium longifolium

From the roots of Leontopotium longifolium, three new bisabolane sesquiterpenes, rel-(1S,4R,5S,6R)-4,5-diacetoxy-6-[(R)-1,5-dimethylhexa-3,5-dienyl]-3-methylcyclohex-2-enyl (Z)-2-methylbut-2-enoate (1), rel-(1S,4R,5S,6R)-4,5-diacetoxy-6-[(R)-5-hydroxy-1,5-dimethylhex-3-enyl]-3-methylcyclohex-2-enyl (Z)-2-methylbut-2-enoate (2), rel-(1R,2S,4R,5S)-4-acetoxy-2-[(R)-5-hydroxy-1,5-dimethylhex-3-enyl]-5-methylcyclohexyl (Z)-2-methylbut-2-enoate (3), and a new coumarin, 2,3-dihydro-5-hydroxy-2-(1-methylethenyl)-7H-pyrano[2,3-g][1,4]benzodioxin-7-one (4) together with nine known compounds have been isolated. The structures of these compounds were established by spectroscopic methods. Compounds 1 and 2 exhibited moderate cytotoxic activities against human promyelocytic leukemia (HL-60) cells.     

Cytochrome P-450-catalyzed stereoselective epoxidation at the K region of benz[a]anthracene and benzo[a]pyrene

The enantiomers of K-region benz[a]anthracene (BA) 5,6-epoxide and benzo[a]pyrene (BP) 4,5-epoxide were resolved by chiral stationary-phase high-performance liquid chromatography (CSP-HPLC). The K-region epoxides formed in the metabolism of BA by liver microsomes from untreated (control), phenobarbital (PB)-treated, and 3-methylcholanthrene (MC)-treated male Sprague-Dawley rats were determined by CSP-HPLC to have a 5R,6S/5S,6R enantiomer ratio of 25:75, 21:79, and 4:96, respectively. The K-region 4,5-epoxide formed in the metabolism of BP by the same rat liver microsomal preparations contained a 4R,5S/4S,5R enantiomer ratio of 48:52 (control), 40:60 (PB), and 5:95 (MC), respectively. The results indicate that various cytochrome P-450 isozymes of rat liver exhibit different stereoselective properties in catalyzing the epoxidation reactions at the K region of BA and of BP.     

Synthesis, structural studies, and cytostatic evaluation of 5,6-di-O-modified L-ascorbic acid derivatives

The 5,6-di-O-tosylated derivative of l-ascorbic acid was synthesized by selective protection and deprotection of 2,3- and 5,6-dihydroxy functional groups involving 5,6-ditosylation in the final step, while the novel 6-acetoxy, 6-hydroxy, and 6-chloro derivatives of 4,5-didehydro-l-ascorbic acid were obtained by reaction of ditosylated compound with nucleophilic reagents. The analysis of 3JH-4-H-5 homonuclear coupling constants shows that all l-ascorbic acid derivatives except for epoxy and 4,5-didehydro compounds exist in high population as gauche conformers across C-4-C-5 bonds, while 3JC-3-H-5 heteronuclear coupling constants in 4,5-didehydro derivatives indicate cis geometry along C-4-C-5 double bond. The X-ray crystal structure analysis of 2,3-di-O-benzyl-5,6-epoxy- and 5,6-isopropylidene-l-ascorbic acid shows that the oxygen atoms attached at positions 2 and 3 of the lactone ring are disposed in a synperiplanar fashion. Besides that, the dioxolane ring adopts half-chair conformation. The molecules of epoxy derivative are joined into infinite chains by one weak hydrogen bond of C-H...O type. Two O-H...O, and C-H...O hydrogen bonds link the molecules of 5,6-di-O-isopropylidene compound into two-dimensional network. 6-Chloro derivative of 2,3-di-O-benzyl-l-ascorbic acid showed the best cytostatic effects against all tested malignant tumor cells (IC50: approximately 18 microM).     

Structural basis of protein-bound endogenous aldehydes. Chemical and immunochemical characterizations of configurational isomers of a 4-hydroxy-2-nonenal-histidine adduct

4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure. In the present study, we characterized configurational isomers of a HNE-N(alpha)-acetylhistidine adduct by NMR spectroscopy and by molecular orbital calculations. In addition, we raised monoclonal antibodies against (R)-HNE-histidine and (S)-HNE-histidine adducts, characterized their specificities, and examined in vivo localizations of each adduct under oxidative stress. To facilitate structural characterization of the configurational isomers of an HNE-histidine adduct, we prepared the (R)-HNE-histidine and (S)-HNE-histidine adducts by incubating N(alpha)-acetylhistidine with each HNE enantiomer, both of which provided two peaks (Ra and Rb from (R)-HNE-histidine and Sa and Sb from (S)-HNE-histidine adducts) in reversed-phase high-performance liquid chromatography. The NMR analysis showed that each peak was a mixture of two diastereomers. In addition, the analysis of the nuclear Overhauser effect enabled the determination of configurations of the eight isomers. The relative amounts of these isomers in the NMR analysis correlated with the relative energies calculated by molecular orbital methods. On the other hand, using (R)-HNE-modified and (S)-HNE-modified keyhole limpet hemocyanins as the antigens, we raised the monoclonal antibodies, mAbR310 and mAbS412, which enantioselectively recognized the (R)-HNE-histidine and (S)-HNE-histidine adducts, respectively. Among the mixtures (Ra, Rb, Sa, and Sb) of diastereomers, mAbR310 showed the highest immunoreactivity to Rb (the mixture of 2R,4S,5R and 2S,4S,5R isomers), whereas mAbS412 preferentially recognized Sa (the mixture of 2R,4S,5S and 2S,4S,5S isomers). The presence of (R)-HNE and (S)-HNE epitopes in vivo was immunohistochemically examined in the kidney of rats exposed to the renal carcinogen, ferric nitrilotriacetate, by which nuclear and cytosolic stainings with mAbR310 and mAbS412, respectively, were detected.     

Syntheses and Proton Magnetic Resonance Studies at 300 MHz of Two New Bromopentachlorocyclohexanes and Three New Bromotrichlorocyclohexenes

Two diastereomers of 6-bromo-1, 2, 3, 4, 5-pentachlorocyclohexane were synthesized from the dl (36/45)-diastereomer of 3, 4, 5, 6-tetrachlorocyclohexene (α-BTC) and the dl (346/5)-diastereomer (γ-BTC) by several stepwise routes. Both of these new products were shown to have the configuration of lindane by NMR studies at 300 MHz and by the synthetic routes. Three diastereomers of 6-bromo-3, 4, 5-trichlorocyclohexene were also prepared and the configurations determined partly by means of 300 MHz NMR.     

Eusiderins and other neolignans from an Aniba species

A previous report disclosed the presence of benzodioxan and bicyclo[3.2.1]octanoid neolignans in the benzene extract of the trunk wood of an Amazonian Aniba (Lauraceae) species. The chloroform extract of the same material contains additionally two new benzodioxan neolignans [rel-(7S,8R)-Δ^8′-7-hydroxy-3,4,5,5′-tetramethoxy-7.0.3′,8.0.4′-neolignan; rel-(7R,8R)-Δ^7′-3,4,5,5′-tetramethoxy-9′-oxo-7.0.3′,8.0.4′-neolignan], two new bicyclo[3.2.1]-octanoid neolignans [(7R,8S,1′S,2′S,3′S,4′R)-Δ^8′-2′,4′-dihydroxy-3,3′-dimethoxy-4,5-methylenedioxy-1′,2′,3′,4′,5′,6′-hexahydro-5′-oxo-7.3′,8.1′-neolignan; (7R,8S,1′R,2′S,3′S)-Δ^8′-2′-hydroxy-3,3′,5′-trimethoxy-4,5-methylenedioxy-1′,2′,3′,4′-tetrahydro-4′-oxo-7.3′,8.1′-neolignan] and a hydrobenzofuranoid neolignan [(7S,8R,1′S,5′S)-Δ^8′-3,3′,5′-tri-methoxy-4,5-methylenedioxy-1′,4′,5′,6′-tetrahydro-4′-oxo-7.0.2′,8.1-neolignan].     

Molecular Determinants of Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Binding to Transient Receptor Potential V1 (TRPV1) Channels

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P₂ is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P₂ binding site located between the TRP box and the S4-S5 linker. At this site, PI(4,5)P₂ interacts with the amino acid residues Arg-575 and Arg-579 in the S4-S5 linker and with Lys-694 in the TRP box. We confirmed that PI(4,5)P₂ behaves as a channel agonist and found that Arg-575, Arg-579, and Lys-694 mutations to alanine reduce PI(4,5)P₂ binding affinity. Additionally, in silico mutations R575A, R579A, and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P₂ in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P₂ binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which cause an opening of the lower TRPV1 channel gate.