Lipophilic Pocket Research Articles

Synthesis and pharmacological evaluation of hydrophobic esters and ethers of butorphanol at opioid receptors

We synthesized several hydrophobic esters and ethers of butorphanol and assessed their affinities at opioid receptors in CHO cell membranes. Tested compounds displayed moderate to high affinities to the μ and κ receptors. The findings accord with previous evidence of a lipophilic binding pocket in the opioid receptors that can be accessed to afford good binding affinity without the need for a phenolic hydrogen-bond donor group. The most potent ( K i = 61 pM at μ and 48 pM at κ) novel agent was (−)- N-cyclobutylmethylmorphinan-3-yl-14-ol phenoxyacetate ( 4d).

Synthesis, induced-fit docking investigations, and in vitro aldose reductase inhibitory activity of non-carboxylic acid containing 2,4-thiazolidinedione derivatives

In continuation of our studies, we here report a series of non-carboxylic acid containing 2,4-thiazolidinedione derivatives, analogues of previously synthesized carboxylic acids which we had found to be very active in vitro aldose reductase (ALR2) inhibitors. Although the replacement of the carboxylic group with the carboxamide or N-hydroxycarboxamide one decreased the in vitro ALR2 inhibitory effect, this led to the identification of mainly non-ionized derivatives with micromolar ALR2 affinity. The 5-arylidene moiety deeply influenced the activity of these 2,4-thiazolidinediones. Our induced-fit docking studies suggested that 5-(4-hydroxybenzylidene)-substituted derivatives may bind the polar recognition region of the ALR2 active site by means of the deprotonated phenol group, while their acetic chain and carbonyl group at position 2 of the thiazolidinedione ring form a tight net of hydrogen bonds with amino acid residues of the lipophilic specificity pocket of the enzyme.

2-Amino-1,3-thiazol-4(5H)-ones as Potent and Selective 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitors: Enzyme−Ligand Co-Crystal Structure and Demonstration of Pharmacodynamic Effects in C57Bl/6 Mice

11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) has attracted considerable attention during the past few years as a potential target for the treatment of diseases associated with metabolic syndrome. In our ongoing work on 11beta-HSD1 inhibitors, a series of new 2-amino-1,3-thiazol-4(5 H)-ones were explored. By inserting various cycloalkylamines at the 2-position and alkyl groups or spirocycloalkyl groups at the 5-position of the thiazolone, several potent 11beta-HSD1 inhibitors were identified. An X-ray cocrystal structure of human 11beta-HSD1 with compound 6d (Ki=28 nM) revealed a large lipophilic pocket accessible by substitution off the 2-position of the thiazolone. To increase potency, analogues were prepared with larger lipophilic groups at this position. One of these compounds, the 3-noradamantyl analogue 8b, was a potent inhibitor of human 11beta-HSD1 (Ki=3 nM) and also inhibited 11beta-HSD1 activity in lean C57Bl/6 mice when evaluated in an ex vivo adipose and liver cortisone to cortisol conversion assay.

Co-Crystal Structures of 7-Azaindole Inhibitors of Wild-Type and T315I Imatinib-Resistant Mutant Forms of the BCR-ABL Tyrosine Kinase.

Chronic myelogenous leukemia (CML) arises from uncontrolled cell growth driven by a constitutively active BCR-ABL fusion protein tyrosine kinase, which is the product of the pathognomonic Philadelphia chromosomal translocation. Imatinib mesylate (Gleevec) is a BCR-ABL inhibitor used as a first line treatment of CML. Although imatinib is highly effective in chronic phase CML, in advanced disease patients frequently relapse due to the emergence of drug resistance. Approximately two-thirds of resistance is caused by point mutations in the BCR-ABL kinase domain, which give rise to active mutant forms of the enzyme that are insensitive to Gleevec. The T315I mutation represents one of the most common causes of resistance, is resistant to the second generation BCR-ABL inhibitors dasatinib and nilotinib, and represents an important and challenging target for discovery of next generation targeted CML treatments. We have applied X-ray crystallographic screening of our FAST™ fragment library and structure-guided hit-to-lead optimization to identify potent inhibitors of both wild-type and T315I mutant BCR-ABL. These efforts yielded a 7-azaindole compound series that exhibits binding to and inhibition of both wild-type and T315I BCR-ABL.Methods: Wild-type (with Y393F) and T315I Abl kinase domain protein were expressed in E. coli and purified to homogeneity. These proteins were crystallized in the presence of a reference inhibitor followed by addition of the 7-azaindole series compounds soaked into the preformed crystals to displace the reference compound, giving the desired co-crystal. X-ray diffraction data were recorded at the company's proprietary synchrotron beamline SGX-CAT at the Advanced Photon Source. Three-dimensional enzyme-inhibitor co-crystal structures were determined by molecular replacement and refined to permit modeling of bound ligand.Results: Both wild-type and T315I Abl structures revealed enzyme in the active conformation with inhibitors bound to the kinase hinge region. The crystal structure of 2-amino-5-[3-(1-ethyl-1H-pyrazol-5-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl]-N,N-dimethylbenzamide in complex with T315I, illustrates the typical binding mode which is independent of the 315 residue, and therefore accounts for the compound inhibiting the T315I mutant form of BCR-ABL (see figure). The inhibitor binds to the hinge region of ABL utilizing hydrogen bonding to backbone carbonyl of Glu316 and NH of Met318, with the pyrazole ring stacking in a lipophilic pocket between Phe382 and Tyr253. In addition, the benzamide carbonyl participates in a hydrogen bond interactioin with the backbone-NH of Glu249 of the p-loop.Conclusions: X-ray crystallographic fragment screening and co-crystal structure studies have been successfully employed in discovery/optimization of 7-azaindole series compounds, yielding potent, selective inhibitors of both wild-type and imatinib-resistant forms of BCR-ABL. [Display omitted]

Evaluation of in vitro aldose redutase inhibitory activity of 5-arylidene-2,4-thiazolidinediones

A number of 5-arylidene-2,4-thiazolidinediones containing a hydroxy or a carboxymethoxy group in their 5-benzylidene moiety have been synthesised and evaluated as in vitro aldose reductase (ALR2) inhibitors. Most of them exhibited strong inhibitory activity, with IC 50 values in the range between 0.20 and 0.70 μM. Molecular docking simulations into the ALR2 active site highlighted that the phenolic or carboxylic substituents of the 5-benzylidene moiety can favourably interact, in alternative poses, either with amino acid residues lining the lipophilic pocket of the enzyme, such as Leu300, or with the positively charged recognition region of the ALR2 active site.

Molecular Recognition of Ligands in Dipeptidyl Peptidase IV

The serine protease dipeptidyl peptidase IV (DPP-IV) is a clinically validated target for the treatment of type II diabetes and has received considerable interest from the pharmaceutical industry over the last years. Concomitant with a large variety of published small molecule DPP-IV inhibitors almost twenty co-crystal structures have been released to the public as of May 2006. In this review, we discuss the structural characteristics of the DPP-IV binding site and use the available X-ray information together with published structure-activity relationship data to identify the molecular interactions that are most important for tight enzyme-inhibitor binding. Optimized interactions with the two key recognition motifs, i.e. the lipophilic S1 pocket and the negatively charged Glu 205/206 pair, result in large gains in binding free energy, which can be further improved by additional favorable contacts to side chains that flank the active site. First examples show that the lessons learned from the X-ray structures can be successfully incorporated into the design of novel DPP-IV inhibitors.

PH-tuneable binding of 2'-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study.

The crystal structure of Escherichia coli ketopantoate reductase in complex with 2'-monophosphoadenosine 5'-diphosphoribose, a fragment of NADP+ that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ;reversed binding mode' observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes.

The synthesis and structure–activity relationship studies of selective acetyl-CoA carboxylase inhibitors containing 4-(thiazol-5-yl)but-3-yn-2-amino motif: Polar region modifications

The structure–activity relationship study focused on the polar region of the HTS hit A-80040 ( 1) producing several series of potent and selective ACC2 inhibitors. The SAR suggests a compact lipophilic pocket that does not tolerate polar and ionic groups. Replacement of the hydroxyurea group with isoxazoles improves ACC2 selectivity while maintaining potency. Variations at the propargylic site of 11a reduce ACC2 potency.

Synthesis and biological evaluation of novel 5( H)-phenanthridin-6-ones, 5( H)-phenanthridin-6-one diketo acid, and polycyclic aromatic diketo acid analogs as new HIV-1 integrase inhibitors

A new series of phenanthridinone derivatives, and diketo acid analogs, as well as related phenanthrene and anthracene diketo acids have been synthesized and evaluated as HIV integrase (IN) inhibitors. Several new β-diketo acid analogs with the phenanthridinone scaffold replaced by phenanthrene, anthracene or pyrene exhibited the highest IN inhibitory potency. There is a general selectivity against the integrase strand transfer step. The most potent IN was 2,4-dioxo-4-phenanthren-9-yl-butyric acid ( 27f) with an IC 50 of 0.38 μM against integrase strand transfer. The phenanthrene diketo acids 27d– f were more potent (IC 50 = 2.7–0.38 μM) than the corresponding phenanthridinone diketo acid 16 (IC 50 = 65 μM), suggesting that the polar amide bridge in the phenanthridinone system decreases inhibitory activity relative to the more lipophilic phenanthrene system. This might have to do with the possible binding of the aryl group of the compounds binding to a lipophilic pocket at the integrase active site as suggested by the docking simulations. Molecular modeling also suggested that effectiveness of chelation of the active site Mg 2+ contributes to IN inhibitory potency. Finally, some of the potent compounds inhibited HIV-1 replication in human peripheral blood mononuclear cells (PBMC) with EC 50 down to 8 μM for phenanthrene-3-(2,4-dioxo)butyric acid ( 27d), with a selectivity index of 10 against PBMCs.

Synthesis and in vitro evaluation of salvinorin A analogues: Effect of configuration at C(2) and substitution at C(18)

κ-opioid receptor ligands have raised interest for their apparent effects on mood. The potent and selective κ-agonist salvinorin A has short-lasting (15 min) depressive-like effects in rats in behavioral models used to study mood disorders. Two series of salvinorin derivatives modified at C(2) and C(18), respectively, were synthesized and their κ-opioid receptor affinities, potencies, and efficacies were evaluated using in vitro receptor binding and biochemical functional assays. Modification at C(2) yielded potent κ-agonists that are predicted to have improved metabolic stability ( 14a, 15a) or increased water solubility ( 10b). Our preliminary SAR study at C(18) suggested that this part of the molecule interacts with a tight lipophilic pocket of the κ-receptor.

Structural and Molecular Interactions of CCR5 Inhibitors with CCR5

We have characterized the structural and molecular interactions of CC-chemokine receptor 5 (CCR5) with three CCR5 inhibitors active against R5 human immunodeficiency virus type 1 (HIV-1) including the potent in vitro and in vivo CCR5 inhibitor aplaviroc (AVC). The data obtained with saturation binding assays and structural analyses delineated the key interactions responsible for the binding of CCR5 inhibitors with CCR5 and illustrated that their binding site is located in a predominantly lipophilic pocket in the interface of extracellular loops and within the upper transmembrane (TM) domain of CCR5. Mutations in the CCR5 binding sites of AVC decreased gp120 binding to CCR5 and the susceptibility to HIV-1 infection, although mutations in TM4 and TM5 that also decreased gp120 binding and HIV-1 infectivity had less effects on the binding of CC-chemokines, suggesting that CCR5 inhibition targeting appropriate regions might render the inhibition highly HIV-1-specific while preserving the CC chemokine-CCR5 interactions. The present data delineating residue by residue interactions of CCR5 with CCR5 inhibitors should not only help design more potent and more HIV-1-specific CCR5 inhibitors, but also give new insights into the dynamics of CC-chemokine-CCR5 interactions and the mechanisms of CCR5 involvement in the process of cellular entry of HIV-1.

Insights from modeling the 3D structure of H5N1 influenza virus neuraminidase and its binding interactions with ligands

The highly pathogenic H5N1 influenza virus, which is rapidly mutating and becoming increasingly drug-resistant, was investigated by means of structure–activity relationship between NA (neuraminidase) and three inhibitors, i.e., DANA (2,3-didehydro-2-deoxy- N-acetylneuraminic acid), zanamivir, and oseltamivir. A homology model of the H5N1-NA from the highly pathogenic chicken H5N1 A viruses isolated during the 2003–2004 influenza outbreaks in Japan was built based on the crystal structure of N9-NA complexed with DANA (PDB code: 1F8B). It was found that the traditional constituent residues around the active site of NA family are highly conserved in the H5N1-NA. However, a partially lipophilic pocket composed by Ala248 and Thr249 in N9-NA becomes a hydrophilic pocket because the two residues in the H5N1-NA are replaced by hydrophilic residues Ser227 and Asn228, respectively. On the other hand, two hydrophilic residues Asn347 and Asn348 in the N9-NA are replaced by two lipophilic residues Ala323 and Tyr324 in the H5N1-NA, respectively, leading to the formation of a new lipophilic pocket. This kind of subtle variation not only destroys the original lipophilic environment but also changes the complement interaction between the H5N1-NA and DANA. Such a finding might provide insights into the secret why some of H5N1 strains bear high resistance for existing NA inhibitors, and stimulate new strategies for designing new drugs against these viruses.

ANTI-HIV STRATEGY

AN AMALGAM OF COMPUTER modeling and experiment has yielded insights on the inner workings of a protein vulnerable to HIV and the molecules that can block the virus. The protein, known as CCR5, belongs to the family of G-protein-coupled receptors (GPCRs). HIV binds to CCR5 to gain access to immune cells. At last week's ACS national meeting in Atlanta, Debananda Das, a computational chemist at the National Institutes of Health, reported that molecules that inhibit CCR5 predominately bind in a large lipophilic pocket on the upper transmembrane portion of the protein (J. Biol. Chem., published online Feb. 13, dx.doi. org/10.1074/jbc.M512688200). Das, in a group headed by NIH retrovirologist Hiroaki Mitsuya, studied the binding of three CCR5 inhibitors, including the promising drug aplaviroc, with dozens of CCR5 mutants. They used that data to develop a computer model of the structure of the CCR5 binding site/inhibitor complex and the key binding interactions. Because no crystal ...

Synthesis, Biological Evaluation, and Crystallographic Studies of Extended Guanine‐Based (lin‐Benzoguanine) Inhibitors for tRNA‐Guanine Transglycosylase (TGT)

AbstractThis paper describes the rational design, synthesis, and biological evaluation of a new generation of inhibitors of the bacterial enzyme tRNA‐guanine transglycosylase (TGT), which has been identified as a new target in the fight against bacillary dysentery (Shigellosis). The enzyme catalyzes the exchange of guanine in the anticodon wobble position of tRNA by the modified base preQ1, a guanine derivative, according to a ping‐pong mechanism involving a covalent TGT‐tRNA intermediate (Fig. 2). Based on computer modeling (Fig. 3), lin‐benzoguanine (6‐aminoimidazol[4,5‐g]quinazolin‐8(7H)‐one (2)) was selected as an extended central scaffold, to form up to seven in‐plane intermolecular H‐bonds with the protein while sandwiching between Tyr106 and Met260. Versatile synthetic protocols were developed for the synthesis of 2, and derivatives with phenyl, benzyl, and 2‐phenylethyl side chains (i.e., 16, 17a, and 12a, 12b, 13, 17, resp.) to reach into the lipophilic pocket lined by Val282, Val45, and Leu68 (Schemes 1–3). To account for the limited solubility of the new ligands and in consequence of a recently developed detailed understanding of the mechanism of TGT catalysis (Fig. 2), the enzyme kinetic assay was completely redesigned, providing competitive (Kic) and uncompetitive (Kiu) inhibition constants with respect to tRNA binding by TGT. The modifications of the various parameters in the new assay are described in detail. Binding affinities of the new inhibitors were found to be in the single‐digit micromolar range (Kic values, Fig. 8). Decoration of the lin‐benzoguanine scaffold with lipophilic residues only gave a modest improvement in biological activity which was explained on structural grounds with the help of four crystal structures (Fig. 10) obtained by soaking the protein with inhibitors 2 and 12a–12c. Both biochemical and biostructural analyses reported in this paper provide a fertile basis for the development of more potent future generations of TGT inhibitors.

Replacement of Isobutyl by Trifluoromethyl in Pepstatin A Selectively Affects Inhibition of Aspartic Proteinases

Two bis-trifluoromethyl pepstatin A analogues, carboxylic acid 1 and its methyl ester 2, have been synthesised in order to probe the properties and size of the trifluoromethyl (Tfm) group and compare it to the "bigger" isobutyl that is present in pepstatin A. The results demonstrate that Tfm can effectively replace the isobutyl chain as far as inhibitory activity against plasmepsin II (PM II), an aspartic proteinase from Plasmodium falciparum, is concerned. On the other hand, replacement of isobutyl by Tfm selectively affected activity against other aspartic proteinases tested. Two lines of evidence led to these conclusions. Firstly, compounds 1 and 2 retained single-digit nanomolar inhibitory activity against PM II, but were markedly less active against PM IV, cathepsin D and cathepsin E. Secondly, the X-ray crystal structures of the three complexes of PM II with 1, 2 and pepstatin A were obtained at 2.8, 2.4 and 1.7 A resolution, respectively. High overall similarity among the three complexes indicated that the central Tfm was well accommodated in the lipophilic S1 pocket of PM II, where it was involved in tight hydrophobic contacts. The interaction of PM II with Phe111 appeared to be crucial. Comparison of the crystal structures presented here, with X-ray structures or structural models of PM IV and cathepsin D, allowed an interpretation of the inhibition profiles of pepstatin A and its Tfm variants against these three enzymes. Interactions of the P1 side chain with amino acids that point into the S1 pocket appear to be critical for inhibitory activity. In summary, Tfm can be used to replace an isobutyl group and can affect the selectivity profile of a compound. These findings have implications for the design of novel bioactive molecules and synthetic mimics of natural compounds.

Structure–activity relationships and enzyme inhibition of pantothenamide-type pantothenate kinase inhibitors

A set of novel pantothenamide-type analogues of the known Staphylococcus aureus pantothenate kinase ( SaPanK) inhibitors, N-pentyl, and N-heptylpantothenamide, was synthesized in three series. The first series of analogues ( 1– 3) were designed as molecular probes of the PanK binding site to elucidate important structure–activity relationships (SAR). The second series of analogues ( 4– 16) were designed using structural information obtained from the Escherichia coli PanK ( EcPanK) structure by targeting the pantothenate binding site and the adjacent phenylalanine-lined lipophilic pocket. Insight into the antimicrobial effect of N-pentylpantothenamide (N5-Pan) through its conversion to the antimetabolite ethyldethia-CoA and further incorporation into an inactive acyl carrier protein analogue drove the development of the third series of analogues ( 17– 25) to enhance this effect using substrate-like substitutions. Each of the analogues was screened for enzyme inhibition activity against a panel of pantothenate kinases consisting of EcPanK, Aspergillus nidulans ( AnPanK), SaPanK, and the murine isoform ( MmPanK1α). Series 1 demonstrated only modest inhibitory activity, but did reveal some important SAR findings including stereospecific binding. Series 2 demonstrated a much higher inhibition rate for the entire series and significant inhibition was seen with analogues containing alkyl substituents. Series 3 demonstrated the most preferential inhibition profile, with the highest inhibitory activity against the SaPanK and MmPanK1α. The MmPanK1α protein was inhibited by a broad spectrum of the compounds, whereas the E. coli enzyme showed greater selectivity. The overall activity data from these analogues suggest a complex and non-enzyme specific SAR for pantothenamide substrate/inhibitors of the different PanK enzymes.

First QSAR report on FSH receptor antagonistic activity: Quantitative investigations on physico-chemical and structural features among 6-amino-4-phenyltetrahydroquinoline derivatives

A quantitative attempt has been made to correlate the structure–activity relationship (SAR) among the recently reported 6-amino-4-phenyltetrahydroquinoline derivatives as antagonists for the Gs-protein-coupled human follicle-stimulating hormone (FSH) receptor. The compounds used for the present study have been reported to show high antagonistic efficacy in vitro using a CHO-hFSHR(luc) assay. Our QSAR investigations revealed a hydrophobic type of interactions between these ligands and the FSH receptor, hence confirming the presence of a lipophilic pocket on the active site of the target structure. The positive coefficient of C log P variable in our derived QSAR model suggests that more hydrophobic ligands are crucial for their FSH receptor antagonistic efficacy. In exploring the structural requirements among these congeners, we found an amide linkage as conducive to their FSH receptor antagonistic activity. Also, an unsubstituted 4-phenyl ring of the tetrahydroquinoline scaffold is favorable for their FSH receptor antagonistic activity. The results discussed herein could be useful in understanding the nature of interactions of these newly identified ligands as FSH receptor antagonists and in designing more potent ligands based on this novel 6-amino-4-phenyltetrahydroquinoline scaffold.

“Reversine” and Its 2-Substituted Adenine Derivatives as Potent and Selective A3 Adenosine Receptor Antagonists

The dedifferentiation agent "reversine" [2-(4-morpholinoanilino)-N(6)-cyclohexyladenine 2] was found to be a moderately potent antagonist for the human A(3) adenosine receptor (AR) with a K(i) value of 0.66 microM. This result prompted an exploration of the structure-activity relationship of related derivatives, synthesized via sequential substitution of 6-chloro-2-fluoropurine with selected nucleophiles. Optimization of substituents at these two positions identified 2-(phenylamino)-N(6)-cyclohexyladenine (12), 2-(phenylamino)-N(6)-cycloheptyladenine (19), and 2-phenylamino-N(6)-endo-norbornyladenine (21) as potent A(3) AR ligands with K(i) values of 51, 42, and 37 nM, respectively, with 30-200-fold selectivity in comparison to A(1) and A(2A) ARs. The most selective A(3) AR antagonist (>200-fold) was 2-(phenyloxy)-N(6)-cyclohexyladenine (22). 9-Methylation of 12, but not 19, was well-tolerated in A(3) AR binding. Extension of the 2-phenylamino group to 2-benzyl- and 2-(2-phenylethylamino) reduced affinity. In the series of 2-(phenylamino), 2-(phenyloxy), and 2-(phenylthio) substitutions, the order of affinity at the A(3) AR was oxy > or = amino > thio. Selected derivatives, including reversine (K(B) value of 466 nM via Schild analysis), competitively antagonized the functional effects of a selective A(3) AR agonist, i.e., inhibition of forskolin-stimulated cAMP production in stably transfected Chinese hamster ovary (CHO) cells. These results are in agreement with other studies suggesting the presence of a lipophilic pocket in the AR binding site that is filled by moderately sized cycloalkyl rings at the N(6) position of both adenine and adenosine derivatives. Thus, the compound series reported herein comprise an important new series of selective A(3) AR antagonists. We were unable to reproduce the dedifferentiation effect of reversine, previously reported, or to demonstrate any connection between A(3) AR antagonist effects and dedifferentiation.

Characterization of the Sulfhydryl-Sensitive Site in the Enzyme Responsible for Hydrolysis of 2- Arachidonoyl-Glycerol in Rat Cerebellar Membranes

We have previously reported that the endocannabinoid, 2-arachidonoyl-glycerol (2-AG), is hydrolyzed in rat cerebellar membranes by monoglyceride lipase (MGL)-like enzymatic activity. The present study shows that, like MGL, 2-AG-degrading enzymatic activity is sensitive to inhibition by sulfhydryl-specific reagents. Inhibition studies of this enzymatic activity by N-ethylmaleimide analogs revealed that analogs with bulky hydrophobic N-substitution were more potent inhibitors than hydrophilic or less bulky agents. Interestingly, the substrate analog N-arachidonylmaleimide was found to be the most potent inhibitor. A comparison model of MGL was constructed to get a view on the cysteine residues located near the binding site. These findings support our previous conclusion that the 2-AG-degrading enzymatic activity in rat cerebellar membranes corresponds to MGL or MGL-like enzyme and should facilitate further efforts to develop potent and more selective MGL inhibitors.

Cut-off phenomenon in the protective effect of alcohols against lysophosphatidylcholine-induced calcium overload

We studied the effect of chain length on the protective effect of alcohols against lysophosphatidylcholine (LPC)-induced Ca2+ overload in neonatal rat cardiomyocytes. We previously found that ethanol retards Ca2+ elevation. Cells were loaded with the Ca2+-sensitive fluorophore fura-2, and changes in fluorescence were followed. The addition of 10 microM LPC increased Ca2+, which reached a plateau after an 8-10 min delay. The presence of 88 mM n-propanol, n-butanol, tert-butanol, or 2,2-dimethylpropanol significantly increased the delay by 94-213%. However, n-pentanol at 2 mM or 88 mM had no protective effect. Among n-alcohols, the increase in lag time was inversely proportional to the length of the carbon chain. Chain length, rather than molecular weight determines the effect, because 2,2-dimethylpropanol had a protective effect. The influence of alcohols on LPC micelle formation was estimated from the increase in octadecyl rhodamine B fluorescence; the increase by n-alcohols was directly proportional to chain length, indicating that micelle formation was not involved in the extension of lag time. The absence of the protective effect when the alcohol aliphatic chain exceeds four carbons suggests that the effect of ethanol may be mediated via a small lipophilic pocket on a protein, or to lateral pressure perturbation in the membrane.