Biochemical Pharmacology of the Sigma-1 Receptor.

The sigma-1 receptor (S1R) is a 223 amino acid two transmembrane (TM) pass protein. It is a non-ATP-binding nonglycosylated ligand-regulated molecular chaperone of unknown three-dimensional structure. The S1R is resident to eukaryotic mitochondrial-associated endoplasmic reticulum and plasma membranes with broad functions that regulate cellular calcium homeostasis and reduce oxidative stress. Several multitasking functions of the S1R are underwritten by chaperone-mediated direct (and indirect) interactions with ion channels, G-protein coupled receptors and cell-signaling molecules involved in the regulation of cell growth. The S1R is a promising drug target for the treatment of several neurodegenerative diseases related to cellular stress. In vitro and in vivo functional and molecular characteristics of the S1R and its interactions with endogenous and synthetic small molecules have been discovered by the use of pharmacologic, biochemical, biophysical, and molecular biology approaches. The S1R exists in monomer, dimer, tetramer, hexamer/octamer, and higher oligomeric forms that may be important determinants in defining the pharmacology and mechanism(s) of action of the S1R. A canonical GXXXG in putative TM2 is important for S1R oligomerization. The ligand-binding regions of S1R have been identified and include portions of TM2 and the TM proximal regions of the C terminus. Some client protein chaperone functions and interactions with the cochaperone 78-kDa glucose-regulated protein (binding immunoglobulin protein) involve the C terminus. Based on its biochemical features and mechanisms of chaperone action the possibility that the S1R is a member of the small heat shock protein family is discussed.

Sigma Receptor Binding Assays.

Sigma receptors, both Sigma-1(S1R) and Sigma-2 (S2R), are small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated sites. A number of drugs bind to sigma receptors, including the antipsychotic haloperidol and (+)-pentazocine, an opioid analgesic. Sigma receptors are implicated in many central nervous system disorders, in particular Alzheimer's disease and conditions associated with motor control, such as Amyotrophic Lateral Sclerosis (ALS). Described in this unit are radioligand binding assays used for the pharmacological characterization of S1R and S2R. Methods detailed include a radioligand saturation binding assay for defining receptor densities and a competitive inhibition binding assay employing [³H]-(+)-pentazocine for identifying and characterizing novel ligands that interact with S1R. Procedures using [³H]-1,3-di(2-tolyl)guanidine ([³H]-DTG), a nonselective sigma receptor ligand, are described for conducting a saturation binding and competitive inhibition assays for the S2R site. These protocols are of value in drug discovery in identifying new sigma ligands and in the characterization of these receptors.

Endothelial protective genes induced by statin are mimicked by ERK5 activation as triggered by a drug combination of FTI-277 and GGTI-298.

BACKGROUND: Statins are potent inhibitors of cholesterol biosynthesis and are clinically beneficial in preventing cardiovascular diseases, however, the therapeutic utility of these drugs is limited by myotoxicity. Here, we explored the mechanism of statin-mediated activation of ERK5 in the human endothelium with the goal of identifying compounds that confer endothelial protection but are nontoxic to muscle. METHODS: An ERK5-one hybrid luciferase reporter transfected into COS-7 cells with pharmacological and molecular manipulations dissected the signaling pathway leading to statin activation of ERK5. qRT-PCR of HUVEC cells documented the transcriptional activation of endothelial-protective genes. Lastly, morphological and cellular ATP analysis, and induction of atrogin-1 in C2C12 myotubes were used to assess statin-induced myopathy. RESULTS: Statin activation of ERK5 is dependent on the cellular reduction of GGPPs. Furthermore, we found that the combination of FTI-277 (inhibitor of farnesyl transferase) and GGTI-298 (inhibitor of geranylgeranyl transferase I) mimicked the statin-mediated activation of ERK5. FTI-277 and GGTI-298 together recapitulated the beneficial effects of statins by transcriptionally upregulating anti-inflammatory mediators such as eNOS, THBD, and KLF2. Finally, C2C12 skeletal myotubes treated with both FTI-277 and GGTI-298 evoked less morphological and cellular changes recognized as biomarkers of statin-associated myopathy. CONCLUSIONS: Statin-induced endothelial protection and myopathy are mediated by distinct metabolic intermediates and co-inhibition of farnesyl transferase and geranylgeranyl transferase I confer endothelial protection without myopathy. GENERAL SIGNIFICANCE: The combinatorial FTI-277 and GGTI-298 drug regimen provides a promising alternative avenue for endothelial protection without myopathy.

The Sigma-2 Receptor and Progesterone Receptor Membrane Component 1 are Different Binding Sites Derived From Independent Genes.

The sigma-2 receptor (S2R) is a potential therapeutic target for cancer and neuronal diseases. However, the identity of the S2R has remained a matter of debate. Historically, the S2R has been defined as (1) a binding site with high affinity to 1,3-di-o-tolylguanidine (DTG) and haloperidol but not to the selective sigma-1 receptor ligand (+)-pentazocine, and (2) a protein of 18-21 kDa, as shown by specific photolabeling with [(3)H]-Azido-DTG and [(125)I]-iodoazido-fenpropimorph ([(125)I]-IAF). Recently, the progesterone receptor membrane component 1 (PGRMC1), a 25 kDa protein, was reported to be the S2R (Nature Communications, 2011, 2:380). To confirm this identification, we created PGRMC1 knockout NSC34 cell lines using the CRISPR/Cas9 technology. We found that in NSC34 cells devoid of or overexpressing PGRMC1, the maximum [(3)H]-DTG binding to the S2R (Bmax) as well as the DTG-protectable [(125)I]-IAF photolabeling of the S2R were similar to those of wild-type control cells. Furthermore, the affinities of DTG and haloperidol for PGRMC1 (KI = 472 µM and 350 µM, respectively), as determined in competition with [(3)H]-progesterone, were more than 3 orders of magnitude lower than those reported for the S2R (20-80 nM). These results clarify that PGRMC1 and the S2R are distinct binding sites expressed by different genes.

The oligomeric states of the purified sigma-1 receptor are stabilized by ligands.

Sigma-1 receptor (S1R) is a mammalian member of the ERG2 and sigma-1 receptor-like protein family (pfam04622). It has been implicated in drug addiction and many human neurological disorders, including Alzheimer and Parkinson diseases and amyotrophic lateral sclerosis. A broad range of synthetic small molecules, including cocaine, (+)-pentazocine, haloperidol, and small endogenous molecules such as N,N-dimethyltryptamine, sphingosine, and steroids, have been identified as regulators of S1R. However, the mechanism of activation of S1R remains obscure. Here, we provide evidence in vitro that S1R has ligand binding activity only in an oligomeric state. The oligomeric state is prone to decay into an apparent monomeric form when exposed to elevated temperature, with loss of ligand binding activity. This decay is suppressed in the presence of the known S1R ligands such as haloperidol, BD-1047, and sphingosine. S1R has a GXXXG motif in its second transmembrane region, and these motifs are often involved in oligomerization of membrane proteins. Disrupting mutations within the GXXXG motif shifted the fraction of the higher oligomeric states toward smaller states and resulted in a significant decrease in specific (+)-[(3)H]pentazocine binding. Results presented here support the proposal that S1R function may be regulated by its oligomeric state. Possible mechanisms of molecular regulation of interacting protein partners by S1R in the presence of small molecule ligands are discussed.

Noncompetitive inhibition of indolethylamine-N-methyltransferase by N,N-dimethyltryptamine and N,N-dimethylaminopropyltryptamine.

Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were determined. After Michaelis-Menten and Lineweaver-Burk analyses had been applied to study inhibition, DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against tryptamine. The novel tryptamine derivative, N-[2-(1H-indol-3-yl)ethyl]-N',N'-dimethylpropane-1,3-diamine (propyl dimethyl amino tryptamine or PDAT), was shown to inhibit rabINMT by a pure noncompetitive mechanism when measured against tryptamine with a Ki of 84 µM. No inhibition by PDAT was observed at 2 mM when it was tested against structurally similar Class 1 methyltransferases, such as human phenylethanolamine-N-methyltransferase (hPNMT) and human nicotinamide-N-methyltransferase (hNNMT), indicating selectivity for INMT. The demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an inhibitory allosteric site. In silico analyses using the computer modeling software Autodock and the rabINMT sequence threaded onto the human INMT (hINMT) structure (Protein Data Bank entry 2A14 ) identified an N-terminal helix-loop-helix non-active site binding region of the enzyme. The energies for binding of DMT and PDAT to this region of rabINMT, as determined by Autodock, were -6.34 and -7.58 kcal/mol, respectively. Assessment of the allosteric control of INMT may illuminate new biochemical pathway(s) underlying the biology of INMT.

Photoaffinity labeling of the sigma-1 receptor with N-[3-(4-nitrophenyl)propyl]-N-dodecylamine: evidence of receptor dimers.

The sigma-1 receptor is a ligand-regulated endoplasmic reticulum (ER) resident chaperone involved in the maintenance of cellular homeostasis. Coupling of the sigma-1 receptor with various ER and/or plasma membrane ion channels is associated with its ability to regulate the locomotor activity and cellular proliferation produced in response to sigma-1 receptor ligands. A number of endogenous small molecules bind to the sigma-1 receptor and have been shown to regulate its activity; these include progesterone, N,N-dimethyltryptamine, d-erythro-sphingosine, and/or other endogenous lipids. We previously reported the synthesis of long chain N-alkylamine derivatives and the characterization of the structure-activity relationship between the chain length of N-alkylamine and affinities at the sigma-1 receptor. Here, we present data demonstrating the photoincorporation of one of these N-alkylamine derivatives, N-[3-(4-nitrophenyl)propyl]-N-dodecylamine (4-NPPC12), to the sigma-1 receptor. Matrix-assisted laser desorption ionization time-of-flight and tandem mass spectrometry showed that 4-NPPC12 photoinserted at histidine 154 of the derivatized population of the sigma-1 receptor. Interestingly, light-dependent photoinsertion of 4-NPPC12 resulted in an enhanced electrophoretic mobility of only 50% of the derivatized receptor molecules as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proposed binding and reactivity of 4-NPPC12 evoke a ligand binding model for the sigma-1 receptor that likely involves a receptor dimer and/or oligomer.

The ligand binding region of the sigma-1 receptor: studies utilizing photoaffinity probes, sphingosine and N-alkylamines.

The sigma-1 receptor is a 26 kDa endoplasmic reticulum resident membrane protein that has been shown to have chaperone activity in addition to its promiscuous binding to pharmacological agents. Ligand binding domain(s) of the sigma-1 receptor have been identified using the E. coli expressed and purified receptor protein and novel radioiodinated azido photoaffinity probes followed by proteolytic and chemical cleavage strategies. The outcome of these experiments indicates that the sigma-1 receptor ligand binding regions are formed primarily by juxtaposition of its second and third hydrophobic domains, regions where the protein shares considerable homology with the fungal enzyme, sterol isomerase that is essential for the biosynthesis of ergosterol. Data indicate that these hydrophobic steroid binding domain like (SBDL) regions on the sigma-1 receptor are likely to interact selectively with N-alkyl amines such as the endogenous sphingolipids and with synthetic N-alkylamines and N-aralkylamines derivatives. A proposed model for the sigma-1 receptor is presented.

Characterization of interactions of 4-nitrophenylpropyl-N-alkylamine with ς receptors.

Sigma receptors are small membrane proteins implicated in a number of pathophysiological conditions, including drug addiction, psychosis, and cancer; thus, small molecule inhibitors of sigma receptors have been proposed as potential pharmacotherapeutics for these diseases. We previously discovered that endogenous monochain N-alkyl sphingolipids, including d-erythro-sphingosine, sphinganine, and N,N-dimethylsphingosine, bind to the sigma-1 receptor at physiologically relevant concentrations [Ramachandran, S., et al. (2009) Eur. J. Pharmacol. 609, 19-26]. Here, we investigated several N-alkylamines of varying chain lengths as sigma receptor ligands. Although the K(I) values for N-alkylamines were found to be in the micromolar range, when N-3-phenylpropyl and N-3-(4-nitrophenyl)propyl derivatives of butylamine (1a and 1b, respectively), heptylamine (2a and 2b, respectively), dodecylamine (3a and 3b, respectively), and octadecylamine (4a and 4b, respectively) were evaluated as sigma receptor ligands, we found that these compounds exhibited nanomolar affinities with both sigma-1 and sigma-2 receptors. A screen of high-affinity ligands 2a, 2b, 3a, and 3b against a variety of other receptors and/or transporters confirmed these four compounds to be highly selective mixed sigma-1 and sigma-2 ligands. Additionally, in HEK-293 cells reconstituted with K(v)1.4 potassium channel and the sigma-1 receptor, these derivatives were able to inhibit the outward current from the channel, consistent with sigma receptor modulation. Finally, cytotoxicity assays showed that 2a, 2b, 3a, and 3b were highly potent against a number of cancer cell lines, demonstrating their potential utility as mixed sigma-1 and sigma-2 receptor anticancer agents.

In vitro interaction of tubulin with the photoreceptor cGMP phosphodiesterase gamma-subunit.

The alpha and beta tubulins compose the microtubule cytoskeleton which is involved in many cellular processes such as vesicular transport. The photoreceptor cells in the retina are neurons specialized for phototransduction. Here we report a novel interaction between tubulin and the photoreceptor cGMP phosphodiesterase (PDE6) gamma subunit (PDE gamma). The specificity and molecular details of the PDE gamma:tubulin interaction were analyzed through the experiments of pull down, microtubule co-sedimentation, and NMR spectroscopy. The tubulin-interacting site was identified to be in the PDE gamma C-terminal I67-G85 region, and the interaction interface appeared to be distinct from those with the other PDE gamma targets in phototransduction. We also observed that PDE gamma interacted with tubulin in a GTP-dependent manner. Our findings offer implications for non-phototransduction role(s) of PDE gamma in the photoreceptor neurons.

Synthesis and characterization of N,N-dialkyl and N-alkyl-N-aralkyl fenpropimorph-derived compounds as high affinity ligands for sigma receptors.

The sigma-1 receptor is a unique non-opioid, non-PCP binding site that has been implicated in many different pathophysiological conditions including psychosis, drug addiction, retinal degeneration and cancer. Based on the structure of fenpropimorph, a high affinity (K(i)=0.005 nM)(1) sigma-1 receptor ligand and strong inhibitor of the yeast sterol isomerase (ERG2), we previously deduced a basic sigma-1 receptor pharmacophore or chemical backbone composed of a phenyl ring attached to a di-substituted nitrogen atom via an alkyl chain.(2) Here, we report the design and synthesis of various N,N-dialkyl or N-alkyl-N-aralkyl derivatives based on this pharmacophore as well as their binding affinities to the sigma-1 receptor. We introduce three high affinity sigma-1 receptor compounds, N,N-dibutyl-3-(4-fluorophenyl)propylamine (9), N,N-dibutyl-3-(4-nitrophenyl)propylamine (3), and N-propyl-N'-4-aminophenylethyl-3-(4-nitrophenyl)propylamine (20) with K(i) values of 17.7 nM, 0.36 nM, and 6 nM, respectively. In addition to sigma receptor affinity, we show through cytotoxicity assays that growth inhibition of various tumor cell lines occurs with our high affinity N,N-dialkyl or N-alkyl-N-aralkyl derivatives.

The sigma1 receptor interacts with N-alkyl amines and endogenous sphingolipids.

The sigma1 receptor is distinguished for its ability to bind various pharmacological agents including drugs of abuse such as cocaine and methamphetamine. Some endogenous ligands have been identified as putative sigma1 receptor regulators. High affinity ligands for the sigma1 receptor contain a nitrogen atom connected to long alkyl chains. We found that long alkyl chain primary amines including endogenous amines belonging to the sphingolipid family such as D-erythro-sphingosine and sphinganine bind with considerable affinity to the sigma1 receptor but not to the sigma2 receptor. The binding of D-erythro-sphingosine to the sigma1 receptor appears to be competitive in nature as assessed against the radioligand [3H]-(+)-pentazocine. Interestingly, the well studied sphingolipid mediator sphingosine-1 phosphate did not bind to the sigma1 or the sigma2 receptor. Sphingosine is converted to sphingosine-1 phosphate by a family of sphingosine kinases that regulate the relative levels of these two bioactive lipids in the cell. The selective binding of sphingosine but not sphingosine-1 phosphate to the sigma1 receptor suggests a mechanism for regulation of sigma1 receptor activity by the sphingosine kinase. We have successfully reconstituted this hypothetical model in HEK-293 cells overexpressing both the sigma1 receptor and sphingosine kinase-1. The data presented here strongly supports sphingosine as an endogenous modulator of the sigma1 receptor.

Probing the steroid binding domain-like I (SBDLI) of the sigma-1 receptor binding site using N-substituted photoaffinity labels.

Radioiodinated photoactivatable photoprobes can provide valuable insights regarding protein structure. Previous work in our laboratory showed that the cocaine derivative and photoprobe 3-[ (125)I]iodo-4-azidococaine ([ (125)I]IACoc) binds to the sigma-1 receptor with 2-3 orders of magnitude higher affinity than cocaine [Kahoun, J. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1393-1397]. Using this photoprobe, we demonstrated the insertion site for [ (125)I]IACoc to be Asp188 [Chen, Y. (2007) Biochemistry 46, 3532-3542], which resides in the proposed steroid binding domain-like II (SBDLII) region (amino acids 176-194) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. An additional photoprobe based on the sigma-1 receptor ligand fenpropimorph, 1- N-(2-3-[ (125)I]iodophenyl)propane ([ (125)I]IAF), was found to label a peptide in both the SBDLII and steroid binding domain-like I (SBDLI) (amino acids 91-109) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. In this report, we describe two novel strategically positioned carrier-free, radioiodinated photoaffinity labels specifically designed to probe the putative "nitrogen interacting region" of sigma-1 receptor ligands. These two novel photoprobes are (-)-methyl 3-(benzoyloxy)-8-2-(4-azido-3-[ (125)I]iodobenzene)-1-ethyl-8-azabicyclo[3.2.1]octane-2-carboxylate ([ (125)I]-N-IACoc) and N-propyl- N-(4-azido-3-iodophenylethyl)-3-(4-fluorophenyl)propylamine ([ (125)I]IAC44). In addition to reporting their binding affinities to the sigma-1 and sigma-2 receptors, we show that both photoaffinity labels specifically and covalently derivatized the pure guinea pig sigma-1 receptor (26.1 kDa) [Ramachandran, S. (2007) Protein Expression Purif. 51, 283-292]. Cleavage of the photolabeled sigma-1 receptor using Endo Lys C and cyanogen bromide (CNBr) revealed that the [ (125)I]-N-IACoc label was located primarily in the N-terminus and SBDLI-containing peptides of the sigma-1 receptor, while [ (125)I]IAC44 was found in peptide fragments consistent with labeling of both SBDLI and SBDLII.

Juxtaposition of the steroid binding domain-like I and II regions constitutes a ligand binding site in the sigma-1 receptor.

sigma-1 receptors represent unique binding sites that are capable of interacting with a wide range of compounds to mediate different cellular events. The composition of the ligand binding site of this receptor is unclear, since no NMR or crystal structures are available. Recent studies in our laboratory using radiolabeled photoreactive ligands suggested that the steroid binding domain-like I (SBDLI) (amino acids 91-109) and the steroid binding domain-like II (SBDLII) (amino acids 176-194) regions are involved in forming the ligand binding site(s) ( Chen, Y., Hajipour, A. R., Sievert, M. K., Arbabian, M., and Ruoho, A. E. (2007) Biochemistry 46, 3532-3542 ; Pal, A., Hajipour, A. R., Fontanilla, D., Ramachandran, S., Chu, U. B., Mavlyutov, T., and Ruoho, A. E. (2007) Mol. Pharmacol. 72, 921-933 ). In this report, we have further addressed this issue by utilizing our previously developed sulfhydryl-reactive, cleavable, radioiodinated photocross-linking reagent: methanesulfonothioic acid, S-((4-(4-amino-3-[125I]iodobenzoyl) phenyl)methyl) ester (Guo, L. W., Hajipour, A. R., Gavala, M. L., Arbabian, M., Martemyanov, K. A., Arshavsky, V. Y., and Ruoho, A. E. (2005) Bioconjugate Chem. 16, 685-693). This photoprobe was shown to derivatize the single cysteine residues as mixed disulfides at position 94 in the SBDLI region of the wild type guinea pig sigma-1 receptor (Cys94) and at position 190 in the SBDLII region of a mutant guinea pig sigma-1 receptor (C94A,V190C), both in a sigma-ligand (haloperidol or (+)-pentazocine)-sensitive manner. Significantly, photocross-linking followed by Endo Lys-C cleavage under reducing conditions and intramolecular radiolabel transfer from the SBDLI to the SBDLII region in the wild type receptor and, conversely, from the SBDLII to the SBDLI region in the mutant receptor were observed. These data support a model in which the SBDLI and SBDLII regions are juxtaposed to form, at least in part, a ligand binding site of the sigma-1 receptor.

Identification of regions of the sigma-1 receptor ligand binding site using a novel photoprobe.

sigma Receptors, once considered a class of opioid receptors, are now regarded as a unique class of receptors that contain binding sites for a wide range of ligands, including the drug 1-N(2',6'-dimethylmorpholino)3-(4-t-butylpropylamine) (fenpropimorph), a yeast sterol isomerase inhibitor. Because fenpropimorph has high-binding affinity to the sigma-1 receptor, we have synthesized a series of fenpropimorph-like derivatives with varying phenyl ring substituents and have characterized their binding affinities to the sigma-1 receptor. In addition, we have synthesized a carrier-free, radioiodinated fenpropimorph-like photoaffinity label, 1-N-(2',6'-dimethyl-morpholino)-3-(4-azido-3-[(125)I]iodo-phenyl)propane ([(125)I]IAF), which covalently derivatized the sigma-1 receptor (25.3 kDa) in both the rat liver and guinea pig liver membranes and the sigma-2 receptor (18 kDa) in rat liver membranes with high specificity. Furthermore, after cleaving the specific [(125)I]IAF-photolabeled sigma-1 receptor in guinea pig and rat liver membranes and the pure guinea pig sigma-1 receptor with EndoLys-C and cyanogen bromide, the [(125)I]IAF label was identified both in a peptide containing steroid binding domain-like I (SBDLI) (amino acids 91-109) and in a peptide containing steroid binding domain-like II (SBDLII) (amino acids 176-194). Because a single population of binding sites (R(2) = 0.992) for [(125)I]IAF interaction with the sigma-1 receptor was identified by (+)-[(3)H]pentazocine competitive binding with nonradioactive [(127)I]IAF, it was concluded that SBDLI (amino acids 91-109) and SBDLII (amino acids 176-194) comprises, at least in part, regions of the sigma-1 receptor ligand binding site(s).

A valid method of laparoscopic simulation training and competence assessment.

BACKGROUND: The purpose of our study was to evaluate the construct validity of laparoscopic technical performance measures and the face validity of three laparoscopic simulations. MATERIALS AND METHODS: Subjects (N = 27) of varying levels of surgical experience performed three laparoscopic simulations, representing appendectomy (LA), cholecystectomy (LC), and inguinal hemiorrhaphy (LH). Five laparoscopic surgeons, blinded to the identity of the subjects, rated the subjects on procedural competence on a binary scale and in four skills categories on a 5-point scale: clinical judgment, dexterity, serial/simultaneous complexity, and spatial orientation. Using a task-specific checklist, non-clinical staff assessed the technical errors. The level of surgical experience was correlated with the ratings, the technical errors, and the time for each procedure. Subject responses to a survey regarding the utility of the inanimate models were evaluated. RESULTS: Years of experience directly correlated with the skills ratings (all P < 0.001) and with the competence ratings across the three procedures (P < 0.01). Experience inversely correlated with the time for each procedure (P < 0.01) and the technical error total across the three models (P < 0.05). Nearly all subjects agreed that the corresponding procedures were well represented by the simulations (LA 96%, LC 96%, LH 100%). CONCLUSION: The laparoscopic simulations demonstrated both face and construct validity. Regardless of the level of surgical experience, the subjects found the models to be suitable representations of actual laparoscopic procedures. Task speed improved with surgical experience. More importantly, the quality of performance increased with experience, as shown by the improvement in the skills assessments by expert laparoscopic surgeons.