Rational design, synthesis, and biological evaluation of rigid pyrrolidone analogues as potential inhibitors of prostate cancer cell growth.

In view of its role in tumor promotion and signal transduction, protein kinase C (PKC) has proven to be an exciting target for cancer therapy. With the aid of molecular modeling, we rationally designed and stereoselectively synthesized a new class of rigidified pyrrolidone-based PKC activators. Pyrrolidone 15 was found to exhibit reasonable affinity for PKCdelta, with lower affinity for the other isozymes tested. Pyrrolidone 2 causes the dose-dependent induction of apoptosis in LNCaP prostate cancer cells. This apoptotic effect could be markedly potentiated by the use of LNCaP cells overexpressing the PKCalpha or delta isozymes.

Molecular modeling studies of the Akt PH domain and its interaction with phosphoinositides.

The serine-threonine protein kinase Akt is a direct downstream target of phosphatidylinositol 3-kinase (PI3-K). The PI3-K-generated phospholipids regulate Akt activity via directly binding to the Akt PH domain. The binding of PI3-K-generated phospholipids is critical to the relocalization of Akt to the plasma membrane, which plays an important role in the process of Akt activation. Activation of the PI3-K/Akt signaling pathway promotes cell survival. To elucidate the structural basis of the interaction of PI3-K-generated phospholipids with the Akt PH domain with the objective of carrying out structure-based drug design, we modeled the three-dimensional structure of the Akt PH domain. Comparative modeling-based methods were employed, and the modeled Akt structure was used in turn to construct structural models of Akt in complex with selected PI3-K-generated phospholipids using the computational docking approach. The model of the Akt PH domain consists of seven beta-strands forming two antiparallel beta-sheets capped by a C-terminal alpha-helix. The beta1-beta2, beta3-beta4, and beta6-beta7 loops form a positively charged pocket that can accommodate the PI3-K-generated phospholipids in a complementary fashion through specific hydrogen-bonding interactions. The residues Lys14, Arg25, Tyr38, Arg48, and Arg86 form the bottom of the binding pocket and specifically interact with the 3- and 4-phophate groups of the phospholipids, while residues Thr21 and Arg23 are situated at the wall of the binding pocket and bind to the 1-phosphate group. The predicted binding mode is consistent with known site-directed mutagenesis data, which reveal that mutation of these crucial residues leads to the loss of Akt activity. Moreover, our model can be used to predict the binding affinity of PI3-K-generated phospholipids and rationalize the specificity of the Akt PH domain for PI(3,4)P2, as opposed to other phospholipids such as PI(3)P and PI(3,4,5)P3. Taken together, our modeling studies provide an improved understanding of the molecular interactions present between the Akt PH domain and the PI3-K-generated phospholipids, thereby providing a solid structural basis for the design of novel, high-affinity ligands useful in modulating the activity of Akt.

Structural basis of Bloom syndrome (BS) causing mutations in the BLM helicase domain.

BACKGROUND: Bloom syndrome (BS) is characterized by mutations within the BLM gene. The Bloom syndrome protein (BLM) has similarity to the RecQ subfamily of DNA helicases, which contain seven conserved helicase domains and share significant sequence and structural similarity with the Rep and PcrA DNA helicases. We modeled the three-dimensional structure of the BLM helicase domain to analyze the structural basis of BS-causing mutations. MATERIALS AND METHODS: The sequence alignment was performed for RecQ DNA helicases and Rep and PcrA helicases. The crystal structure of PcrA helicase (PDB entry 3PJR) was used as the template for modeling the BLM helicase domain. The model was used to infer the function of BLM and to analyze the effect of the mutations. RESULTS: The structural model with good stereochemistry of the BLM helicase domain contains two subdomains, 1A and 2A. The electrostatic potential of the model is highly negative over most of the surface, except for the cleft between subdomains 1A and 2A which is similar to the template protein. The ATP-binding site is located inside the model between subdomains 1A and 2A; whereas, the DNA-binding region is situated at the surface cleft, with positive potential between 1A and 2A. CONCLUSIONS: The three-dimensional structure of the BLM helicase domain was modeled and applied to interpret BS-causing mutations. The mutation I841T is likely to weaken DNA binding, while the mutations C891R, C901Y, and Q672R presumably disturb the ATP binding. In addition, other critical positions are discussed.

3-(Hydroxymethyl)-bearing phosphatidylinositol ether lipid analogues and carbonate surrogates block PI3-K, Akt, and cancer cell growth.

Phosphatidylinositol 3-kinase (PI3-K) phosphorylates the 3-position of phosphatidylinositol to give rise to three signaling phospholipids. Binding of the pleckstrin homology (PH) domain of Akt to membrane PI(3)P's causes the translocation of Akt to the plasma membrane bringing it into contact with membrane-bound Akt kinase (PDK1 and 2), which phosphorylates and activates Akt. Akt inhibits apoptosis by phosphorylating Bad, thus promoting its binding to and blockade of the activity of the cell survival factor Bcl-x. Herein we present the synthesis and biological activity of several novel phosphatidylinositol analogues and demonstrate the ability of the carbonate group to function as a surrogate for the phosphate moiety. Due to a combination of their PI3-K and Akt inhibitory activities, the PI analogues 2, 3, and 5 proved to be good inhibitors of the growth of various cancer cell lines with IC(50) values in the 1-10 microM range. The enhanced Akt inhibitory activity of the axial hydroxymethyl-bearing analogue 5 compared to its equatorial counterpart 6 is rationalized based upon postulated differences in the H-bonding patterns of these compounds in complex with a homology modeling generated structure of the PH domain of Akt. This work represents the first attempt to examine the effects of 3-modified PI analogues on these two crucial cell signaling proteins, PI3-K and Akt, in an effort to better understand their cell growth inhibitory properties.

Structural basis of Wiskott-Aldrich syndrome causing mutations in the WH1 domain.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immunodeficiency disease associated with eczema, hemorrhagic episodes, and recurrent severe infections. The N-terminus of the cytoplasmic WAS protein (WASP) has similarity to WH1 domains, which recognize proline-rich sequences and direct protein localization and formation of multicomponent assemblies. About one-half of the WAS-causing mutations affect the WH1 domain, but this forms only about one-fifth of the length of the protein. To understand the structural and functional effects of WAS-causing mutations within the WH1 domain, the three-dimensional model of the WASP WH1 domain was constructed based on the crystal structures of the Mena and Ev1 EVH1 (WH1) domains. Based on the model, the protein structural effects of the mutations were evaluated and putative ligand-binding regions identified. Mutations in the WASP WH1 domain were found to influence both the function and structure of the WASP. The amino acid substitutions cause general and local structural changes because of steric clashes and changes to the positions of adjacent strands and the fold of the protein. Some mutations alter the electrostatics and interactions with partners and other domains of WASP.

Molecular modeling on solvent effect and interaction mechanism of fentanyl analogs to mu-opioid receptor.

AIM: To do theoretical study about solvation effect and interaction mechanism of fentanyl analogs (FA) to mu opioid receptor (microOR). METHODS: Flexible docking (FlexiDock) was performed by using the possible active conformations of FA and optimized 3D structure of mu opioid receptor. Binding energies were calculated. Comparative molecular force field analysis (CoMFA) and quantitative structure activity relationship (QSAR) studies were carried out based on results of flexible docking. Solvation effects were considered by studying interaction of FA with water molecules. Partial least square (PLS) analysis was used to calculate regression equation for analgesic activities using binding energies as descriptive factor. RESULTS: 1) Binding conformations of these analogs derived by flexible docking were reasonable. 2) It was most possible for the FA to exist in water solution in the form of binding conformations. 3) Energetic calculation and QSAR analysis showed a good correlation between the calculated binding energies of FA and their analgesic activities. 4) Based on the 3D-model, the possible interaction mechanism of FA with mu opioid receptor can be illustrated reasonably. CONCLUSION: The nature of the correlation between the binding affinities and analgesic activities of FA was explained by our modeling result.

Molecular modeling of mu opioid receptor and receptor-ligand interaction.

AIM: To construct the 3D structural model of mu opioid receptor (mu OR) and study the interaction between mu OR and fentanyl derivatives. METHODS: The 3D structure of mu OR was modeled using the bacteriorhodopsin (bRh) as a template, in which the alignments of transmembrane (TM) of bRh and mu OR were achieved by scoring the alignment between the amino acid sequence of mu OR and the structure of bRh. The fentanyl derivatives were docked into the 7 helices of mu OR and the binding energies were calculated. RESULTS: (1) The receptor-ligand interaction models were obtained for fentanyl derivatives. (2) In these models, the fundamental binding sites were possibly Asp147 and His297. The negatively charged oxygen of Asp147 and the positively charged ammonium group of ligand formed the potent electrostatic and hydrogen-binding interactions. Whereas the interactions between the positively charged nitrogen of His297 and the carbonyl oxygen of ligand were weak. In addition, there were some pi-pi interactions between the receptor and the ligand. (3) The binding energies of the receptor-ligand complexes had a good correlation with the analgesic activities (-lg ED50) of the fentanyl derivatives. CONCLUSION: This model is helpful for understanding the receptor-ligand interaction and for designing novel mu OR selective ligands.

Molecular modeling of interaction between delta opioid receptor and 3-methylfentanylisothiocyanate.

AIM: To construct a 3D structural model of delta opioid receptor (delta OR) and study its interaction with 3-methylfentanylisothiocyanate (SuperFIT). METHODS: Using the bacteriohodopsin as a template, the 3D structure of delta OR was modeled; SuperFIT was docked into its inside. RESULTS: The interaction model between delta OR and (3R, 4S)-SuperFIT was achieved, in which the important binding sites possibly were Asp128, Ser106, Phe104, Tyr308, and Pro315. Asp128 formed the electrostatic and hydrogen-binding interactions with the protonated nitrogen on piperidine of the ligand. Ser106 formed the electrostatic interaction with the N atom of isothiocyano group of the ligand; whereas Phe104, Tyr308, and Pro315 formed the hydrophobic interactions with the S atom of isothiocyano group. In addition, there were some other interactions between delta OR and the ligand. CONCLUSION: The residues Phe104, Tyr308, Pro315, and Ser106 of delta OR are crucial to the delta selectivity of the ligand, which is beneficial for designing novel delta-selective ligand.

Interaction models of 3-methylfentanyl derivatives with mu opioid receptors.

AIM: To study the interaction model of 3-methylfentanyl derivatives with mu opioid receptor. METHODS: After a systematic conformational search, a three-dimensional quantitative structure-activity relationship study was carried out with comparative molecular field analysis (CoMFA). RESULTS: 1) The 6 CoMFA models had good predictive values and each model corresponded to the minimum-energy conformations of 13 compounds studied; 2) The important geometric parameters of mu pharmacophore d1 (A), d2 (A), d3 (A), d4 (A), d5 (A), and d6 (A) were 5.2, 5.4, 4.9, 10.6, 10.2, and 5.8 in Model A; 5.2, 6.5, 3.6, 10.6, 11.6, and 5.8 in Model B; 5.2, 4.6, 4.9, 11.6, 9.2, and 6.5 in Model C; 5.2, 5.4, 4.9, 10.5, 10.3, and 5.8 in Model D; 3.6, 5.4, 4.9, 5.7, 7.5, and 5.7 in Model E; 5.2, 4.7, 4.9, 11.2, 9.5, and 6.4 in Model F, respectively. CONCLUSIONS: The several bioactive conformations of fentanyl analogs possibly existed and did not need to be the absolute minimum-energy conformation, each of which was involved in the interaction with mu opioid receptor.

Structure-activity relationship of channel binding affinity of quaternary ammonium ions.

AIM: To explore the structure-activity relationship of quaternary ammonium (QA) ions at the external binding site of K+ channel. METHODS: InsightII and MOPAC 6.0 molecular modeling package were used to calculate the free energy of hydration (delta Ghydration), the energy of the highest occupied orbital (EHOMO), and the energy of the lowest unoccupied orbital (ELUMO) for each QA ion, respectively. The partial least square method was used to analyze the relationship between the binding free energy and these descriptive parameters. RESULTS: Generally, the higher the ELUMO of a QA ion was, the weaker its solvation was and accordingly the stronger binding affinity. For a QA ion larger than tetraethylammonium (TEA), its large size was unfavorable to its channel binding affinity. CONCLUSION: The binding affinity of all QA ions correlated well with delta Ghydration and ELUMO.