Molecular Mode Of Interaction Research Articles

Discovery of Pyrrolo[3,2- d]pyrimidin-4-one Derivatives as a New Class of Potent and Cell-Active Inhibitors of P300/CBP-Associated Factor Bromodomain.

Herein, we report the discovery of a series of new P300/CBP-associated factor (PCAF) bromodomain (BRD) inhibitors, which were obtained through a hit discovery process and subsequent structure-based optimization and structure-activity relationship analyses toward a retrieved hit compound (12). Among these inhibitors, ( R, R)-36n is the most potent one with an IC50 of 7 nM in homogeneous time-resolved fluorescence assay and a KD of 78 nM in isothermal titration calorimetry assay. This compound also exhibited activity against GCN5 and FALZ, but weak or no activity against other 29 BRD proteins and 422 kinases, indicating considerable selectivity. X-ray cocrystal structure analysis revealed the molecular interaction mode and the precise stereochemistry required for bioactivity. Cellular activity, preliminary RNA-seq analysis, and pharmacokinetic properties were also examined for this compound. Collectively, this study provides a versatile tool molecule to explore molecular mechanisms of PCAF BRD regulation and also offers a new lead compound for drug discovery targeting PCAF.

Cyclins B1, T1, and H differ in their molecular mode of interaction with cytomegalovirus protein kinase pUL97

Human cytomegalovirus (HCMV) is a common β-herpesvirus causing life-long latent infections. HCMV replication interferes with cell cycle regulation in host cells because the HCMV-encoded cyclin-dependent kinase (CDK) ortholog pUL97 extensively phosphorylates the checkpoint regulator retinoblastoma protein. pUL97 also interacts with cyclins B1, T1, and H, and recent findings have strongly suggested that these interactions influence pUL97 substrate recognition. Interestingly, here we detected profound mechanistic differences among these pUL97-cyclin interactions. Our study revealed the following. (i) pUL97 interacts with cyclins B1 and H in a manner dependent on pUL97 activity and HCMV-specific cyclin modulation, respectively. (ii) The phosphorylated state of both proteins is an important determinant of the pUL97-cyclin B1 interaction. (iii) Activated phospho-Thr-315 cyclin H is up-regulated during HCMV replication. (iv) Thr-315 phosphorylation is independent of intracellular pUL97 or CDK7 activity. (v) pUL97-mediated in vitro phosphorylation is detectable for cyclin B1 but not H. (vi) Mutual transphosphorylation between pUL97 and CDK7 is not detectable, and an MS-based phosphosite analysis indicated that pUL97 might unexpectedly not be phosphorylated in its T-loop. (vii) The binary complexes pUL97-cyclin H and CDK7-cyclin H as well as the ternary complex pUL97-cyclin-H-CDK7 are detectable in an assembly-based CoIP approach. (viii) pUL97 self-interaction can be bridged by the transcriptional cyclins T1 or H but not by the classical cell cycle-regulating B1 cyclin. Combined, our findings unravel a number of cyclin type-specific differences in pUL97 interactions and suggest a multifaceted regulatory impact of cyclins on HCMV replication.

Solid-State FTIR Spectroscopic Study of Two Binary Mixtures: Cefepime-Metronidazole and Cefoperazone-Sulbactam

The structural information of the pharmaceuticals and insights on the modes of molecular interactions are very important aspects in drug development. In this work, two cephalosporins and antimicrobial combinations, cefepime-metronidazole and cefoperazone-sulbactam, were studied in the solid state using FTIR spectroscopy for the first time. Quantitation of the studied drugs and their binary mixtures was performed by integrating the peak areas of the characteristic well-resolved bands:υ(C=O) band at 1773 cm−1for cefepime and ring torsion band at 826 cm−1for metronidazole andυ(C=O) band at 1715 cm−1for cefoperazone and ring torsion band at 1124 cm−1for sulbactam. The results of this work were compared with the relevant spectrophotometric reported methods. This study provides data that can be used for the preparative process monitoring of the studied drugs in various dosage forms.

Heat Shock Protein 90 kDa (Hsp90) Has a Second Functional Interaction Site with the Mitochondrial Import Receptor Tom70

To accomplish its crucial role, mitochondria require proteins that are produced in the cytosol, delivered by cytosolic Hsp90, and translocated to its interior by the translocase outer membrane (TOM) complex. Hsp90 is a dimeric molecular chaperone and its function is modulated by its interaction with a large variety of co-chaperones expressed within the cell. An important family of co-chaperones is characterized by the presence of one TPR (tetratricopeptide repeat) domain, which binds to the C-terminal MEEVD motif of Hsp90. These include Tom70, an important component of the TOM complex. Despite a wealth of studies conducted on the relevance of Tom70·Hsp90 complex formation, there is a dearth of information regarding the exact molecular mode of interaction. To help fill this void, we have employed a combined experimental strategy consisting of cross-linking/mass spectrometry to investigate binding of the C-terminal Hsp90 domain to the cytosolic domain of Tom70. This approach has identified a novel region of contact between C-Hsp90 and Tom70, a finding that is confirmed by probing the corresponding peptides derived from cross-linking experiments via isothermal titration calorimetry and mitochondrial import assays. The data generated in this study are combined to input constraints for a molecular model of the Hsp90/Tom70 interaction, which has been validated by small angle x-ray scattering, hydrogen/deuterium exchange, and mass spectrometry. The resultant model suggests that only one of the MEEVD motifs within dimeric Hsp90 contacts Tom70. Collectively, our findings provide significant insight on the mechanisms by which preproteins interact with Hsp90 and are translocated via Tom70 to the mitochondria.

Effect of Molecular Interactions and Homologue Number on UV Absorption Spectra of Liquid Crystalline Alkyl Cyanobiphenyl Dimers: DFT Calculations

The ultraviolet (UV) absorption spectra of the liquid crystalline dimer complexes of 4′-n-alkyl-4-cyanobiphenyls (nCB: n = 3, 4, 5 where n is the number of carbon atoms in the alkyl chain) have been presented. The nCB structures have been optimized using the density functional Becke3-Lee-Yang-Parr (B3LYP) hybrid functional with 6-31+G (d) basis set using the crystallographic geometry as input. The electronic structures of the dimer molecules have been computed using the optimized geometries. The spectra of the dimer molecules have been calculated by employing the density functional theory (DFT). The features of electronic transitions and excited states have been calculated via configuration interaction singles (CIS) with the semiempirical Hamiltonian Zerner intermediate neglect of differential overlap (ZINDO). The various modes of molecular interactions and the homologue number are found to be structural parameters affecting the formation of mesophases, UV absorption spectral characteristics, and photo stability of the compounds. These results offer a hint for the calculations involving the different modes of molecular interactions, and separations between dimers, to model photo stability or in tuning the absorbing chromophore to match the wavelength of desired application.

A Novel Role for a Major Component of the Vitamin D Axis: Vitamin D Binding Protein-Derived Macrophage Activating Factor Induces Human Breast Cancer Cell Apoptosis through Stimulation of Macrophages

The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH)(2)D3), its two binding proteins that are the vitamin D receptor (VDR) and the vitamin D-binding protein-derived macrophage activating factor (GcMAF). In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This al1ows 1,25(OH)(2)D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects.

Competing positive and negative feedbacks mediated by PH domain ligand interactions regulate Itk activation kinetics in T Cells (178.3)

Abstract Inositol phosphate second messengers often regulate crosstalk between receptor signaling and lipid metabolic networks critically affecting cell decision processes, however, molecular mechanisms underlying such cross-regulation are poorly understood. Pairing mathematical modeling and experiments we elucidate these mechanisms in thymocyte activation initiated by T cell receptor (TCR) and antigen interactions. Thymocyte activation is carefully controlled by transient activation kinetics of the Tec-family protein tyrosine kinase Itk generated by TCR signaling, and, production of the membrane lipid phosphatidylinositol(3,4,5)trisphosphate (PIP3) and soluble inositol(1,3,4,5) tetrakisphosphate (IP4). By combining PLC-g (substrate of active Itk) activation kinetics in experiments with maximum entropy based computational approaches we show multiple possible in-silico models describing different modes of molecular interactions between Itk, PIP3, and IP4, can be distinguished. We show that models displaying maximum robustness share a cooperative-allosteric mode of Itk regulation by IP4 involving oligomeric Itk PH domains which induces dueling positive and negative feedbacks in Itk activation. Models lacking the feedbacks or containing monomeric Itk are significantly less robust. We also elucidate key mechanisms regulating the "shape" of the transient Itk kinetics that can be manipulated in experiments for developing therapeutic strategies targeting TCR signaling.

Vibrational spectroscopic investigation of polymorphs and cocrystals of indomethacin

Context:Identification of optimal solid form of an active pharmaceutical ingredient and form control are very important in drug development. Thus, the structural information of these forms and in-depth insight on the modes of molecular interactions are necessary, and vibrational spectroscopic methods are well suited for this purpose.Objective:In-depth structural analysis of different solid forms of indomethacin (IND) using Raman and infrared (IR) spectroscopy is the objective. We have investigated the modes of molecular interactions in polymorphs (α and γ), amorphous and discovered cocrystals of IND with nicotinamide (NIC) and trans-cinnamic acid (CIN) coformers.Materials and methods: The solid forms of IND have been prepared; their purity has been verified by differential scanning calorimetry and powder X-ray diffractometry and then studied in the solid-state by Raman and IR spectroscopy. The modes of the interactions were closely investigated from the vibrational data.Results: The key vibrational features of IND solid forms have been specified. The IR (C=O) band at 1713 cm−1 attributed to cyclic acid dimer of γ IND has disappeared in IND–NIC/CIN whilst retained in IND–SAC cocrystal.Discussion:IND cocrystallizes in different conformations and crystal lattices with different coformers. The cyclic acid dimer of IND has been kept on its cocrystallization with saccharin and it could have been broken with NIC and CIN.Conclusions: The complementary nature of Raman and IR spectroscopy allowed unambiguous investigation of the chemical composition of pharmaceutical materials which is of particular importance in the absence of detailed structural information, as in the case of IND–NIC and IND–CIN.

Estimation of Configurational Probability and Phase Behaviour of N-n-undecyl-D-gluconamide having V-Shaped Conformation – A Molecular Simulation Approach

The present article deals with the configurational probabilities of a smectogen N-n-undecyl-Dgluconamide (GAM11). The complete neglect differential overlap (CNDO/2) method has been employed to compute the net atomic charge and atomic dipole moment components at each atomic center. The modified Rayleigh-Schr¨odinger perturbation theory along with the multicentered-multipole expansion method has been employed to evaluate the long-range intermolecular interactions, while a ‘6-exp’ potential function has been assumed for short-range interactions. The total interaction energy values obtained during different modes of molecular interactions have been used as input to calculate the configurational probability at room temperature (300 K), and smectic-isotropic transition temperature (429:7 K) using the Maxwell-Boltzmann formula. The various possible geometrical arrangements of molecular pairs have been considered. Further, an attempt has been made to explain the smectic behaviour of GAM11 in terms of their relative order based on the molecular parameters introduced in this paper

Effect of Non-Mesogenic Solvent on Molecular Conformations and Interactions of a Nematogen: A Probabilistic Approach

A probabilistic approach has been carried out on molecular conformations and interactions of a monotropic nematogen 4-n-hexyl-N-(4-cyanophenyl) piperidine with respect to translational and orientational motions. The atomic net charge and dipole moment components at each atomic center have been evaluated using the complete neglect differential overlap method. The modified Rayleigh–Schrodinger perturbation theory along with the multicentered–multipole expansion method has been employed to evaluate the long-range intermolecular interactions, while a “6-exp” potential function has been assumed for short-range interactions. The interaction energy values obtained during the different modes of molecular interactions have been taken as input to calculate the configurational probability at room temperature (300 K). On the basis of stacking, in-plane, and terminal interaction energy calculations, all possible geometrical arrangements between the molecular pairs have been considered. Furthermore, the effect of a non-mesogenic solvent (benzene) on molecular conformations during different modes of interactions has been analyzed. The adopted framework provides valuable information on configurational freedom of a molecule that may be useful in understanding the mesomorphic behavior/transitions.

Role of configurational entropy on ordering and phase organization of nematic liquid crystals—A molecular model

The temperature dependence of configurational entropy for two mesogens p– n-heptyloxybenzoic acid (7OBAC) and p–n-octyloxybenzoic acid (8OBAC) has been estimated. The atomic net charge and dipole moment at each atomic center have been evaluated using the complete neglect differential overlap (CNDO/2) method. The modified Rayleigh–Schrodinger perturbation theory along with multicentered–multipole expansion method has been employed to evaluate the long-range intermolecular interactions, while a ‘6-exp’ potential function has been assumed for short-range interactions. The interaction energy values obtained with respect to translational and orientational motions during the different modes of molecular interactions have been taken as input to estimate the configurational entropy and Helmholtz free energy at room temperature, nematic–isotropic transition temperature, and above transition temperature. An attempt has been made to understand the role of configurational entropy on ordering and phase organization of nOBAC ( n = 7, 8) molecules. The flexibility of a particular configuration at different temperatures has been analyzed in the light of thermodynamic parameters introduced in this paper. These parameters are helpful to develop a new and interesting model for structure-phase stability relationship at the molecular level.

Electronic Transition Oscillator Strength and UV Stability of Nematogenic Cyanobiphenyls—Role of Alkyl Chains

The electronic transitions in the ultraviolet (UV)/visible (VIS) range of nematogenic 4′-n-alkyl-4-cyanobiphenyl (nCB) with butyl (4CB) and hexyl (6CB) groups have been studied. The UV/VIS and circular dichroism (CD) spectra of nCB (n = 4, 6) molecules have been simulated using the TDDFT/B3LYP/6-31+G (d) method. Mulliken atomic charges for each molecule have been compared with Loewdin atomic charges to analyze the molecular charge distribution and phase stability. The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies corresponding to the electronic transitions in the UV/VIS range have been reported. Excited states have been calculated via CI-singles (CIS) with semiempirical Hamiltonian ZINDO. Furthermore, two types of calculations have been performed for model systems containing single and double molecules of nCB. The dimer complexes during the different modes of molecular interactions have also been taken into consideration in order to investigate the most energetically stable configuration. These computations provide valuable information regarding the role and flexibility of end chains in a particular phase behavior and UV stability.

Computational analysis of molecular properties and spectral characteristics of cyano-containing liquid crystals: Role of alkyl chains

The electronic transitions in the uv-visible range of 4'-n-alkyl-4-cyanobiphenyl (nCB) with propyl, pentyl, and heptyl groups, which are of commercial and application interests, have been studied. The uv-visible and circular dichroism spectra of nCB (n = 3,5,7) molecules have been simulated using the time dependent density functional theory Becke3-Lee-Yang-Parr hybrid functional-6-31 + G (d) method. Mulliken atomic charges for each molecule have been compared with Loewdin atomic charges to analyze the molecular charge distribution and phase stability. The highest occupied molecular orbital and lowest unoccupied molecular orbital energies corresponding to the electronic transitions in the uv-visible range have been reported. Excited states have been calculated via the configuration interaction single level with a semiempirical Hamiltonian (intermediate neglect of differential overlap method, as parametrized by Zerner and co-workers). Further, two types of calculations have been performed for model systems containing single and double molecules of nCB. Furthermore, the dimer complexes during the different modes of molecular interactions have also been studied. The interaction energies of dimer complexes have been taken into consideration in order to investigate the most energetically stable configuration. These studies are helpful for understanding the role and flexibility of end chains, in particular, phase behavior and stability.

Molecular Structure and Conformational Behavior of a Nematogen—A Statistical Approach

Molecular structure and conformational behavior of a nematogen p-ethoxy salicylidene-n-butyl aniline (ESBA) has been studied with respect to translational and orientational motions. The atomic net charge and dipole moment components at each atomic center have been evaluated using the complete neglect differential overlap (CNDO/2) method. The modified Rayleigh-Schrodinger perturbation theory along with multicentered-multipole expansion method has been employed to evaluate the long-range intermolecular interactions, and a 6-exp potential function has been assumed for short-range interactions. The interaction energy values obtained during the different modes of molecular interactions have been taken as input to calculate the configurational probability at room temperature (300 K). On the basis of stacking, in-plane, and terminal interaction energy calculations, all possible geometrical arrangements between the molecular pairs have been considered. The effect of nonmesogenic and noninteracting solvent (benzene) has also been studied. Further, the analysis of conformational behavior provides valuable information on configurational freedom of a molecule that may be useful in understanding the structure of phases and their transitions.

Thermodynamic Stability of the Nematic Phase and Configurational Entropy of Mesogens: A Molecular Simulation Approach

The thermodynamic stability of the nematic phase and the configurational entropy of mesogens–p-n-pentylbenzoic acid (5BAC) and p-n-hexylbenzoic acid (6BAC)–have been studied. The atomic net charge and dipole moment components at each atomic center have been evaluated using the complete neglect differential overlap (CNDO/2) method. The modified Rayleigh–Schrodinger perturbation theory along with multicentered-multipole expansion method has been employed to evaluate the long-range intermolecular interactions, while a “6-exp” potential function has been assumed for short-range interactions. The interaction energy values obtained with respect to translational and orientational motions during the different modes of molecular interactions have been taken as input to estimate the configurational entropy and Helmholtz free energy at room temperature, nematic–isotropic transition temperature, and above transition temperature. The observed difference in free energy of the molecules between the mesophase and the crystal phase during in-plane interactions suggests the thermodynamic stability of the nematic phase. Further, the comparable values of translational entropy during stacking and in-plane interactions are helpful to understand the relative flexibility/stability of one configuration over the other.

Estimation of configurational entropy and stability of nematic phase in mesogens — Role of molecular interactions

The temperature dependence of configurational entropy for two mesogens p- n-Octylbenzoic acid (8BAC) and p- n-Nonylbenzoic acid (9BAC) has been estimated. The atomic net charge and dipole moment at each atomic center have been evaluated using the complete neglect differential overlap (CNDO/2) method. The modified Rayleigh–Schrodinger perturbation theory along with multicentered-multipole expansion method has been employed to evaluate the long-range intermolecular interactions, while a ‘6 -exp’ potential function has been assumed for short-range interactions. The interaction energy values obtained with respect to translational and orientational motions during the different modes of molecular interactions have been taken as input to estimate the configurational entropy and Helmholtz free energy at room temperature, nematic–isotropic transition temperature and above transition temperature. Further, an attempt has been made to analyze the stability of nematic phase at phase transition temperature. This study is essential in understanding the flexibility of a configuration in a particular phase and in employing molecular models to yield insight into structural organization of the compounds.

Relationship between Conformational Changes in the Dopamine Transporter and Cocaine-Like Subjective Effects of Uptake Inhibitors

Cocaine exerts its stimulatory effect by inhibiting the dopamine transporter (DAT). However, novel benztropine- and rimcazole-based inhibitors show reduced stimulant effects compared with cocaine, despite higher affinity and selectivity for DAT. To investigate possible mechanisms, we compared the subjective effects of different inhibitors with their molecular mode of interaction at the DAT. We determined how different inhibitors affected accessibility of the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate to an inserted cysteine (I159C), which is accessible when the extracellular transporter gate is open but inaccessible when it is closed. The data indicated that cocaine analogs bind an open conformation, whereas benztropine and rimcazole analogs bind a closed conformation. Next, we investigated the changes in inhibition potency of [(3)H]dopamine uptake of the compounds at a mutant DAT (Y335A) characterized by a global change in the conformational equilibrium. We observed a close relationship between the decrease in potencies of inhibitors at this mutant and cocaine-like responding in rats trained to discriminate cocaine from saline injections. Our data suggest that chemically different DAT inhibitors stabilize distinct transporter conformations and that this in turn affects the cocaine-like subjective effects of these compounds in vivo.

Modern High Resolution NMR for the Study of Structure, Dynamics and Interactions of Biological Macromolecules

High resolution liquid state nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for in vitro studies of structure and dynamics of soluble biological macromolecules under physiological conditions. The unique combination of atomically resolved structural data with both local and global dynamic features covering the entire range of time scales from picoseconds to seconds makes NMR the method of choice in a very diverse and rapidly growing array of biochemical, biomedical, and pharmaceutical applications. After briefly introducing the basic principles of liquid state NMR we review recent methodological and instrumental advances in the field of biologically focused high resolution NMR. The main emphasis of the second part is on molecular interactions. Such interactions are fundamental for the function of proteins in living systems, e.g. for signal transduction, enzymatic catalysis, and immune defense. The tremendous opportunities of high resolution NMR in the identification and detailed characterization of the sites and modes of molecular interactions will be demonstrated.

Tachyphylactic properties of angiotensin II analogs with bulky and hydrophobic substituents at the N-terminus.

Tachyphylaxis, defined as the acute loss of response of some smooth muscles upon repeated stimulations with angiotensin II (Ang II), has been shown to be dependent mainly on the N-terminal region of the ligand. To further study the structural requirements for the induction of tachyphylaxis we have synthesized Ang II analogs containing the bulky and very lipophilic substituents 9-fluorenylmethyloxycarbonyl (Fmoc) and 9-fluorenylmethyl ester (OFm) at the alpha-amino (Nalpha-Fmoc-Ang II) or the beta-carboxyl ([Asp(OFm)1]-Ang II) groups of the Asp1 residue, respectively. In binding assays with Chinese hamster ovary cells transfected with the AT1 Ang II receptor, Nalpha-Fmoc-Ang II bound with high affinity, whereas [Asp(OFm)1]-Ang II showed lower affinity. In biological assays, these two analogs were full agonists and showed 30 and 3%, respectively, of the Ang II potency in contracting the guinea-pig ileum smooth muscle. The two analogs induced tachyphylaxis, in spite of the lack of a free amino group in Nalpha-Fmoc-Ang II. Thus, analogs with Fmoc- or OFm-type groups coupled to the Asp1 residue, whether at the amino or carboxyl functions, induce tachyphylaxis through an unreported mechanism. Based in these findings and those available from the literature, an alternate molecular interaction mode between Ang II N-terminal portion and the AT1 receptor is proposed to explain the tachyphylactic phenomenon.

Studies of the solvent effects on the internal reorganization energy for electron transfer of uracil and its anion with ONIOM

In this paper, the aqueous adiabatic electron affinity (AEA) of Uracil (U) and internal reorganization energy λ i of the self-exchange electron transfer (ET) reaction between Uracil and Uracil anion radical (U − ) in aqueous solution were studied. The effect of the solvation was studied with the recently developed hybrid quantum molecular chemical method, ONIOM. In all calculations, the geometrical optimization for U and U − was performed at B3LYP/6-31++G(d) level. As for the solvent surroundings, the seven water molecules as the first hydration shell were adopted and treated with B3LYP, PM3 and AMBER methods, namely, ONIOM (B3LYP:B3LYP), ONIOM (B3LYP:PM3) and ONIOM (B3LYP:Amber) methods, respectively. The values of AEA for Uracil, predicted by the above three methods, are small positive ones. The geometrical differences between neutral and anion radical molecules of U originate mainly from those of dihedral angles. According to the corresponding dipole moment values, the excess electron in U − should be trapped dominantly by dipole-bound way. The calculated λ i values by ONIOM (B3LYP:B3LYP) and ONIOM (B3LYP:PM3) are close to each other within 0.89%. The λ i value from ONIOM (B3LYP:Amber) is in agreement with the one from SCRF-CPCM very well. Finally, the calculation results of the detailed geometries and molecular interaction mode effect of U and U − and related water molecules in the hydration shell were discussed.