An explorative study on potent Gram-negative specific LpxC inhibitors: CoMFA,CoMSIA, HQSAR and molecular docking

Pathogenic Gram-negative bacteria are responsible for nearly half of the serious human infections. Hologram quantitative structure–activity relationships (HQSAR), comparative molecular field analysis (CoMFA), and comparative molecular similarity index analysis (CoMSIA) were implemented on a group of 32 of potent Gram-negative LpxC inhibitors. The most effective HQSAR model was obtained using atoms, bonds, donor, and acceptor as fragment distinction. The cross-validated correlation coefficient (q²), non-cross-validated correlation coefficient (r²), and predictive correlation coefficient (r²_Pred) for test set of HQSAR model were 0.937, 0.993, and 0.892, respectively. The generated models were found to be statistically significant as the CoMFA model had (r²?=?0.967, q²?=?0.804, r²_Pred?=?0.827); the CoMSIA model had (r²?=?0.963, q²?=?0.752, r²_Pred?=?0.857). Molecular docking was employed to validate the results of the HQSAR, CoMFA, and CoMSIA models. Based on the obtained information, six new LpxC inhibitors have been designed.     

Modulation of Ca Activity in Cardiomyocytes through Caveolae-Gαq Interactions

Cardiomyocytes have a complex Ca²⁺ behavior and changes in this behavior may underlie certain disease states. Intracellular Ca²⁺ activity can be regulated by the phospholipase Cβ–Gα_q pathway localized on the plasma membrane. The plasma membranes of cardiomycoytes are rich in caveolae domains organized by caveolin proteins. Caveolae may indirectly affect cell signals by entrapping and localizing specific proteins. Recently, we found that caveolin may specifically interact with activated Gα_q, which could affect Ca²⁺ signals. Here, using fluorescence imaging and correlation techniques we show that Gα_q-Gβγ subunits localize to caveolae in adult ventricular canine cardiomyoctyes. Carbachol stimulation releases Gβγ subunits from caveolae with a concurrent stabilization of activated Gα_q by caveolin-3 (Cav3). These cells show oscillating Ca²⁺ waves that are not seen in neonatal cells that do not contain Cav3. Microinjection of a peptide that disrupts Cav3-Gα_q association, but not a control peptide, extinguishes the waves. Furthermore, these waves are unchanged with rynaodine treatment, but not seen with treatment of a phospholipase C inhibitor, implying that Cav3-Gα_q is responsible for this Ca²⁺ activity. Taken together, these studies show that caveolae play a direct and active role in regulating basal Ca²⁺ activity in cardiomyocytes.     

Correlation of antibody production rate with glucose and lactate metabolism in Chinese hamster ovary cells

A linear relationship was found between the antibody production rate (q _mAb) and the glucose and lactate consumption rate (q _GL) in Chinese hamster ovary cells. Under a series of q _mAb-perturbing conditions, q _GL was determined and a linear relationship between q _mAb and q _GL was further established (R ²  = 0.914). Mitochondrial dehydrogenase activity was monitored in all the q _mAb-perturbing conditions and showed a linear correlation with q _GL (R ² = 0.874) as well as with q _mAb. Taken collectively, our results establish that the metabolic parameter, q _GL, is linearly correlated with q _mAb; this finding strengthens our current understanding of process optimization for antibody production.     

Virtual identification of novel PPARα/γ dual agonists by 3D-QSAR,molecule docking and molecular dynamics studies

Abstract

Peroxisome proliferator-activated receptors (PPARs) are considered important targets for the treatment of Type 2 diabetes (T2DM). To accelerate the discovery of PPAR α/γ dual agonists, the comparative molecular field analysis (CoMFA) were performed for PPARα and PPARγ, respectively. Based on the molecular alignment, highly predictive CoMFA model for PPARα was obtained with a cross-validated q² value of 0.741 and a conventional r² of 0.975 in the non-cross-validated partial least-squares (PLS) analysis, while the CoMFA model for PPARγ with a better predictive ability was shown with q² and r² values of 0.557 and 0.996, respectively. Contour maps derived from the 3D-QSAR models provided information on main factors towards the activity. Then, we carried out structural optimization and designed several new compounds to improve the predicted biological activity. To investigate the binding modes of the predicted compounds in the active site of PPARα/γ, a molecular docking simulation was carried out. Molecular dynamic (MD) simulations indicated that the predicted ligands were stable in the active site of PPARα/γ. Therefore, combination of the CoMFA and structure-based drug design results could be used for further structural alteration and synthesis and development of novel and potent dual agonists. Abbreviations DM diabetes mellitus

T2DM type 2 diabetes

PPARs peroxisome proliferator-activated receptors

LBDD ligand based drug design

3D-QSAR three-dimensional quantitative structure activity relationship

CoMFA comparative molecular field analysis

PLS partial least square

LOO leave-one-out

q² cross-validated correlation coefficient

ONC optimal number of principal components

r² non-cross-validated correlation coefficient

SEE standard error of estimate

F the Fischer ratio

r²_pred predictive correlation coefficient

DBD DNA binding domain

MD molecular dynamics

RMSD root-mean-square deviation

RMSF root mean square fluctuations

Communicated by Ramaswamy H. Sarma     

Soil hydrological properties as influenced by long‐term nitrogen application and landscape positions under switchgrass seeded to a marginal cropland

Switchgrass (Panicum virgatum L.) has the potential to recover the soil hydrological properties of marginal lands. Nitrogen (N) and landscape position are the key factors in impacting these soil properties under switchgrass. The specific objective of this study was to investigate the responses of N rate (low, 0 kg N/ha and high, 112 kg N/ha) and landscape positions (shoulder and footslope) on near‐surface soil hydrological properties that included: infiltration rate (q_s), saturated hydraulic conductivity (K_sat), bulk density (ρ_b), penetration resistance (SPR), water retention (SWR), pore‐size distribution (PSD), and carbon (C) and nitrogen (N) fractions under switchgrass production. Data showed that, in general, the N and landscape position significantly influenced soil hydrological properties. Higher N rate decreased ρ_b (1.23 and 1.36 g/cm³ at 0–5 and 5–15 cm, respectively) and SPR (1.06 and 1.53 MPa at 0–5 and 5–15 cm, respectively) at both depths and increased the q_s, K_sat and Green–Ampt estimated sorptivity (S) and hydraulic conductivity (K_s) parameters, and SWR (0–5 cm depth) at 0 and ?0.4 kPa matric potentials (ψ_m). Furthermore, footslope position significantly decreased ρ_b, SPR at 0–5 and 5–15 cm depths, and increased the q_s, K_sat, S, K_s, and SWR (0–5 cm depth) at every ψ_m ranged from 0 to ?30.0 kPa. The higher N rate increased the coarse mesopores (60–1,000 μm) and total pores, whereas, footslope position increased the coarse mesopores, micropores (<60 μm), and total pores. Data from this study showed that planting switchgrass with 112 kg N/ha under footslope position helped in improving the soil hydrological properties, those can be beneficial in enhancing the biomass yield under marginal lands.     

Relationship between cell size and specific thrombopoietin productivity in chinese hamster ovary cells during dihydrofolate reductase-mediated gene amplification

When parental Chinese hamster ovary (CHO) cell clones that are capable of producing thrombopoietin (TPO) were subjected to high methotrexate (MTX) concentrations, clonal variations in cell growth were apparent. In the clones that had no significant enhancement in specific TPO productivity (q _Tpo) when a higher level of MTX was administered, their growth was not depressed significantly nor their cell size changed significantly. On the other hand, those clones that showed a significant enhancement inq _Tpo at higher a MTX dosage, cell growth was depressed initially but recovered during successive sub-cultures. Furthermore, their cell size increased, which suggested that changes in cell size may be indicative of an enhancedq _Tpo. When the enhancement of theq _Tpo of 9 clones after a high MTX dosage was plotted against the extent of the increase of their size, there was a linear correlation (r ²=0.80,P<0.001, ANOVA), which suggested that an enhancement ofq _Tpo after high MTX administration can be measured by the increase in their cell size. Taken together, our data demonstrate that the selection of amplified CHO cell clones with enhancedq _Tpo can be done based upon their increased size and growth pattern. This facilitates the development of highly productive recombinant CHO cell lines.     

CoMFA and CoMSIA 3D-QSAR analysis on hydroxamic acid derivatives as urease inhibitors

Urease (EC 3.5.1.5) serves as a virulence factor in pathogens that are responsible for the development of many diseases in humans and animals. Urease allows soil microorganisms to use urea as a source of nitrogen and aid in the rapid break down of urea-based fertilizers resulting in phytopathiCIT000y. It has been well established that hydroxamic acids are the potent inhibitors of urease activity. The 3D-QSAR studies on thirty five hydroxamic acid derivatives as known urease inhibitors were performed by Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) methods to determine the factors required for the activity of these compounds. The CoMFA model produced statistically significant results with cross-validated (q²) 0.532 and conventional (r²) correlation coefficients 0.969.The model indicated that the steric field (70.0%) has greater influence on hydroxamic acid inhibitors than the electrostatic field (30.0%). Furthermore, five different fields: steric, electrostatic, hydrophobic, H-bond donor and H-bond acceptor assumed to generate the CoMSIA model, which gave q² 0.665 and r² 0.976.This model showed that steric (43.0%), electrostatic (26.4%) and hydrophobic (20.3%) properties played a major role in urease inhibition. The analysis of CoMFA and CoMSIA contour maps provided insight into the possible modification of the hydroxamic acid derivatives for improved activity.     

Cytolytic T cells activated by H-2-controlled E molecules cross-react with A molecules

Cell-mediated lymphocytotoxicity was generated in four strain combinations differing only by the cell-surface expression of the class II E molecule controlled by the H-2 complex. The four combinations were: B10.D2(R107) anti-B10.A(3R), B10.A(4R) anti-B10.A(2R), B10.GD anti-B10.D2(R101), and B10.S(7R) anti-B10.S(9R). In all four of these combinations, the stimulator expresses E molecules on the cell surface, while the responder does not. The cytolytic T lymphocytes generated in the B10.D2(R107) anti-B10.A(3R) and B10.A(4R) anti-B10.A(2R) combinations reacted not only with the stimulator but also with strains that do not express cell-surface E molecules, in particular, strains carrying the H-2 ^f and H-2 ^q haplotypes. The cross-reactivity with E-negative strains could be blocked by monoclonal antibodies specific for the A^f or A^q molecules but not by antibodies recognizing determinants on E or class I (K) molecules. The anti-H-2^f cross-reactivity could be inhibited by H-2 ^q cold targets and, reciprocally, the anti-H-2^q reactivity could be blocked by H-2 ^f cold targets. These findings are interpreted as indicating that the cytolytic T lymphocytes stimulated by E molecules can recognize and lyse cells lacking E molecules but expressing A molecules. The observed E-A cross-reactivity supports the notion of structural and functional relatedness between the A and E molecules and suggests a common evolutionary origin of the A- and E-encoding loci.     

Changes in in vivo fluorescence quenching in rye and barley as a function of reduced PSII light harvesting antenna size

The relationship between the size of the light harvesting antenna to photosystem II (LHCII) and quenching of non-photochemical and dark level fluorescence was studied in wild-type rye (Secale cereale L. cv. Musketeer) and barley (Hordeum vulgare L. cv. Gunilla) as well as in the barley chlorophyll b-less chlorina F2 mutant (H. vulgare L. cv. Dornaria, chlorina-F2). Exposure for 10 min to an irradiance of 500 μmol m^?2 s^?1 resulted in a strong (0.71–0.73) non-photochemical (q_s) quenching of the fluorescence yield in wild-type (WT) material, while the barley chlorina F2-mutant was quenched to 75% of this level. Relaxation of q_s in darkness revealed a fast initial decay, related to relaxation of the high-energy-state dependent (q_E) part of q_s. Etiolated seedlings of rye and barley exposed to intermittent light (IML) for 36 cycles of 2 min light and 118 min darkness had suppressed Chl b and LHCII-production in both WT rye and barley, while the barley chlorina F2-mutant became totally devoid of all LHCII-polypeptides. It was found that the levels of q_s and q_s were similar in control grown barley chlorina F2 and IML-grown WT rye and barley, but q_s was reduced by 30 to 35% and q_s by 50 to 65%, respectively, as compared to control-grown. WT plants. No significant q_s could be detected in IML-grown barley chlorina F2. It is clear, from these changes in in vivo fluorescence quenching in rye and barley that a significant level of q_s is detectable even in the absence of LHCII. Only when the proximal antennae are totally absent, does q_E completely disappear. We conclude that the presence of LHCII is not an absolute requirement for q_E-quenching and suggest that distal as well as proximal antenna may contribute to q_E in vivo.     

Polarization of counterions in polyelectrolytes

A theory of the polarization of counterions bound to a polyion, such as a DNA, in low and high electric field strengths is developed using statistical mechanics of inhomogeneous systems. For low fields, one finds that the polarizability p is (Zq)² ρ0βL³/(12[1 + Lρ0σ(L, b, ζ, Z, I, ρ0)]J), where σ = ∫¹₀ (λ′ − λ₀ {dc(λ − λ′)/dλ}_{λ = λ0} dλ′J), Z and L are the valence and the length of the polyion, respectively, q is the proton charge, β = 1/k_BT, T is the temperature, k_B is the Boltzmann constant, I is the ionic strength, λ = x/L and λ₀ = x₀/L are scaled distances, x₀ is a reference point such that the inhomogeneous counterion density at x₀ is equal to ρ₀—the uniform density in the absence of an electric field E—and c(x) is the direct correlation function of the homogeneous counterion-polyion phase, which includes attractive and repulsive interactions. If Lσ(L, .) is much less than one, then the polarizability is proportional to L³. If the term Lσ(L, .) is much larger than one, the polarizability scales as L². The induced dipole moment saturates and its value is the same as that of Mandel-Manning theories. The onset of the saturation, however, depends critically on the direct correlation function and hence polyelectrolyte effects. In the formalism, the polarization of the counterions is the equilibrium response to an electric field provided E is less than E_saturated. A dynamical scheme that incorporates the fact that in high fields the bound counterions conduct is discussed. © 1996 John Wiley & Sons, Inc.     

Effect of Intracluster Correlation on the R-Square Statistic

R²-statistic is a popular and very widely used statistic in regression analysis to estimate the square multiple correlation (SMC), ρ², between a response variable Y and p predictor variables, _X1, …, _Xp. Numerous articles are available in the statistical literature on the properties of R² as an estimator of ρ² when the observations are uncorrelated. However, relatively little is known about the behavior of R² when the available observations are correlated such as the data that result from complex sampling schemes. In this paper, we study the behavior R² in the presence of two-stage sampling data. An approximate expressions for the variance and the bias of R² in the presence of two-stage cluster sampling data with positive intracluster correlation (ρ*) are obtained. It is evident from these formulas and from a simulation study that R² is a poor estimator of ρ² except when ρ* is small. As such, we consider several alternative estimators of ρ² and evaluate their theoretical properties and finite sample performance using a simulation study.     

Analysis of theD-region products ofH-2444using monoclonal antibodies reveals the expression of a new class I-like molecule

To analyze how many D-region-encoded molecules could be detected inH-2 ^q , we produced a panel of nine monoclonal antibodies from AKR (K^kD^k) anti-AKR.M (K^kD^q) immunizations. All of the D_q region antibodies cross-reacted on D^d and/or L^d, and all except one cross-reacted on D^b, confirming the previously observed serologic and amino acid sequence homology between theD-region products ofH-2 ^d ,H-2 ^b , andH-2 ^q . All of these monoclonal antibodies precipitated 46 000 dalton molecules from both cell-surface-labeled and biosynthetically labeled BIO.AKM spleen cells, indicating that all were reactive with class I-like molecules. Sequential immunoprecipitation analysis with one of these antibodies, 66-3-5, reveals the presence of a previously unidentified class I-like molecule. Tryptic peptide map analysis reveals that this molecule may be the product of a newly describedH-2D ^q -region gene.     

Metabolic quotient of the soil microflora in relation to plant succession

Summary In this study we propose the hypothesis that ecosystem succession is accompanied by a decrease in the metabolic quotient qCO₂ (respiration-to-biomass ratio) of the soil microflora. The qCO₂ is calculated from basal respiration (CO₂-C·h^-1) per unit microbial biomass carbon (C_mier). The hypothesis was tested by studying two primary successions on recessional moraines of the Rotmoos Ferner (Austria) and the Athabasca Glacier (Canada). For both soil seres (0->200 years) it was shown that the qCO₂ decreased with time, which corroborated the hypothesis. In addition, the short term development of the qCO₂ was demonstrated with a revegetation trial. We observed a rise in qCO₂ for the first two years after reclamation, followed by a subsequent decrease.     

Elucidation of binding mode and three dimensional quantitative structure–activity relationship studies of a novel series of protein kinase B/Akt inhibitors

Protein kinase B (PKB; also known as Akt kinase) is located downstream in the PI-3 kinase pathway. Overexpression and constitutive activation of PKB/Akt leads to human prostate, breast and ovarian carcinomas. A series of 69 PKB/Akt inhibitors were examined to explore their binding modes using FlexX, and three-dimensional quantitative structure–activity relationship (3D-QSAR) studies based on comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed to provide structural insights into these compounds. CoMFA produced statistically significant results, with cross-validated q ² and non-cross validated correlation r ² coefficients of 0.53 and 0.95, respectively. For CoMSIA, steric, hydrophobic and hydrogen bond acceptor fields jointly yielded ‘leave one out’ q ²  = 0.51 and r ²  = 0.84. The predictive power of CoMFA and CoMSIA was determined using a test set of 13 molecules, which gave correlation coefficients, of 0.58 and 0.62, respectively. Molecular docking revealed that the binding modes of these molecules in the ATP binding sites of the Akt kinase domain were very similar to those of the co-crystallized ligand. The information obtained from 3D contour maps will allow the design of more potent and selective Akt kinase inhibitors.     

Energetic metabolism during individual development of <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> (Lymnaeidae,gastropoda): III. Late postlarval ontogeny

Changes in the rate and intensity of oxygen consumption during individual ontogeny of 14 specimens of Lymnaea stagnalis in the period from the 10th week after emergence until death was investigated in aquaculture. It was demonstrated that the rate of oxygen consumption increased and the intensity of this process decreased during the whole period of observations. Alterations of these parameters were accompanied with permanent oscillations of their meanings. The correlation between intensity of oxygen consumption (q) and age (t) can be described with the equation q = q _st/(1-exp(−k _g (t+t ₀))). The values of coefficients of this equation do not differ significantly between individuals and, on average, comprise k _g = 0.0696 ± 0.0072 weeks⁻¹; q _st = 60.4 ± 2.6 mcl O₂/(h · g); t ₀ = −3.0 ± 0.7 weeks. The dependence of the rate of oxygen consumption (Q, μl O₂/h) on body weight (M, g) for all data is significantly described with the allometric equation Q = 0.369M ^0.779.     

Dependence of laser light scattering of DNA on NaCl concentration

We show that the persistence length a of DNA, derived from total intensity laser light scattering of linear Col E₁ DNA and corrected for excluded-volume effects, varies from about 68 nm in 0.005M NaCl to about 40 nm in 0.2M NaCl, leveling off to a constant value (about 27 nm) at high NaCl (1–4M) concentration. These observations do not agree with current views on the effect of electrostatic charge and ionic conditions on DNA dimensions. The apparent diffusion constant D_app, determined from laser light scattering autocorrelation as a function of scattering vector q, at NaCl concentrations 0.005–4M, correctly yields the translational diffusion coefficient D_t at low values of q and scales with molecular dimensions rather than segment length at high values of q; thus, D_app/D_t yields a universal curve when plotted against q²R, where R_g is the radius of the gyration. The sedimentation coefficients s at 0.1 and 0.2M NaCl concentration closely agree with the well-tested empirical relations, and a combination of s, D_t, and the appropriate density increments yield correct molar masses over the whole salt concentration range. Approximate constancy of D_tR_g indicates limited draining in translational flow. We present some observations and thoughts on the regimes in which a dependence of the correlation decay times on q³ rather than q² applies. We conclude that quasielastic laser light scattering discloses little information about dynamics of internal motion of DNA chains.     

Virtual identification of novel peroxisome proliferator-activated receptor (PPAR) α/δ dual antagonist by 3D-QSAR,molecule docking,and molecule dynamics simulation

Abstract

The therapeutic potential of PPARs antagonists extends beyond diabetes. PPARs antagonists represent a new drug class that holds promise as a broadly applicable therapeutic approach for cancer treatment. Thus, there is a strong need to develop a rational design strategy for creating PPARs antagonists. In this study, three-dimensional quantitative structure-activity relationship (3D-QSAR) models of PPARα receptor (CoMFA-1, q ² = 0.636, r ² = 0.953; CoMSIA-1, q ² = 0.779, r ² = 0.999) and PPARδ receptor (CoMFA-2, q ² = 0.624, r ² = 0.906; CoMSIA-2, q ² = 0.627, r ² = 0.959) were successfully constructed using 35 triazolone ring derivatives. Contour map analysis revealed that the electrostatic and hydrophobic fields played vital roles in the bioactivity of dual antagonists. Molecular docking studies suggested that the hydrogen bonding, electrostatic and hydrophobic interactions all influenced the binding of receptor-ligand complex. Based on the information obtained above, we designed a series of compounds. The docking results were mutually validated with 3D-QSAR results. Three-dimensional-QSAR and absorption, distribution, metabolism, excretion and toxicity (ADMET) predictions indicated that 19 newly designed compounds possessed excellent biological activity and physicochemical properties. In summary, this research could provide theoretical guidance for the structural optimization of novel PPARα and δ dual antagonists.

Communicated by Ramaswamy H. Sarma     

H2-A polymorphism contributes to H2-Eβ-mediated protection in collagen-induced arthritis

 Collagen-induced arthritis (CIA) is an animal model of auto-immune inflammatory polyarthritis which has features similar to rheumatoid arthritis (RA). Much like RA, susceptibility to mouse CIA is influenced by the major histocompatibility complex (MHC) and is restricted to the H2 haplotypes q and r. In previous experiments, we have found that the introduction of an H2-Eb ^d transgene in H2-A^q CIA-susceptible mice was able to protect these mice against disease development. More recently, we have proposed that the polymorphism of the first domain of the Eβ molecule modulates this protection, and that the presentation of a peptide from the third hypervariable region of the Eβ chain by the H2-A^q molecule plays an important role in this mechanism. In the present report, we investigated whether the H2-E-mediated protection is H2-A^q-specific and whether the source of collagen has any influence. While the source of collagen had no effect on the protection, our results showed that the H2-E molecule failed to protect B10.RIII (H2^r) mice against CIA. Further, the H2 haplotype r exerted a negative effect on the Eβ^d-mediated protection in H2-A^q-restricted disease. Our results provide additional proof that self-MHC-derived peptides, such as Eβ peptides, may play an important role in the T-cell repertoire education and/or modulation of the T-cell response in the periphery. Received: 29 May 1996 / Revised: 26 June 1996