Practical application of ligand efficiency metrics in lead optimisation

The use of composite metrics that normalise biological potency values in relation to markers of physicochemical properties, such as size or lipophilicity, has gained a significant amount of traction with many medicinal chemists in recent years. However, there is no consensus on best practice in the area and their application has attracted some criticism. Here we present our approach to their application in lead optimisation projects, provide an objective discussion of the principles we consider important and illustrate how our use of lipophilic ligand efficiency enabled the progression of a number of our successful drug discovery projects. We derive, from this and some recent literature highlights, a set of heuristic guidelines for lipophilicity based optimisation that we believe are generally applicable across chemical series and protein targets.     

Discovery of triazolopyrimidine-based PDE8B inhibitors: Exceptionally ligand-efficient and lipophilic ligand-efficient compounds for the treatment of diabetes

PDE8B is a cAMP-specific isoform of the broader class of phosphodiesterases (PDEs). As no selective PDE8B inhibitors had been reported, a high throughput screen was run with the goal of identifying selective tools for exploring the potential therapeutic utility of PDE8B inhibition. Of the numerous hits, one was particularly attractive since it was amenable to rapid deconstruction leading to inhibitors with very high ligand efficiency (LE) and lipophilic ligand efficiency (LLE). These triazolopyrimidines were optimized for potency, selectivity and ADME properties ultimately leading to compound 42. This compound was highly potent and selective with good bioavailability and advanced into pre-clinical development.     

Investigation of the pyrazinones as PDE5 inhibitors: evaluation of regioisomeric projections into the solvent region

We describe the design, synthesis and profiling of a novel series of PDE5 inhibitors. We take advantage of an alternate projection into the solvent region to identify compounds with excellent potency, selectivity and pharmacokinetic profiles.     

Capacitance of bilayers in the presence of lipophilic ions

The capacitance of glycerolmonooleate and egg phosphatidylcholine bilayer membranes in the presence of NaCl solutions containing tetraphenylborate, tetraphenylarsonium or dipicrylamine ions has been measured using alternating current techniques over a wide range of frequencies (1–200 kHz). The concentrations of ions corresponded to the lower limits of conductance saturation. Similar determinations were also made with solutions containing no lipophilic ions. The experimental method used in this work requires correction of admittance measurements for the solution resistance in series with the membrane, as well as careful area determinations. In all cases membrane capacitance levels off at sufficiently high frequencies to values which are independent of frequency. The high-frequency capacitance, which is regarded as the ‘geometrical capacitance’ due to dielectric polarization, is practically unaffected by the presence of lipophilic ions. The results support the assumption made in other studies, such as in charge pulse investigations, that the adsorption of lipophilic ions at concentrations up to the saturation range does not have an important effect on the dielectric properties of bilayers.     

Discovery of a series of potent and selective human H4 antagonists using ligand efficiency and libraries to explore structure-activity relationship (SAR)

We describe the identification of a potent, selective lead series that shows antagonism against the human histamine H4 receptor from thirteen actives identified in an HTS as part of a hit to lead program. By focusing on ligand efficiency and concurrently using a diversity based approach, compounds based around 2,4-diaminopyrimidine were identified with compound 25 being quickly shown to be a good lead. It also had the highest ligand efficiency in the series.     

Regeneration of lipophilic antioxidants by NAD(P)H:quinone oxidoreductase 1

Summary.  The aim of this work was to study the activity of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) in the regeneration of lipophilic antioxidants, alpha-tocopherol, and reduced-coenzyme Q analogs. First, we tested whether or not two isoforms of the NAD(P)H:(quinone acceptor) oxidoreductase 1 designated as “hydrophilic” and “hydrophobic” (H. J. Prochaska and P. Talalay, Journal of Biological Chemistry 261: 1372–1378, 1986) show differential enzyme activities towards hydrophilic or hydrophobic ubiquinone homologs. By chromatography on phenyl Sepharose, we purified the two isoforms from pig liver cytosol and measured their reduction of several ubiquinone homologs of different side chain length. We also studied by electron paramagnetic resonance the effect of NAD(P)H:(quinone acceptor) oxidoreductase 1 on steady-state levels of chromanoxyl radicals generated by linoleic acid and lipooxygenase and confirmed the enzyme's ability to protect alpha-tocopherol against oxidation induced with H₂O₂-Fe²⁺. Our results demonstrated that the different hydrophobicities of the isoforms do not reflect different reactivities towards ubiquinones of different side chain length. In addition, electron paramagnetic resonance studies showed that in systems containing the reductase plus NADH, levels of chromanoxyl radicals were dramatically reduced. Morever, in the presence of oxidants, alpha-tocopherol was preserved by NAD(P)H:(quinone acceptor) oxidoreductase 1, supporting our hypothesis that regeneration of alpha-tocopherol may be one of the physiologic functions of this enzyme. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio Severo Ochoa, Campus de Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

Restriction enzyme cleavage of fluorescently labeled DNA fragments--analysis of the method and its usage in examination of digestion completeness

Restriction enzymes have proven to be among the most valuable tools in molecular biology. In this work, we demonstrate that the cleavage of fluorescently labeled, PCR-amplified DNA can be used as a simple and highly sensitive technique for detection of sequences present in a percentage as low as 0.6% in a DNA pool. Due to the fact that fluorescent labeling of DNA fragments enables such sensitive detection and quantification of restriction enzyme cleavage, the method was further exploited in monitoring of the enzymatic digestion completeness and in determination of factors that influence restriction enzyme effectiveness. We analyzed the activity of six restriction endonucleases; the percentage of uncleaved DNA fragments predominantly ranged between 2.0 and 2.5 and the highest value was 8.00%. We conclude that, since the enzymatic digestion completeness may not always be assured, each assay based on restriction enzyme cleavage that is intended to be used in investigations of heterogeneity in a DNA pool should be constructed so that the presence of cleaved sequences is the indication of pool nonuniformity. When the presence of uncleaved sequences indicates pool heterogeneity, the results could be misleading due to possible incompleteness of enzymatic cleavage.     

The use of different models for the estimation of residual feed intake (RFI) as a measure of feed efficiency in meat sheep

The concept of residual feed intake (RFI), in determining differences among animals in converting feed into body tissue, was first raised in 1963. Feed efficiency is typically calculated as a function of liveweight gain (LWG) and feed intake (FI). Historically two versions of the same model were proposed, one where FI was adjusted for liveweight (LW) and LWG, and the other where LWG was adjusted for FI and LW. Variation in LWG or FI could then be partitioned into two parts; that which is expected and can be attributed to differences in FI or LWG; and that which is the residual portion, which is the deviation from the expected value based on regression, and therefore not accounted for by differences in FI or LWG. Based on this definition, it is the residual portion which is the measure of efficiency. Both within a livestock industry and between different livestock industries there is no set model for calculating RFI. This paper evaluated four models used to calculate RFI and one model used to calculate residual LWG (RLWG) at a standard level of nutrition. They were the main model currently in use in the Australian beef cattle industry (RFI_B), the original models proposed in 1963 (RFI₁₉₆₃; RLWG₁₉₆₃); a French model which included ultrasound measures of muscle and fat depth (RFI_F) and the use of the Australian feeding standards to calculate predicted intake and thus RFI (RFI_SCA). Using feed intake, liveweight and body composition data generated from the same group of sheep (n = 52) at two ages (6 mo, 13 mo), the relative merits of each model were evaluated and compared to the other models, to determine the most appropriate model to calculate RFI for sheep. For all the models except that used to calculate RLWG, over half of the variation in FI could be explained by the model. The amount of variation in FI accounted for depended on the parameters included and the dataset, with less variation in FI explained by the specific models in the older animals. The RFI_F model, which included measures of body composition, accounted for the greatest proportion of the variation in FI and as such suggests that the inclusion of body composition parameters is likely to more accurately reflect true biological efficiency.     

Enthalpy/entropy compensation phenomena in the reduction thermodynamics of electron transport metalloproteins

Compensation phenomena between the enthalpy and entropy changes of the reduction reaction for all classes of electron transport metalloproteins, namely cytochromes, iron-sulfur, and blue copper proteins, are brought to light. This is the first comprehensive report on such effects for biological redox reactions. Following Grunwalds approach for the interpretation of H/S compensation for solution reactions, it is concluded that reduction-induced solvent reorganization effects involving the hydration shell of the molecule dominate the reduction thermodynamics in these species, although they have no net effect on the E° values, owing to exact compensation. Thus the reduction potentials of these species are primarily determined by the selective enthalpic stabilization of one of the two oxidation states due to ligand binding interactions and electrostatics at the metal site and by the entropic effects of reduction-induced changes in protein flexibility.     

Physiological basis of QTLs for boron efficiency in Arabidopsis thaliana

Boron (B) is an essential micronutrient for higher plants, but the adaptability of plants to B deficiency varies widely both between and within species. On the basis of quantitative trait loci (QTL) analysis of the B efficiency coefficient (BEC) detected in an Arabidopsis thaliana Ler × Col recombinant inbred (RI) population, B efficiency was evaluated in the original parents (Ler and Col-4) and two F₈ lines (1938 and 1961), both of which were selected on the basis of phenotype and genotype of the RI population. The parent Ler and F₈ progeny 1938 had higher BEC and B utilization efficiency (BUE) values than those calculated for parent Col-4 and F₈ progeny 1961, respectively, when grown in nutrient solutions containing three different concentrations of B. The magnitude of the BEC and BUE-values was correlated closely with the combined phenotypic effect of the corresponding QTLs among the four genotypes. The F₈ line, 1938, inherited all four B-efficient QTLs, AtBE1-1, AtBE1-2, AtBE2 and AtBE5, from its two original parents. The four QTLs accounted for 65.2% of the total variation in BEC and 1938 showed the highest BEC (0.74) and BUE (10.5) values among the four genotypes when grown in nutrient solution that contained 0.324 μM B. Only one minor-effect QTL (AtBE1-1) was found in the parent, Col-4. This QTL accounted only for 8.8% of total BEC variation and resulted in the lowest BEC (0.39) and BUE (0.76) in Col-4 when it was grown in nutrient solution that contained 0.324 μM B. Phenotypic profile analysis showed that 1938 not only inherited the B utilization and distribution characteristics found in the silique of Ler, but also acquired the low-B requirement for root and shoot growth from Col-4. As a result, this genotype displayed the strongest tolerance to B deficiency. In addition, both B-efficient genotypes, 1938 and Ler, possessed the QTL (AtBE1-2) and both plants had high-seed yields and high-B distributions in their siliques. Therefore, we hypothesize that QTL AtBE1-2 plays a role in the utilization and/or the distribution of B to the silique when plants suffer from B deficiency. A close correlation between the B-efficient phenotype and the corresponding QTLs indicated that phenotypic differences depend on the genetic variation. Responsible Editor: Richard W. Bell.     

春小麦水分利用效率日变化及其生理生态基础的研究

采用光合速率和蒸腾速率之比 (Pn/Tr)表示植物水分利用效率 (WUE) ,发现在两种不同水分处理下春小麦拔节期 .WUE日变化中都有上午高于下午值 ,且 8∶0 0～ 1 0∶0 0为明显峰值的特征 ,这种特征与Pn、Tr日变化存在的上、下午值不对称性和反向性 (Pn为上午高于下午 ,Tr相反 )紧密相关 ,水分胁迫处理中 ,Pn、Tr值均有降低 ,但不同品种的WUE反应不一 .研究表明 ,叶水势 ( ψw)、气孔阻力 (Rs)等生理因素和空气相对湿度 (RH)、光照 (Q)及冠层温度 (Tc)等生态因子 ,通过对Tr和Pn的不同影响而同WUE显著相关 ,40 %RH是WUE变化的一个重要阈值 .     

Biomarkers of antioxidant capacity in the hydrophilic and lipophilic compartments of human plasma

Antioxidants located in both the hydrophilic and lipophilic compartments of plasma are actively involved as a defense system against reactive oxygen species (ROS), which are continuously generated in the body due to both normal metabolism and disease. However, when the production of ROS is not controlled, it leads to cellular lipid, protein, and DNA damage in biological systems. Several assays to measure 'total' antioxidant capacity of plasma have been developed to study the involvement of oxidative stress in pathological conditions and to evaluate the functional bioavailability of dietary antioxidants. Conventional assays to determine antioxidant capacity primarily measure the antioxidant capacity in the aqueous compartment of plasma. Consequently, water-soluble antioxidants such as ascorbic acid, uric acid and protein thiols mainly influence these assays, whereas fat-soluble antioxidants such as tocopherols and carotenoids play only a minor role. However, there are active interactions among antioxidants located in the hydrophilic and lipophilic compartments of plasma. Therefore, new approaches to define the 'true' total antioxidant capacity of plasma should reflect the antioxidant network between water- and fat-soluble antioxidants in plasma. Revelation of the mechanism of action of antioxidants and their true antioxidant potential will help us to optimize the antioxidant defenses in the body.     

植物的养分利用效率(NUE)及植物对养分胁迫环境的适应策略

提高养分利用效率（ＮＵＥ）是植物适应贫瘠生境的一种重要的竞争策略。养分利用效率的概念从提出到现在,曾用多个参数描述,其间经历了一个不断完善的发展过程。通过综述近年来相关的研究结果,可以初步得出以下结论：⑴不同种,不同生活型植物,乃至同株植物不同器官的ＮＵＥ存在不同程度的差异。⑵ＮＵＥ受多种因素影响。其中,养分有效性的影响研究较多,但争议较大,综合考虑,它对ＮＵＥ的影响不甚显著;叶片脱落持续时间的影     

Revised algorithm of ecosystem water use efficiency for semi-arid steppe in the Loess Plateau of China

Aims We evaluated the applicability of different measures of water use efficiency through analyzing the coupled dynamics of GPP and evapotranspiration in the semi-arid steppe in the Loess Plateau of China. Our objective is to explore the applicability of two quantitative measures of ecosystem water use efficiency—inherent water use efficiency (IWUE) and underlying water use efficiency (uWUE) —for the semi-arid steppe and to endeavor necessary modifications.Methods The consistency and stability of three indices of water use efficiency formulations (i.e. WUE, IWUE, uWUE) were calculated and compared at hourly, daily and annual time scales before proposing an optimal water use efficiency (oWUE). These indices were additionally used to quantify their importances in modeling the diel change of gross primary production (GPP). The yielded-accuracy of the prediction was used for justifying their uses.Important findings IWUE and uWUE appeared suitable for examining the coupled water-carbon characteristics of vegetation at hourly and daily scales, whereas WUE was more plausible on the annual and interannual scales. The optimized water use efficiency index did not improve the prediction of the coupled water-carbon characteristics as compared with uWUE, but it improved the prediction of GPP and its dynamics. oWUE and uWUE improved the predictions of GPP in the peak growing period, while WUE predicted the GPP better at the early and late growing season. Interestingly, we found that IWUE was not suitable for predicting GPP and its dynamics. The results will be of great importance in modeling the effects of climate change on the carbon assimilation and water cycle for the future.     

Influencing factors of dsDNA dye (high-resolution) melting curves and improved genotype call based on thermodynamic considerations

High-resolution melting of dsDNA using suitable dyes is a simple and cost-effective alternative for mutation scanning. Analytical variation can result from salt and template concentration (C_T). To overcome this problem the van’t Hoff transition enthalpy ΔH_vH from dsDNA melting curves was estimated and used for robust genotype calling or mutation scanning. Model calculations show the effect of salt, C_T, and temperature resolution on (1) T_m, (2) difference plots, (3) melting peaks, and (4) calculated ΔH_vH. Using the LightCycler480, the influence of dye (ResoLight) and scanning speed was assessed. The model calculations show that only ΔH_vH is not influenced by salt and C_T. Higher amplicon enthalpy ameliorates the ability to discriminate mutations. Temperature resolution is important for peak- but not for curve-based genotyping. ResoLight increases T_m by 3.4 °C, while lowering ΔH_vH. Using a 4-bp deletion in a 200-bp amplicon as a model, the miscalling rate improved substantially, when using ΔH_vH instead of difference plots. Melting curves of duplex DNA are influenced by dye and salt and less so by duplex concentrations. As predicted from theory, ΔH_vH is a robust measure for mutation detection in two-state melting. The influence of dyes on enthalpy is of general impact for PCR assays.     

Complexes of the highly preorganized ligand PDALC (2,9-bis(hydroxymethyl)-1,10-phenanthroline) with trivalent lanthanides. A thermodynamic and crystallographic study

Metal ion complexing properties of the highly preorganized tetradentate ligand PDALC (2,9-bis(hydroxymethyl)-1,10-phenanthroline) are presented. The structure of [Gd(PDALC)(NO₃)₃]·H₂O (1) is reported: triclinic, , a = 7.545(12), b = 10.811(17), c = 11.909(18) Å, α = 97.71(2)°, β = 91.56(2)°, γ = 109.06(2)°, V = 907(2) Å³, Z = 2, R = 0.0354. The Gd is 10-coordinate, with the coordination sphere comprising the four donor atoms of the PDALC plus the six O-donors of three chelated nitrates. Comparison with structures in the literature suggests that the Gd-L (L = ligand) bond lengths, particularly those to the alcoholic O-donors of PDALC, are a little short. It was suggested that the short Gd-L bond lengths in 1 were due to the efficiency of packing of the nitrates around the Gd, with the short ‘bite’ distances of the nitrate ligand. Formation constants (log K₁) were measured spectroscopically in 0.1 M NaClO₄ at 25 °C by monitoring the variation of the π-π∗ transitions of 2 × 10⁻⁵ M PDALC in the range 200-350 nm as a function of pH, in the presence of 1:1 concentrations of the lanthanide(III) (Ln(III)) metal ion. The measured log K₁ values varied from 5.34 (La(III)) to 6.40 (Lu(III), which is an unusually small variation across the series of Ln(III) ions. Values of log K₁ with PDALC were also measured for Y(III) (5.85) and Sc(III) (6.02). The small amount of variation in log K₁ for PDALC across the series of Ln(III) ions was rationalised in terms of the effect of neutral oxygen donors on complex stability, which promotes selectivity for larger metal ions such as La(III). It was discussed how the small amount of variation in log K₁ across the Ln(III) series might lead to optimal selectivity for the Am(III) ion relative to the Ln(III) ions as a group.     

Physiological foundation for the differences of long-term water use efficiency among Populus deltoides clones

With randomized complete block design, long-term water use efficiency (WUE_L), photosynthetic parameters (net photosynthetic rate Pn, transpiration rate Tr, stomatal conductance Gs, instantaneous water use efficiency WUE_i, maximum photosynthetic rate P max, light saturation pointLSP, carboxylic efficiency Ce), stomatal properties (frequency, size, superior/inferior stoma amount ratio) and root/shoot ratio of twelve Populus deltoids clones were studied under water stress in a greenhouse. Four water treatments were designed, namely, well-watered condition, slight water stress, moderate water stress, and severe water stress. Although the volumes of irrigated water for different treatments were the same, the irrigation intervals were strictly controlled. The results showed that long-term water use efficiency (WUE_L) differences among tested clones were obvious and became greater with the increase in water stress. J₂, J₆, J₇, J₈, and J₉ were excellent clones with high WUE_L. Stomatal frequency, stomatal size, Gs, Tr, Pn, and root/shoot ratio of J₂et al were moderate, whereas the superior/inferior ratio, P_max, LSP, Ce of J2et al were higher than other clones. The interrelations between WUE_L and the physiological parameters were analyzed. The results revealed that the WUE_L differences were induced by a series of physiological parameters, and clones with higher WUE_L always had strong photosynthetic capacity, higher WUE_i, and optimum root/shoot ratio.     

Modulation of i-motif thermodynamic stability by the introduction of UNA (unlocked nucleic acid) monomers

The influence of acyclic RNA derivatives, UNA (unlocked nucleic acid) monomers, on i-DNA thermodynamic stability has been investigated. The 22 nt human telomeric fragment was chosen as the model sequence for stability studies. UNA monomers modulate i-motif stability in a position-depending manner. The largest destabilization is observed for position C14, while UNA placed in position A12 causes significant increase of i-DNA thermodynamic stability. CD curves of UNA-modified variants imply no structural changes relative to the native i-motif.     

农田氮肥利用率计算方法研究进展

氮肥利用率是农学和环境科学等领域普遍关注的焦点,合理计算氮肥利用率是实现农田氮素优化管理的重要前提和基础. 近年来,鉴于我国农田氮肥利用率测定值偏低,且存在很大变幅的事实,国内学者针对传统氮肥利用率的概念和计算方法进行了深入的思考和探讨,并提出了一些改进的尝试. 本文首先综述了传统氮肥利用率在概念和计算方法上存在的种种缺陷,然后较为系统地介绍了目前学者提出的计算农田氮肥利用率的几种常见的替代方法,并对今后的研究方向进行了展望.建议在正确理解氮肥用量、作物产量、氮肥利用和损失之间辩证关系的基础上,逐步建立起符合现阶段我国农业生产现状的农田氮肥利用状况评价体系.