Congwei Niu

Rational design based on bioactive conformation analysis of pyrimidinylbenzoates as acetohydroxyacid synthase inhibitors by integrating molecular docking, CoMFA, CoMSIA, and DFT calculations.

Pyrimidinylthiobenzoates constitute an important kind of herbicides targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6), which catalyze the first common step in branched-chain amino acid biosynthesis. Due to the symmetry of 4,6-dimethoxypyrimidyl, there are two kinds of conformation of pyrimidinylthiobenzoates: ones phenyl is left-extending (named conformation-L); the others phenyl is right-extending (named conformation-R). On the basis of the assumption that 3D quantitative structure-activity relationship (QSAR) models derived from the bioactive conformation should give the best result, a strategy of density-functional-theory-based 3D-QSAR was proposed to identify the bioactive conformation of pyrimidinylthiobenzoates by integrating the techniques of molecular docking, comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), and density functional theory calculation. The combination of three criteria of q2, r2, and r2pred obtained from CoMFA and CoMSIA analyses clearly indicated that conformation-R rather than conformation-L might be the bioactive conformation for pyrimidinylthiobenzoates. A further comparison between the two binding modes indicated that pyrimidinylthiobenzoates and sulfonylureas have very similar binding sites, such as Trp586, Arg380, and Pro192. However, Lys251 formed H bonds with sulfonylureas rather than pyrimidinylthiobenzoates. In addition, the orientation of phenyl groups of the two classes of compounds in the binding pocket were revealed to be opposite, which explained why the mutation of Pro192 displayed different sensitivity to sulfonylureas and pyrimidinylthiobenzoates. On the basis of the understanding of interactions between pyrimidinyl-thiobenzoates and AHAS, we designed and synthesized six 8-(4,6-dimethoxypyrimidin-2-yloxy)-4-methylphthalazin-1-one derivatives according to the 3D-QSAR models. The excellent correlation between the tested Ki values against wild-type A. thaliana acetohydroxyacid synthase and the predicted IC50 values demonstrated the high reliability of the established 3D-QSAR models. To our knowledge, this is the first report highlighting the binding mode of herbicidal pyrimidinylthiobenzoates, which consisted of the reported results of herbicide resistance.

Development of a general quantum-chemical descriptor for steric effects : Density functional theory based QSAR study of herbicidal sulfonylurea analogues

Quantitative structure‐activity relationship (QSAR) analysis has become one of the most effective approaches for optimizing lead compounds and designing new drugs. Although large number of quantum‐chemical descriptors were defined and applied successfully, it is still a big challenge to develop a general quantum‐chemical descriptor describing the bulk effects more directly and effectively. In this article, we defined a general quantum‐chemical descriptor by characterizing the volume of electron cloud for specific substituent using the method of density functional theory. The application of our defined steric descriptors in the QSAR analysis of sulfonylurea analogues resulted in four QSAR models with high quality (the best model: q2 = 0.881, r2 = 0.901, n = 35, s = 0.401, F = 68.44), which indicated that this descriptor may provide an effective way for solving the problem how to directly describe steric effect in quantum chemistry‐based QSAR studies.

Design and synthesis of N-2,6-difluorophenyl-5-methoxyl-1,2,4-triazolo[1,5-a]-pyrimidine-2-sulfonamide as acetohydroxyacid synthase inhibitor.

Triazolopyrimidine-2-sulfonamide belongs to a herbicide group called acetohydroxyacid synthase inhibitors. With the aim to discover new triazolopyrimidine sulfonanilide compounds with high herbicidal activity and faster degradation rate in soil, the methyl group of Flumetsulam (FS) was modified into a methoxy group to produce a new herbicidal compound, N-2,6-difluorophenyl-5-methoxy-1,2,4-triazolo[1,5-a]pyrimidine-2-sulfonamide (experimental code: Y6610). The enzymatic kinetic results indicated that compound Y6610 and FS have k(i) values of 3.31x10(-6) M and 3.60x10(-7) M against Arabidopsis thaliana AHAS, respectively. The 10-fold lower enzyme-inhibiting activity of Y6610 was explained rationally by further computational simulations and binding free energy calculations. In addition, compound Y6610 was found to display the same level in vivo post-emergent herbicidal activity as FS against some broad-leaf weeds and good safety to rice, maize, and wheat at the dosages of 75-300 gai/ha. Further determination of the half-lives in soil revealed that the half-life in soil of Y6610 is 3.9 days shorter than that of FS. The experimental results herein showed that compound Y6610 could be regarded as a new potential acetohydroxyacid synthase-inhibiting herbicide candidate for further study.

Computational Design and Discovery of Conformationally Flexible Inhibitors of Acetohydroxyacid Synthase to Overcome Drug Resistance Associated with the W586L Mutation

Acetohydroxyacid synthase (AHAS, also known as acetolactate synthase, EC 2.2.1.6 (formerly EC 4.1.3.18)) has attracted attention for many years as a potential target for inhibitors to be used as herbicides and antibiotics. Despite the great success of commercial AHAS inhibitors over the past decades, drug resistance has become one of the most serious problems to overcome. In most cases, resistance to AHAS-inhibiting products has been shown to be caused by an alteration in the AHAS enzyme itself. Single point mutations that confer resistance to AHAS inhibitors include A117T, P192A, P192S, P192E, P192L, A200V, and W586L (Saccharomyces cerevisiae AHAS residue numbering). Among these, W586L is the most comprehensively characterized mutation, which results in at least 10fold resistance to all types of AHAS inhibitors. Therefore, the design of novel compounds that block the activity of the W586L mutant form has become one of the biggest challenges in this field. Herein we report the first computational design that has led to the discovery of 2-aroxyl-1,2,4-triazoloACHTUNGTRENNUNG[1,5-c]pyrimidines that have the same level of inhibitory activity against both wild-type AHAS and its W586L mutant. The present study demonstrates that the computational design approach based on the analysis of ligand conformational flexibility in the binding pocket holds promise for the rational design of conformationally flexible inhibitors of enzymes to overcome drug resistance. A general understanding of the molecular mechanisms behind some representative commercially available AHAS inhibitors provides useful clues for further molecular design against drug resistance. Three structurally diverse AHAS inhibitors, chlorsulfuron (CS), bispyribac (BP), and flumetsulam (FS) (Figure 1) were considered for this purpose.

Preliminary X-ray crystallographic studies of the catalytic subunit of Escherichia coli AHAS II with its cofactors.

Acetohydroxyacid synthase (AHAS) is the first common enzyme in the branched-chain amino-acid biosynthesis pathway and is the target of several classes of commercial herbicides. In this study, the Escherichia coli ilvG gene that encodes the catalytic subunit of AHAS II was cloned into the pET28a vector and expressed in soluble form at high levels in E. coli strain BL21 (DE3) cells. The protein was purified using Ni(2+)-chelating chromatography followed by size-exclusion chromatography. The catalytic subunit of E. coli AHAS II was cocrystallized with its cofactors Mg(2+), FAD and ThDP using the sitting-drop vapour-diffusion method and the crystals diffracted to 2.80 Å resolution.

Experimental and computational correlation and prediction on herbicide resistance for acetohydroxyacid synthase mutants to Bispyribac

Bispyribac is a widely used herbicide that targets the acetohydroxyacid synthase (AHAS) enzyme. Mutations in AHAS have caused serious herbicide resistance that threatened the continued use of the herbicide. So far, a unified model to decipher herbicide resistance in molecular level with good prediction is still lacking. In this paper, we have established a new QSAR method to construct a prediction model for AHAS mutation resistance to herbicide Bispyribac. A series of AHAS mutants concerned with the herbicide resistance were constructed, and the inhibitory properties of Bispyribac against these mutants were measured. The 3D-QSAR method has been transformed to process the AHAS mutants and proposed as mutation-dependent biomacromolecular QSAR (MB-QSAR). The excellent correlation between experimental and computational data gave the MB-QSAR/CoMFA model (q2 = 0.615, r2 = 0.921, r2pred = 0.598) and the MB-QSAR/CoMSIA model (q2 = 0.446, r2 = 0.929, r2pred = 0.612), which showed good prediction for the inhibition properties of Bispyribac against AHAS mutants. Such MB-QSAR models, containing the three-dimensional molecular interaction diagram, not only disclose to us for the first time the detailed three-dimensional information about the structure-resistance relationships, but may also provide further guidance to resistance mutation evolution. Also, the molecular interaction diagram derived from MB-QSAR models may aid the resistance-evading herbicide design.

Biomacromolecular 3D-QSAR to Decipher Molecular Herbicide Resistance in Acetohydroxyacid Synthases

Being able to quantitatively predict the risk of mutational drug resistance and drug selectivity at molecular level will greatly benefit the drug development. Many efforts have been dedicated towards these issues. Different methods based on energy calculation, statistics, or combination of both have been developed to deal with the prediction of either mutational drug resistance or drug selectivity, such as comparative docking, comparative molecular dynamics, GRID/CPCA, SIFt, COMBINE, AFMoC, selective 3D-QSAR models, proteochemometrics, 2D-QSFR, coevolutionary analysis, and bioinformatic approaches. Though these methods can achieve relatively accurate prediction on either mutational drug resistance or drug selectivity for a given protein mutant at molecular level, a visually chemical-intuitive method that can quickly be adapted to predict both of mutational drug resistance and drug selectivity with the guiding information for future mutational evolution is still needed. The widely spread usage of 3DQSAR for small molecules make it quite an attractive alternative to be adapted for biomacromolecules. 3D-QSAR, such as Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA), can quantitatively establish the structure-activity relationship, based on structural variations of small molecules, with an assumption that a suitable sampling of the molecular field values, surrounding a set of analogical ligand molecules, can provide sufficient information for understanding their observed properties. Considering that the QSAR is the reflection of the intermolecular interaction between small molecules and biomacromolecules, it would be interesting to see if this 3D-QSAR approach can be extended to biomacromolecules through a suitable sampling of the molecular field values in the interesting regions of a series of mutated or homological proteins interacting with a small molecule (drug). In this paper, we have shown 3D-QSAR can be successfully extended into the establishment of the structure-activity relationship, particularly the quantitative description of molecular mutational resistance to herbicide chlorimuron ethyl (CE) by acetohydroxyacid synthase (AHAS). This newly extended approach of 3D-QSAR is thus named as MB-QSAR (Mutationdependent Biomacromolecular Quantitative Structure Activity Relationship) to differentiate from 3D-QSAR for small molecules. The procedure for MB-QSAR method follows standard 3D-QSAR using biomacromolecules instead of small molecules as shown in Figure 1. The structures of the mutated or homological proteins were built up from the solved crystal structures or homology models. Following a proper alignment rule, the molecular field values of the mutated or homological proteins were calculated using different probe atom, and correlated with the experimental biological properties using the partial least square (PLS) method. The MB-QSAR models were thus established showing the quantitative structure-activity relationships for biomacromolecules. In this work, the so called MB-QSAR method was applied to AHAS to decipher the molecular herbicide resistance. AHAS is one of the most important targets for herbicides, fungicides and antimicrobial compounds. AHAS quickly developed resistance to herbicides in the field. In most cases, resistance to AHAS-inhibiting herbicides is caused by mutations on AHAS. Herein, the wild type E. coli AHAS II (EC 2.2.1.6) and its 85 mutants were constructed and overexpressed. The inhibitory properties (the apparent inhibition constant, Ki ) for a commonly used sulfonylurea herbicide, chlorimuron ethyl (CE), against these mutants were measured. The MB-QSAR models were constructed and validated based on these data. The details of the biological experiments and data were shown in Supporting Information. In the MB-QSAR studies, the wild type AHAS and 55 mutants were used as the training set and 30 mutants were used as the test set (see Supporting Information). The 3D structures of these AHAS proteins were modeled in SYBYL6.9 (Tripos, Inc.) using the crystal structure of the wild type E. coli AHAS II as the template. Three alignment rules were investigated in order to obtain reasonable MB-QSAR models: 1) to align the back-

The Minimum Activation Peptide from ilvH Can Activate the Catalytic Subunit of AHAS from Different Species

Acetohydroxyacid synthases (AHASs), which catalyze the first step in the biosynthesis of branched‐chain amino acids, are composed of a catalytic subunit (CSU) and a regulatory subunit (RSU). The CSU harbors the catalytic site, and the RSU is responsible for the activation and feedback regulation of the CSU. Previous results from Chipman and co‐workers and our lab have shown that heterologous activation can be achieved among isozymes of Escherichia coli AHAS. It would be interesting to find the minimum peptide of ilvH (the RSU of E. coli AHAS III) that could activate other E. coli CSUs, or even those of ## species. In this paper, C‐terminal, N‐terminal, and C‐ and N‐terminal truncation mutants of ilvH were constructed. The minimum peptide to activate ilvI (the CSU of E. coli AHAS III) was found to be ΔN14–ΔC89. Moreover, this peptide could not only activate its homologous ilvI and heterologous ilvB (CSU of E. coli AHAS I), but also heterologously activate the CSUs of AHAS from Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana plumbaginifolia. However, this peptide totally lost its ability for feedback regulation by valine, thus suggesting different elements for enzymatic activation and feedback regulation. Additionally, the apparent dissociation constant (Kd) of ΔN14–ΔC89 when binding CSUs of different species was found to be 9.3–66.5 μM by using microscale thermophoresis. The ability of this peptide to activate different CSUs does not correlate well with its binding ability (Kd) to these CSUs, thus implying that key interactions by specific residues is more important than binding ability in promoting enzymatic reactions. The high sequence similarity of the peptide ΔN14–ΔC89 to RSUs across species hints that this peptide represents the minimum activation motif in RSU and that it regulates all AHASs.

Arginine 26 and Aspartic Acid 69 of the Regulatory Subunit are Key Residues of Subunits Interaction of Acetohydroxyacid Synthase Isozyme III from E. coli

Acetohydroxyacid synthase (AHAS), which catalyzes the first step in the biosynthesis of branched‐chain amino acids, is composed of catalytic and regulatory subunits. The enzyme exhibits full activity only when the regulatory subunit (RSU) binds to the catalytic subunit (CSU). However, the crystal structure of the holoenzyme has not been reported yet, and the molecular interaction between the CSU and RSU is also unknown. Herein, we introduced a global‐surface, site‐directed labeling scanning method to determine the potential interaction region of the RSU. This approach relies on the insertion of a bulky fluorescent probe at the designated site on the surface of the RSU to cause a dramatic change in holoenzyme activity by perturbing subunit interaction. Then, the key amino acid residues in the potential interaction regions were identified by site‐directed mutagenesis. Compared to the wild‐type, the single‐point mutants R26A and D69A showed 54 and 64 % activity, respectively, whereas the double mutant (R26A+D69A) gave 14 %, thus suggesting that residues Arg26 and Asp69 are the key residues of subunit interaction with cooperative action. Additionally, the results of GST pull‐down assays and pH‐dependence experiments suggested that polar interaction is the main force for subunits interaction. A plausible protein–protein interaction model of the holoenzyme of Escherichia coli AHAS III is proposed, based on the mutagenesis and protein docking studies. The protocol established here should be useful for the identification of the molecular interactions between proteins.

Computational Design of Novel Inhibitors to Overcome Weed Resistance Associated with Acetohydroxyacid Synthase (AHAS) P197L Mutant

BACKGOUND Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is the first common enzyme in the biosynthetic pathway leading to the branched-chain amino acids in plants and a wide range of microorganisms. With the long-term and wide application of AHAS inhibitors, weed resistance is becoming a global problem, which leads to an urgent demand for novel inhibitors to antagonize both wild-type and resistant AHAS. RESULTS Pyrimidinyl salicylic acid derivatives, as one of the main classes of commercial AHAS herbicides, show potential anti-resistant bioactivity to wild-type and P197L mutant. In current work, a series of novel 2-benzoyloxy-6-pyrimidinyl salicylic acid derivatives were designed through fragment-based drug discovery. Fortunately, the newly synthesized compounds showed good inhibitory activity against both wild-type and P197L mutant. Some compounds not only had a lower resistance factor value but also showed excellent inhibitory activity against wild-type AHAS and P197L mutant. Furthermore, greenhouse experiments showed compound 11m displayed almost 100% inhibition against both wild-type and high-resistant Descurainia sophia at a dosage of 150 g a.i. ha-1 . CONCLUSION The present work indicated that the 2-benzoyloxy-6-pyrimidinyl salicylic acid motif was well worth further optimization. Also, compound 11m could be used as a potential anti-resistant AHAS herbicide, which requires further research.