The Art and Science of Molecular Docking.

Molecular docking has become an essential part of a structural biologist's and medicinal chemist's toolkits. Given a chemical compound and the three-dimensional structure of a molecular target-for example, a protein-docking methods fit the compound into the target, predicting the compound's bound structure and binding energy. Docking can be used to discover novel ligands for a target by screening large virtual compound libraries. Docking can also provide a useful starting point for structure-based ligand optimization or for investigating a ligand's mechanism of action. Advances in computational methods, including both physics-based and machine learning approaches, as well as in complementary experimental techniques, are making docking an even more powerful tool. We review how docking works and how it can drive drug discovery and biological research. We also describe its current limitations and ongoing efforts to overcome them.

GSScore: a novel Graphormer-based shell-like scoring method for protein-ligand docking.

Protein-ligand interactions (PLIs) are essential for cellular activities and drug discovery. But due to the complexity and high cost of experimental methods, there is a great demand for computational approaches to recognize PLI patterns, such as protein-ligand docking. In recent years, more and more models based on machine learning have been developed to directly predict the root mean square deviation (RMSD) of a ligand docking pose with reference to its native binding pose. However, new scoring methods are pressingly needed in methodology for more accurate RMSD prediction. We present a new deep learning-based scoring method for RMSD prediction of protein-ligand docking poses based on a Graphormer method and Shell-like graph architecture, named GSScore. To recognize near-native conformations from a set of poses, GSScore takes atoms as nodes and then establishes the docking interface of protein-ligand into multiple bipartite graphs within different shell ranges. Benefiting from the Graphormer and Shell-like graph architecture, GSScore can effectively capture the subtle differences between energetically favorable near-native conformations and unfavorable non-native poses without extra information. GSScore was extensively evaluated on diverse test sets including a subset of PDBBind version 2019, CASF2016 as well as DUD-E, and obtained significant improvements over existing methods in terms of RMSE, $R$ (Pearson correlation coefficient), Spearman correlation coefficient and Docking power.

PGBind: pocket-guided explicit attention learning for protein-ligand docking.

As more and more protein structures are discovered, blind protein-ligand docking will play an important role in drug discovery because it can predict protein-ligand complex conformation without pocket information on the target proteins. Recently, deep learning-based methods have made significant advancements in blind protein-ligand docking, but their protein features are suboptimal because they do not fully consider the difference between potential pocket regions and non-pocket regions in protein feature extraction. In this work, we propose a pocket-guided strategy for guiding the ligand to dock to potential docking regions on a protein. To this end, we design a plug-and-play module to enhance the protein features, which can be directly incorporated into existing deep learning-based blind docking methods. The proposed module first estimates potential pocket regions on the target protein and then leverages a pocket-guided attention mechanism to enhance the protein features. Experiments are conducted on integrating our method with EquiBind and FABind, and the results show that their blind-docking performances are both significantly improved and new start-of-the-art performance is achieved by integration with FABind.

Characterization of lignin-degrading enzyme PmdC, which catalyzes a key step in the synthesis of polymer precursor 2-pyrone-4,6-dicarboxylic acid.

Pyrone-2,4-dicarboxylic acid (PDC) is a valuable polymer precursor that can be derived from the microbial degradation of lignin. The key enzyme in the microbial production of PDC is 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase, which acts on the substrate CHMS. We present the crystal structure of CHMS dehydrogenase (PmdC from Comamonas testosteroni) bound to the cofactor NADP, shedding light on its three-dimensional architecture, and revealing residues responsible for binding NADP. Using a combination of structural homology, molecular docking, and quantum chemistry calculations, we have predicted the binding site of CHMS. Key histidine residues in a conserved sequence are identified as crucial for binding the hydroxyl group of CHMS and facilitating dehydrogenation with NADP. Mutating these histidine residues results in a loss of enzyme activity, leading to a proposed model for the enzyme's mechanism. These findings are expected to help guide efforts in protein and metabolic engineering to enhance PDC yields in biological routes to polymer feedstock synthesis.

Functional Characterization Supports Multiple Evolutionary Origins of Pheromone Receptors in Bark Beetles.

Chemical communication using pheromones is thought to have contributed to the diversification and speciation of insects. The species-specific pheromones are detected by specialized pheromone receptors (PRs). Whereas the evolution and function of PRs have been extensively studied in Lepidoptera, only a few PRs have been identified in beetles, which limits our understanding of their evolutionary histories and physiological functions. To shed light on these questions, we aimed to functionally characterize potential PRs in the spruce bark beetle Ips typographus ("Ityp") and explore their evolutionary origins and molecular interactions with ligands. Males of this species release an aggregation pheromone comprising 2-methyl-3-buten-2-ol and (4S)-cis-verbenol, which attracts both sexes to attacked trees. Using two systems for functional characterization, we show that the highly expressed odorant receptor (OR) ItypOR41 responds specifically to (4S)-cis-verbenol, with structurally similar compounds eliciting minor responses. We next targeted the closely related ItypOR40 and ItypOR45. Whereas ItypOR40 was unresponsive, ItypOR45 showed an overlapping response profile with ItypOR41, but a broader tuning. Our phylogenetic analysis shows that these ORs are present in a different OR clade as compared to all other known beetle PRs, suggesting multiple evolutionary origins of PRs in bark beetles. Next, using computational analyses and experimental validation, we reveal two amino acid residues (Gln179 and Trp310) that are important for ligand binding and pheromone specificity of ItypOR41 for (4S)-cis-verbenol, possibly via hydrogen bonding to Gln179. Collectively, our results shed new light on the origins, specificity, and ligand binding mechanisms of PRs in beetles.

Exploring ligands that target von Willebrand factor selectively under oxidizing conditions through docking and molecular dynamics simulations.

The blood protein von Willebrand factor (VWF) is a large multimeric protein that, when activated, binds to blood platelets, tethering them to the site of vascular injury and initiating blood coagulation. This process is critical for the normal hemostatic response, but especially under inflammatory conditions, it is thought to be a major player in pathological thrombus formation. For this reason, VWF has been the target for the development of anti-thrombotic therapeutics. However, it is challenging to prevent pathological thrombus formation while still allowing normal physiological blood coagulation, as currently available anti-thrombotic therapeutics are known to cause unwanted bleeding, in particular intracranial hemorrhage. This work explores the possibility of inhibiting VWF selectively under the inflammatory conditions present during pathological thrombus formation. In particular, the A2 domain of VWF is known to inhibit the neighboring A1 domain from binding to the platelet surface receptor GpIbα, and this auto-inhibitory mechanism has been shown to be removed by oxidizing agents released during inflammation. Hence, finding drug molecules that bind at the interface between A1 and A2 only under oxidizing conditions could restore such an auto-inhibitory mechanism. Here, by using a combination of computational docking, molecular dynamics simulations, and free energy perturbation calculations, a ligand from the ZINC15 database was identified that binds at the A1A2 interface, with the interaction being stronger under oxidizing conditions. The results provide a framework for the discovery of drug molecules that bind to a protein selectively in the presence of inflammatory conditions.

FitDock: protein-ligand docking by template fitting.

Protein-ligand docking is an essential method in computer-aided drug design and structural bioinformatics. It can be used to identify active compounds and reveal molecular mechanisms of biological processes. A successful docking usually requires thorough conformation sampling and scoring, which are computationally expensive and difficult. Recent studies demonstrated that it can be beneficial to docking with the guidance of existing similar co-crystal structures. In this work, we developed a protein-ligand docking method, named FitDock, which fits initial conformation to the given template using a hierarchical multi-feature alignment approach, subsequently explores the possible conformations and finally outputs refined docking poses. In our comprehensive benchmark tests, FitDock showed 40%-60% improvement in terms of docking success rate and an order of magnitude faster over popular docking methods, if template structures exist (> 0.5 ligand similarity). FitDock has been implemented in a user-friendly program, which could serve as a convenient tool for drug design and molecular mechanism exploration. It is now freely available for academic users at http://cao.labshare.cn/fitdock/.

Pro197Ser and the new Trp574Leu mutations together with enhanced metabolism contribute to cross-resistance to ALS inhibiting herbicides in Sinapis alba.

White mustard, (Sinapis alba), a problematic broadleaf weed in many Mediterranean countries in arable fields has been detected as resistant to tribenuron-methyl in Tunisia. Greenhouse and laboratory studies were conducted to characterize Target-Site Resistance (TSR) and the Non-Target Site Resistance (NTSR) mechanisms in two suspected white mustard biotypes. Herbicide dose-response experiments confirmed that the two S. alba biotypes were resistant to four dissimilar acetolactate synthase (ALS)-pinhibiting herbicide chemistries indicating the presence of cross-resistance mechanisms. The highest resistance factor (>144) was attributed to tribenuron-methyl herbicide and both R populations survived up to 64-fold the recommended field dose (18.7 g ai ha-1). In this study, the metabolism experiments with malathion (a cytochrome P450 inhibitor) showed that malathion reduced resistance to tribenuron-methyl and imazamox in both populations, indicating that P450 may be involved in the resistance. Sequence analysis of the ALS gene detected target site mutations in the two R biotypes, with amino acid substitutions Trp574Leu, the first report for the species, and Pro197Ser. Molecular docking analysis showed that ALSPro197Ser enzyme cannot properly bind to tribenuron-methyl's aromatic ring due to a reduction in the number of hydrogen bonds, while imazamox can still bind. However, Trp574Leu can weaken the binding affinity between the mutated ALS enzyme and both herbicides with the loss of crucial interactions. This investigation provides substantial evidence for the risk of evolving multiple resistance in S. alba to auxin herbicides while deciphering the TSR and NTSR mechanisms conferring cross resistance to ALS inhibitors.

QUBO Problem Formulation of Fragment-Based Protein-Ligand Flexible Docking.

Protein-ligand docking plays a significant role in structure-based drug discovery. This methodology aims to estimate the binding mode and binding free energy between the drug-targeted protein and candidate chemical compounds, utilizing protein tertiary structure information. Reformulation of this docking as a quadratic unconstrained binary optimization (QUBO) problem to obtain solutions via quantum annealing has been attempted. However, previous studies did not consider the internal degrees of freedom of the compound that is mandatory and essential. In this study, we formulated fragment-based protein-ligand flexible docking, considering the internal degrees of freedom of the compound by focusing on fragments (rigid chemical substructures of compounds) as a QUBO problem. We introduced four factors essential for fragment-based docking in the Hamiltonian: (1) interaction energy between the target protein and each fragment, (2) clashes between fragments, (3) covalent bonds between fragments, and (4) the constraint that each fragment of the compound is selected for a single placement. We also implemented a proof-of-concept system and conducted redocking for the protein-compound complex structure of Aldose reductase (a drug target protein) using SQBM+, which is a simulated quantum annealer. The predicted binding pose reconstructed from the best solution was near-native (RMSD = 1.26 Å), which can be further improved (RMSD = 0.27 Å) using conventional energy minimization. The results indicate the validity of our QUBO problem formulation.

Accurate ligand-protein docking in CASP15 using the ClusPro LigTBM server.

In the ligand prediction category of CASP15, the challenge was to predict the positions and conformations of small molecules binding to proteins that were provided as amino acid sequences or as models generated by the AlphaFold2 program. For most targets, we used our template-based ligand docking program ClusPro ligTBM, also implemented as a public server available at https://ligtbm.cluspro.org/. Since many targets had multiple chains and a number of ligands, several templates, and some manual interventions were required. In a few cases, no templates were found, and we had to use direct docking using the Glide program. Nevertheless, ligTBM was shown to be a very useful tool, and by any ranking criteria, our group was ranked among the top five best-performing teams. In fact, all the best groups used template-based docking methods. Thus, it appears that the AlphaFold2-generated models, despite the high accuracy of the predicted backbone, have local differences from the x-ray structure that make the use of direct docking methods more challenging. The results of CASP15 confirm that this limitation can be frequently overcome by homology-based docking.

Conserved loop residues-Tyr270 and Asn372 near the catalytic site of the lysostaphin endopeptidase are essential for staphylolytic activity toward pentaglycine binding and catalysis.

Lysostaphin endopeptidase cleaves pentaglycine cross-bridges found in staphylococcal cell-wall peptidoglycans and proves very effective in combatting methicillin-resistant Staphylococcus aureus. Here, we revealed the functional importance of two loop residues, Tyr270 in loop 1 and Asn372 in loop 4, which are highly conserved among the M23 endopeptidase family and are found close to the Zn2+-coordinating active site. Detailed analyses of the binding groove architecture together with protein-ligand docking showed that these two loop residues potentially interact with the docked ligand-pentaglycine. Ala-substituted mutants (Y270A and N372A) were generated and over-expressed in Escherichia coli as a soluble form at levels comparable to the wild type. A drastic decrease in staphylolytic activity against S. aureus was observed for both mutants, suggesting an essential role of the two loop residues in lysostaphin function. Further substitutions with an uncharged polar Gln side-chain revealed that only the Y270Q mutation caused a dramatic reduction in bioactivity. In silico predicting the effect of binding site mutations revealed that all mutations displayed a large ΔΔGbind value, signifying requirements of the two loop residues for efficient binding to pentaglycine. Additionally, MD simulations revealed that Y270A and Y270Q mutations induced large flexibility of the loop 1 region, showing markedly increased RMSF values. Further structural analysis suggested that Tyr270 conceivably participated in the oxyanion stabilization of the enzyme catalysis. Altogether, our present study disclosed that two highly conserved loop residues, loop 1-Tyr270 and loop 4-Asn372, located near the lysostaphin active site are crucially involved in staphylolytic activity toward binding and catalysis of pentaglycine cross-links.

GalaxyDock2-HEME: Protein-ligand docking for heme proteins.

Prediction of protein-ligand binding poses is an essential component for understanding protein-ligand interactions and computer-aided drug design. Various proteins involve prosthetic groups such as heme for their functions, and adequate consideration of the prosthetic groups is vital for protein-ligand docking. Here, we extend the GalaxyDock2 protein-ligand docking algorithm to handle ligand docking to heme proteins. Docking to heme proteins involves increased complexity because the interaction of heme iron and ligand has covalent nature. GalaxyDock2-HEME, a new protein-ligand docking program for heme proteins, has been developed based on GalaxyDock2 by adding an orientation-dependent scoring term to describe heme iron-ligand coordination interaction. This new docking program performs better than other noncommercial docking programs such as EADock with MMBP, AutoDock Vina, PLANTS, LeDock, and GalaxyDock2 on a heme protein-ligand docking benchmark set in which ligands are known to bind iron. In addition, docking results on two other sets of heme protein-ligand complexes in which ligands do not bind iron show that GalaxyDock2-HEME does not have a high bias toward iron binding compared to other docking programs. This implies that the new docking program can distinguish iron binders from noniron binders for heme proteins.

Benchmarking applicability of medium-resolution cryo-EM protein structures for structure-based drug design.

Cryo-electron microscopy (cryo-EM) is gaining large attention for high-resolution protein structure determination in solutions. However, a very high percentage of cryo-EM structures correspond to resolutions of 3-5 Å, making the structures difficult to be used in in silico drug design. In this study, we analyze how useful cryo-EM protein structures are for in silico drug design by evaluating ligand docking accuracy. From realistic cross-docking scenarios using medium resolution (3-5 Å) cryo-EM structures and a popular docking tool Autodock-Vina, only 20% of docking succeeded, when the success rate doubles in the same kind of cross-docking but using high-resolution (<2 Å) crystal structures instead. We decipher the reason for failures by decomposing the contribution from resolution-dependent and independent factors. The heterogeneity in the protein side-chain and backbone conformations is identified as the major resolution-dependent factor causing docking difficulty from our analysis, while intrinsic receptor flexibility mainly comprises the resolution-independent factor. We demonstrate the flexibility implementation in current ligand docking tools is able to rescue only a portion of failures (10%), and the limited performance was majorly due to potential structural errors than conformational changes. Our work suggests the strong necessity of more robust method developments on ligand docking and EM modeling techniques in order to fully utilize cryo-EM structures for in silico drug design.

Identification of N-Acyl Hydrazones as New Non-Zinc-Binding MMP-13 Inhibitors by Structure-Based Virtual Screening Studies and Chemical Optimization.

Matrix metalloproteinase 13 plays a central role in osteoarthritis (OA), as its overexpression induces an excessive breakdown of collagen that results in an imbalance between collagen synthesis and degradation in the joint, leading to progressive articular cartilage degradation. Therefore, MMP-13 has been proposed as a key therapeutic target for OA. Here we have developed a virtual screening workflow aimed at identifying selective non-zinc-binding MMP-13 inhibitors by targeting the deep S1' pocket of MMP-13. Three ligands were found to inhibit MMP-13 in the µM range, and one of these showed selectivity over other MMPs. A structure-based analysis guided the chemical optimization of the hit compound, leading to the obtaining of a new N-acyl hydrazone-based derivative with improved inhibitory activity and selectivity for the target enzyme.

Recent Advances in Computer-Aided Structure-Based Drug Design on Ion Channels.

Ion channels play important roles in fundamental biological processes, such as electric signaling in cells, muscle contraction, hormone secretion, and regulation of the immune response. Targeting ion channels with drugs represents a treatment option for neurological and cardiovascular diseases, muscular degradation disorders, and pathologies related to disturbed pain sensation. While there are more than 300 different ion channels in the human organism, drugs have been developed only for some of them and currently available drugs lack selectivity. Computational approaches are an indispensable tool for drug discovery and can speed up, especially, the early development stages of lead identification and optimization. The number of molecular structures of ion channels has considerably increased over the last ten years, providing new opportunities for structure-based drug development. This review summarizes important knowledge about ion channel classification, structure, mechanisms, and pathology with the main focus on recent developments in the field of computer-aided, structure-based drug design on ion channels. We highlight studies that link structural data with modeling and chemoinformatic approaches for the identification and characterization of new molecules targeting ion channels. These approaches hold great potential to advance research on ion channel drugs in the future.

Improving the Modeling of Extracellular Ligand Binding Pockets in RosettaGPCR for Conformational Selection.

G protein-coupled receptors (GPCRs) are the largest class of drug targets and undergo substantial conformational changes in response to ligand binding. Despite recent progress in GPCR structure determination, static snapshots fail to reflect the conformational space of putative binding pocket geometries to which small molecule ligands can bind. In comparative modeling of GPCRs in the absence of a ligand, often a shrinking of the orthosteric binding pocket is observed. However, the exact prediction of the flexible orthosteric binding site is crucial for adequate structure-based drug discovery. In order to improve ligand docking and guide virtual screening experiments in computer-aided drug discovery, we developed RosettaGPCRPocketSize. The algorithm creates a conformational ensemble of biophysically realistic conformations of the GPCR binding pocket between the TM bundle, which is consistent with a knowledge base of expected pocket geometries. Specifically, tetrahedral volume restraints are defined based on information about critical residues in the orthosteric binding site and their experimentally observed range of Cα-Cα-distances. The output of RosettaGPCRPocketSize is an ensemble of binding pocket geometries that are filtered by energy to ensure biophysically probable arrangements, which can be used for docking simulations. In a benchmark set, pocket shrinkage observed in the default RosettaGPCR was reduced by up to 80% and the binding pocket volume range and geometric diversity were increased. Compared to models from four different GPCR homology model databases (RosettaGPCR, GPCR-Tasser, GPCR-SSFE, and GPCRdb), the here-created models showed more accurate volumes of the orthosteric pocket when evaluated with respect to the crystallographic reference structure. Furthermore, RosettaGPCRPocketSize was able to generate an improved realistic pocket distribution. However, while being superior to other homology models, the accuracy of generated model pockets was comparable to AlphaFold2 models. Furthermore, in a docking benchmark using small-molecule ligands with a higher molecular weight between 400 and 700 Da, a higher success rate in creating native-like binding poses was observed. In summary, RosettaGPCRPocketSize can generate GPCR models with realistic orthosteric pocket volumes, which are useful for structure-based drug discovery applications.

Parallel multi-swarm cooperative particle swarm optimization for protein-ligand docking and virtual screening.

BACKGROUND: A high-quality docking method tends to yield multifold gains with half pains for the new drug development. Over the past few decades, great efforts have been made for the development of novel docking programs with great efficiency and intriguing accuracy. AutoDock Vina (Vina) is one of these achievements with improved speed and accuracy compared to AutoDock4. Since it was proposed, some of its variants, such as PSOVina and GWOVina, have also been developed. However, for all these docking programs, there is still large room for performance improvement. RESULTS: In this work, we propose a parallel multi-swarm cooperative particle swarm model, in which one master swarm and several slave swarms mutually cooperate and co-evolve. Our experiments show that multi-swarm programs possess better docking robustness than PSOVina. Moreover, the multi-swarm program based on random drift PSO can achieve the best highest accuracy of protein-ligand docking, an outstanding enrichment effect for drug-like activate compounds, and the second best AUC screening accuracy among all the compared docking programs, but with less computation consumption than most of the other docking programs. CONCLUSION: The proposed multi-swarm cooperative model is a novel algorithmic modeling suitable for protein-ligand docking and virtual screening. Owing to the existing coevolution between the master and the slave swarms, this model in parallel generates remarkable docking performance. The source code can be freely downloaded from https://github.com/li-jin-xing/MPSOVina .

A reinforcement learning approach for protein-ligand binding pose prediction.

Protein ligand docking is an indispensable tool for computational prediction of protein functions and screening drug candidates. Despite significant progress over the past two decades, it is still a challenging problem, characterized by the still limited understanding of the energetics between proteins and ligands, and the vast conformational space that has to be searched to find a satisfactory solution. In this project, we developed a novel reinforcement learning (RL) approach, the asynchronous advantage actor-critic model (A3C), to address the protein ligand docking problem. The overall framework consists of two models. During the search process, the agent takes an action selected by the actor model based on the current location. The critic model then evaluates this action and predict the distance between the current location and true binding site. Experimental results showed that in both single- and multi-atom cases, our model improves binding site prediction substantially compared to a naïve model. For the single-atom ligand, copper ion (Cu2+), the model predicted binding sites have a median root-mean-square-deviation (RMSD) of 2.39 Å to the true binding sites when starting from random starting locations. For the multi-atom ligand, sulfate ion (SO42-), the predicted binding sites have a median RMSD of 3.82 Å to the true binding sites. The ligand-specific models built in this study can be used in solvent mapping studies and the RL framework can be readily scaled up to larger and more diverse sets of ligands.

RDPSOVina: the random drift particle swarm optimization for protein-ligand docking.

Protein-ligand docking is of great importance to drug design, since it can predict the binding affinity between ligand and protein, and guide the synthesis direction of the lead compounds. Over the past few decades, various docking programs have been developed, some of them employing novel optimization algorithms. However, most of those methods cannot simultaneously achieve both good efficiency and accuracy. Therefore, it is worthwhile to pour the efforts into the development of a docking program with fast speed and high quality of the solutions obtained. The research presented in this paper, based on the docking scheme of Vina, developed a novel docking program called RDPSOVina. The RDPSOVina employes a novel search algorithm but the same scoring function of Vina. It utilizes the random drift particle swarm optimization (RDPSO) algorithm as the global search algorithm, implements the local search with small probability, and applies Markov chain mutation to the particles' personal best positions in order to harvest more potential-candidates. To prove the outstanding docking performance in RDPSOVina, we performed the re-docking experiments on two PDBbind datasets and cross-docking experiments on the Sutherland-crossdock-set, respectively. The RDPSOVina exhibited superior protein-ligand docking accuracy and better cross-docking prediction with higher operation efficiency than most of the compared methods. It is available at https://github.com/li-jin-xing/RDPSOVina .

Tribenuron-methyl metabolism and the rare Pro197Phe double mutation together with 2,4-D metabolism and reduced absorption can evolve in Papaver rhoeas with multiple and cross herbicide resistance to ALS inhibitors and auxin mimics.

Multiple resistance mechanisms to ALS inhibitors and auxin mimics in two Papaver rhoeas populations were investigated in wheat fields from Portugal. Dose-response trials, also with malathion (a cytochrome P450 inhibitor), cross-resistance patterns for ALS inhibitors and auxin mimics, alternative herbicides tests, 2,4-D and tribenuron-methyl absorption, translocation and metabolism experiments, together with ALS activity, gene sequencing and enzyme modelling and ligand docking were carried out. Results revealed two different resistant profiles: one population (R1) multiple resistant to tribenuron-methyl and 2,4-D, the second (R2) only resistant to 2,4-D. In R1, several target-site mutations in Pro197 and enhanced metabolism (cytochrome P450-mediated) were responsible of tribenuron-methyl resistance. For 2,4-D, reduced transport was observed in both populations, while cytochrome P450-mediated metabolism was also present in R1 population. Moreover, this is the first P. rhoeas population with enhanced tribenuron-methyl metabolism. This study reports the first case for P. rhoeas of the amino acid substitution Pro197Phe due to a double nucleotide change. This double mutation could cause reduced enzyme sensitivity to most ALS inhibitors according to protein modelling and ligand docking. In addition, this study reports a P. rhoeas population resistant to 2,4-D, apparently, with reduced transport as the sole resistance mechanism.