Target-ligand binding affinity from single point enthalpy calculation and elemental composition.

Reliable target-ligand binding thermodynamics data are essential for successful drug design and molecular engineering projects. Besides experimental methods, a number of theoretical approaches have been introduced for the generation of binding thermodynamics data. However, available approaches often neglect electronic effects or explicit water molecules influencing target-ligand interactions. To handle electronic effects within a reasonable time frame, we introduce a fast calculator QMH-L using a single target-ligand complex structure pre-optimized at the molecular mechanics level. QMH-L is composed of the semi-empirical quantum mechanics calculation of binding enthalpy with predicted explicit water molecules at the complex interface, and a simple descriptor based on the elemental composition of the ligand. QMH-L estimates the target-ligand binding free energy with a root mean square error (RMSE) of 0.94 kcal mol-1. The calculations also provide binding enthalpy values and they were compared with experimental binding thermodynamics data collected from the most reliable isothermal titration calorimetry studies of systems including various protein targets and challenging, large peptide ligands with a molecular weight of up to 2-3 thousand. The single point enthalpy calculations of QMH-L require modest computational resources and are based on short runs with open source and/or free software like Gromacs, Mopac, MobyWat, and Fragmenter. QMH-L can be applied for fast, automated scoring of drug candidates during a virtual screen, enthalpic engineering of new ligands or thermodynamic explanation of complex interactions.

Molecular imaging of bacterial outer membrane vesicles based on bacterial surface display.

The important roles of bacterial outer membrane vesicles (OMVs) in various diseases and their emergence as a promising platform for vaccine development and targeted drug delivery necessitates the development of imaging techniques suitable for quantifying their biodistribution with high precision. To address this requirement, we aimed to develop an OMV specific radiolabeling technique for positron emission tomography (PET). A novel bacterial strain (E. coli BL21(DE3) ΔnlpI, ΔlpxM) was created for efficient OMV production, and OMVs were characterized using various methods. SpyCatcher was anchored to the OMV outer membrane using autotransporter-based surface display systems. Synthetic SpyTag-NODAGA conjugates were tested for OMV surface binding and 64Cu labeling efficiency. The final labeling protocol shows a radiochemical purity of 100% with a ~ 29% radiolabeling efficiency and excellent serum stability. The in vivo biodistribution of OMVs labeled with 64Cu was determined in mice using PET/MRI imaging which revealed that the biodistribution of radiolabeled OMVs in mice is characteristic of previously reported data with the highest organ uptakes corresponding to the liver and spleen 3, 6, and 12 h following intravenous administration. This novel method can serve as a basis for a general OMV radiolabeling scheme and could be used in vaccine- and drug-carrier development based on bioengineered OMVs.

Construction of Histone-Protein Complex Structures by Peptide Growing.

The structures of histone complexes are master keys to epigenetics. Linear histone peptide tails often bind to shallow pockets of reader proteins via weak interactions, rendering their structure determination challenging. In the present study, a new protocol, PepGrow, is introduced. PepGrow uses docked histone fragments as seeds and grows the full peptide tails in the reader-binding pocket, producing atomic-resolution structures of histone-reader complexes. PepGrow is able to handle the flexibility of histone peptides, and it is demonstrated to be more efficient than linking pre-docked peptide fragments. The new protocol combines the advantages of popular program packages and allows fast generation of solution structures. AutoDock, a force-field-based program, is used to supply the docked peptide fragments used as structural seeds, and the building algorithm of Modeller is adopted and tested as a peptide growing engine. The performance of PepGrow is compared to ten other docking methods, and it is concluded that in situ growing of a ligand from a seed is a viable strategy for the production of complex structures of histone peptides at atomic resolution.

The Advances and Limitations of the Determination and Applications of Water Structure in Molecular Engineering.

Water is a key actor of various processes of nature and, therefore, molecular engineering has to take the structural and energetic consequences of hydration into account. While the present review focuses on the target-ligand interactions in drug design, with a focus on biomolecules, these methods and applications can be easily adapted to other fields of the molecular engineering of molecular complexes, including solid hydrates. The review starts with the problems and solutions of the determination of water structures. The experimental approaches and theoretical calculations are summarized, including conceptual classifications. The implementations and applications of water models are featured for the calculation of the binding thermodynamics and computational ligand docking. It is concluded that theoretical approaches not only reproduce or complete experimental water structures, but also provide key information on the contribution of individual water molecules and are indispensable tools in molecular engineering.

Extent of intraspecific trait variability in ecologically central and marginal populations of a dominant alpine plant across European mountains.

BACKGROUND AND AIMS: Studying trait variability and restricted gene flow between populations of species can reveal species dynamics. Peripheral populations commonly exhibit lower genetic diversity and trait variability due to isolation and ecological marginality, unlike central populations experiencing gene flow and optimal conditions. This study focused on Carex curvula, the dominant species in alpine acidic meadows of European mountain regions. The species is sparser in dry areas such as the Pyrenees and Balkans, compared to the Central-Eastern Alps and Carpathians. We hypothesized that distinct population groups could be identified based on their mean functional trait values and their correlation with the environment; we predicted that ecologically marginal populations would have stronger trait correlations, lower within-population trait variability (intraspecific trait variability, ITV) and lower genetic diversity than populations of optimal habitats. METHODS: Sampling was conducted in 34 populations that spanned the entire distribution range of C. curvula. We used hierarchical clustering to identify emergent functional groups of populations, defined by combinations of multiple traits associated with nutrient economy and drought tolerance (e.g. specific leaf area, anatomy). We contrasted the geographical distribution of these groups in relation to environment and genetic structure. We compared pairwise trait relationships, within-population trait variation (ITV) and neutral genetic diversity between groups. KEY RESULTS: Our study identified emergent functional groups of populations. Those in the southernmost ranges, specifically the Pyrenees and Balkan region, showed drought-tolerant trait syndromes and correlated with indicators of limited water availability. While we noted a decline in population genetic diversity, we did not observe any significant changes in ITV in ecologically marginal (peripheral) populations. CONCLUSIONS: Our research exemplifies the relationship between ecological marginality and geographical peripherality, which in this case study is linked to genetic depauperation but not to reduced ITV. Understanding these relationships is crucial for understanding the biogeographical factors shaping trait variation.

Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor.

Introduction: Previous studies have established that endogenous inorganic polysulfides have significant biological actions activating the Transient Receptor Potential Ankyrin 1 (TRPA1) receptor. Organic polysulfides exert similar effects, but they are much more stable molecules, therefore these compounds are more suitable as drugs. In this study, we aimed to better understand the mechanism of action of organic polysulfides by identification of their binding site on the TRPA1 receptor. Methods: Polysulfides can readily interact with the thiol side chain of the cysteine residues of the protein. To investigate their role in the TRPA1 activation, we replaced several cysteine residues by alanine via site-directed mutagenesis. We searched for TRPA1 mutant variants with decreased or lost activating effect of the polysulfides, but with other functions remaining intact (such as the effects of non-electrophilic agonists and antagonists). The binding properties of the mutant receptors were analyzed by in silico molecular docking. Functional changes were tested by in vitro methods: calcium sensitive fluorescent flow cytometry, whole-cell patch-clamp and radioactive calcium-45 liquid scintillation counting. Results: The cysteines forming the conventional binding site of electrophilic agonists, namely C621, C641 and C665 also bind the organic polysulfides, with the key role of C621. However, only their combined mutation abolished completely the organic polysulfide-induced activation of the receptor. Discussion: Since previous papers provided evidence that organic polysulfides exert analgesic and anti-inflammatory actions in different in vivo animal models, we anticipate that the development of TRPA1-targeted, organic polysulfide-based drugs will be promoted by this identification of the binding site.

Interaction of Fumonisin B1, N-Palmitoyl-Fumonisin B1, 5-O-Palmitoyl-Fumonisin B1, and Fumonisin B4 Mycotoxins with Human Serum Albumin and Their Toxic Impacts on Zebrafish Embryos.

Fumonisins are frequent food contaminants. The high exposure to fumonisins can cause harmful effects in humans and animals. Fumonisin B1 (FB1) is the most typical member of this group; however, the occurrence of several other derivatives has been reported. Acylated metabolites of FB1 have also been described as possible food contaminants, and the very limited data available suggest their significantly higher toxicity compared to FB1. Furthermore, the physicochemical and toxicokinetic properties (e.g., albumin binding) of acyl-FB1 derivatives may show large differences compared to the parent mycotoxin. Therefore, we tested the interactions of FB1, N-palmitoyl-FB1 (N-pal-FB1), 5-O-palmitoyl-FB1 (5-O-pal-FB1), and fumonisin B4 (FB4) with human serum albumin as well as the toxic effects of these mycotoxins on zebrafish embryos were examined. Based on our results, the most important observations and conclusions are the following: (1) FB1 and FB4 bind to albumin with low affinity, while palmitoyl-FB1 derivatives form highly stable complexes with the protein. (2) N-pal-FB1 and 5-O-pal-FB1 likely occupy more high-affinity binding sites on albumin. (3) Among the mycotoxins tested, N-pal-FB1 showed the most toxic effects on zebrafish, followed by 5-O-pal-FB1, FB4, and FB1. (4) Our study provides the first in vivo toxicity data regarding N-pal-FB1, 5-O-pal-FB1, and FB4.

Probing the Interactions of 31 Mycotoxins with Xanthine Oxidase: Alternariol, Alternariol-3-Sulfate, and α-Zearalenol Are Allosteric Inhibitors of the Enzyme.

Mycotoxins are frequent toxic contaminants in foods and beverages, causing a significant health threat. Interactions of mycotoxins with biotransformation enzymes (e.g., cytochrome P450 enzymes, sulfotransferases, and uridine 5'-diphospho-glucuronosyltransferases) may be important due to their possible detoxification or toxic activation during enzymatic processes. Furthermore, mycotoxin-induced enzyme inhibition may affect the biotransformation of other molecules. A recent study described the strong inhibitory effects of alternariol and alternariol-9-methylether on the xanthine oxidase (XO) enzyme. Therefore, we aimed to test the impacts of 31 mycotoxins (including the masked/modified derivatives of alternariol and alternariol-9-methylether) on XO-catalyzed uric acid formation. Besides the in vitro enzyme incubation assays, mycotoxin depletion experiments and modeling studies were performed. Among the mycotoxins tested, alternariol, alternariol-3-sulfate, and α-zearalenol showed moderate inhibitory actions on the enzyme, representing more than tenfold weaker impacts compared with the positive control inhibitor allopurinol. In mycotoxin depletion assays, XO did not affect the concentrations of alternariol, alternariol-3-sulfate, and α-zearalenol in the incubates; thus, these compounds are inhibitors but not substrates of the enzyme. Experimental data and modeling studies suggest the reversible, allosteric inhibition of XO by these three mycotoxins. Our results help the better understanding of the toxicokinetic interactions of mycotoxins.

Development of SSR markers for Carex curvula (Cyperaceae) and their importance in investigating the species genetic structure.

BACKGROUND: Microsatellite primers were developed and tested to genotype several populations of Carex curvula s. l. (Cyperaceae), in order to infer the phylogeographic relationships of the populations within species and the boundaries between the two described subspecies: C. curvula subsp. curvula and C. curvula subsp. rosae. METHODS AND RESULTS: Candidate microsatellite loci were isolated based on next-generation sequencing. We tested 18 markers for polymorphism and replicability in seven C. curvula s. l. populations and identified 13 polymorphic loci with dinucleotide repeats. Genotyping results showed the total number of alleles per locus varied from four to 23 (including both infrataxa), and the observed and expected heterozygosity ranged between 0.1 to 0.82 and 0.219 to 0.711, respectively. Furthermore, the NJ tree showed a clear separation between C. curvula subsp. curvula and C. curvula subsp. rosae. CONCLUSION: The development of these highly polymorphic markers proved to be very efficient not only in delineating between the two subspecies, but also in genetic discriminating at population level within each infrataxon. They are promising tools for evolutionary studies in Cariceae section, as well as in providing knowledge on patterns of the species phylogeography.

Testing Serum Albumins and Cyclodextrins as Potential Binders of the Mycotoxin Metabolites Alternariol-3-Sulfate, Alternariol-9-Monomethylether and Alternariol-9-Monomethylether-3-Sulfate.

Alternaria mycotoxins, including alternariol (AOH), alternariol-9-monomethylether (AME), and their masked/modified derivatives (e.g., sulfates or glycosides), are common food contaminants. Their acute toxicity is relatively low, while chronic exposure can lead to the development of adverse health effects. Masked/modified metabolites can probably release the more toxic parent mycotoxin due to their enzymatic hydrolysis in the intestines. Previously, we demonstrated the complex formation of AOH with serum albumins and cyclodextrins; these interactions were successfully applied for the extraction of AOH from aqueous matrices (including beverages). Therefore, in this study, the interactions of AME, alternariol-3-sulfate (AS), and alternariol-9-monomethylether-3-sulfate (AMS) were investigated with albumins (human, bovine, porcine, and rat) and with cyclodextrins (sulfobutylether-ß-cyclodextrin, sugammadex, and cyclodextrin bead polymers). Our major results/conclusions are the following: (1) The stability of mycotoxin-albumin complexes showed only minor species dependent variations. (2) AS and AMS formed highly stable complexes with albumins in a wide pH range, while AME-albumin interactions preferred alkaline conditions. (3) AME formed more stable complexes with the cyclodextrins examined than AS and AMS. (4) Beta-cyclodextrin bead polymer proved to be highly suitable for the extraction of AME, AS, and AMS from aqueous solution. (5) Albumins and cyclodextrins are promising binders of the mycotoxins tested.

The Structural Effects of Phosphorylation of Protein Arginine Methyltransferase 5 on Its Binding to Histone H4.

The protein arginine methyltransferase 5 (PRMT5) enzyme is responsible for arginine methylation on various proteins, including histone H4. PRMT5 is a promising drug target, playing a role in the pathomechanism of several diseases, especially in the progression of certain types of cancer. It was recently proved that the phosphorylation of PRMT5 on T80 residue increases its methyltransferase activity; furthermore, elevated levels of the enzyme were measured in the case of human hepatocellular carcinoma and other types of tumours. In this study, we constructed the complexes of the unmodified human PRMT5-methylosome protein 50 (MEP50) structure and its T80-phosphorylated variant in complex with the full-length histone H4 peptide. The full-length histone H4 was built in situ into the human PRMT5-MEP50 enzyme using experimental H4 fragments. Extensive molecular dynamic simulations and structure and energy analyses were performed for the complexed and apo protein partners, as well. Our results provided an atomic level explanation for two important experimental findings: (1) the increased methyltransferase activity of the phosphorylated PRMT5 when compared to the unmodified type; (2) the PRMT5 methylates only the free form of histone H4 not bound in the nucleosome. The atomic level complex structure H4-PRMT5-MEP50 will help the design of new inhibitors and in uncovering further structure-function relationships of PRMT enzymes.

Exploration of Somatostatin Binding Mechanism to Somatostatin Receptor Subtype 4.

Somatostatin (also named as growth hormone-inhibiting hormone or somatotropin release-inhibiting factor) is a regulatory peptide important for the proper functioning of the endocrine system, local inflammatory reactions, mood and motor coordination, and behavioral responses to stress. Somatostatin exerts its effects via binding to G-protein-coupled somatostatin receptors of which the fourth subtype (SSTR4) is a particularly important receptor mediating analgesic, anti-inflammatory, and anti-depressant effects without endocrine actions. Thus, SSTR4 agonists are promising drug candidates. Although the knowledge of the atomic resolution-binding modes of SST would be essential for drug development, experimental elucidation of the structures of SSTR4 and its complexes is still awaiting. In the present study, structures of the somatostatin-SSTR4 complex were produced using an unbiased, blind docking approach. Beyond the static structures, the binding mechanism of SST was also elucidated in the explicit water molecular dynamics (MD) calculations, and key binding modes (external, intermediate, and internal) were distinguished. The most important residues on both receptor and SST sides were identified. An energetic comparison of SST binding to SSTR4 and 2 offered a residue-level explanation of receptor subtype selectivity. The calculated structures show good agreement with available experimental results and indicate that somatostatin binding is realized via prerequisite binding modes and an induced fit mechanism. The identified binding modes and the corresponding key residues provide useful information for future drug design targeting SSTR4.

Binding Networks Identify Targetable Protein Pockets for Mechanism-Based Drug Design.

The human genome codes only a few thousand druggable proteins, mainly receptors and enzymes. While this pool of available drug targets is limited, there is an untapped potential for discovering new drug-binding mechanisms and modes. For example, enzymes with long binding cavities offer numerous prerequisite binding sites that may be visited by an inhibitor during migration from a bulk solution to the destination site. Drug design can use these prerequisite sites as new structural targets. However, identifying these ephemeral sites is challenging. Here, we introduce a new method called NetBinder for the systematic identification and classification of prerequisite binding sites at atomic resolution. NetBinder is based on atomistic simulations of the full inhibitor binding process and provides a networking framework on which to select the most important binding modes and uncover the entire binding mechanism, including previously undiscovered events. NetBinder was validated by a study of the binding mechanism of blebbistatin (a potent inhibitor) to myosin 2 (a promising target for cancer chemotherapy). Myosin 2 is a good test enzyme because, like other potential targets, it has a long internal binding cavity that provides blebbistatin with numerous potential prerequisite binding sites. The mechanism proposed by NetBinder of myosin 2 structural changes during blebbistatin binding shows excellent agreement with experimentally determined binding sites and structural changes. While NetBinder was tested on myosin 2, it may easily be adopted to other proteins with long internal cavities, such as G-protein-coupled receptors or ion channels, the most popular current drug targets. NetBinder provides a new paradigm for drug design by a network-based elucidation of binding mechanisms at an atomic resolution.

Prerequisite Binding Modes Determine the Dynamics of Action of Covalent Agonists of Ion Channel TRPA1.

Transient receptor potential ankyrin 1 (TRPA1) is a transmembrane protein channeling the influx of calcium ions. As a polymodal nocisensor, TRPA1 can be activated by thermal, mechanical stimuli and a wide range of chemically damaging molecules including small volatile environmental toxicants and endogenous algogenic lipids. After activation by such compounds, the ion channel opens up, its central pore widens allowing calcium influx into the cytosol inducing signal transduction pathways. Afterwards, the calcium influx desensitizes irritant evoked responses and results in an inactive state of the ion channel. Recent experimental determination of structures of apo and holo forms of TRPA1 opened the way towards the design of new agonists, which can activate the ion channel. The present study is aimed at the elucidation of binding dynamics of agonists using experimental structures of TRPA1-agonist complexes at the atomic level applying molecular docking and dynamics methods accounting for covalent and non-covalent interactions. Following a test of docking methods focused on the final, holo structures, prerequisite binding modes were detected involving the apo forms. It was shown how reversible interactions with prerequisite binding sites contribute to structural changes of TRPA1 leading to covalent bonding of agonists. The proposed dynamics of action allowed a mechanism-based forecast of new, druggable binding sites of potent agonists.

Determination of Ligand Binding Modes in Hydrated Viral Ion Channels to Foster Drug Design and Repositioning.

Target-based design and repositioning are mainstream strategies of drug discovery. Numerous drug design and repositioning projects have been launched to fight the ongoing COVID-19 pandemic. The resulting drug candidates have often failed due to the misprediction of their target-bound structures. The determination of water positions of such structures is particularly challenging due to the large number of possible drugs and the diversity of their hydration patterns. To answer this challenge and help correct predictions, we introduce a new protocol HydroDock, which can build hydrated drug-target complexes from scratch. HydroDock requires only the dry target and drug structures and produces their complexes with appropriately positioned water molecules. As a test application of the protocol, we built the structures of amantadine derivatives in complex with the influenza M2 transmembrane ion channel. The repositioning of amantadine derivatives from this influenza target to the SARS-CoV-2 envelope protein was also investigated. Excellent agreement was observed between experiments and the structures determined by HydroDock. The atomic resolution complex structures showed that water plays a similar role in the binding of amphipathic amantadine derivatives to transmembrane ion channels of both influenza A and SARS-CoV-2. While the hydrophobic regions of the channels capture the bulky hydrocarbon group of the ligand, the surrounding waters direct its orientation parallel with the axes of the channels via bridging interactions with the ionic ligand head. As HydroDock supplied otherwise undetermined structural details, it can be recommended to improve the reliability of future design and repositioning of antiviral drug candidates and many other ligands with an influence of water structure on their mechanism of action.

Safety assessment of a novel thermostable phytase.

A novel 6-phytase (Phytase TSP, trade name OptiPhos® PLUS) with improved thermostability has been developed for use in animal feed. The safety of the new phytase was evaluated by testing for genotoxicity and subchronic toxicity. In in vitro and in vivo genotoxicity assays Phytase TSP concentrate was not mutagenic and did not induce biologically or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes. In a subchronic toxicity study, male and female rats administered 100, 500 or 1000 mg/kg body weight/day of Phytase TSP concentrate via oral gavage for 90 days had no mortalities, and no treatment-related effects on body weight, food consumption, clinical observations or ophthalmology. Furthermore, there were no changes in haematology, clinical chemistry, urinalysis, gross pathology, organ weights or histopathology that could be attributed to the test article. Several endpoints exhibited statistically significant effects, but none was dose-related or considered to be of toxicological relevance. Based on these results, Phytase TSP concentrate (OptiPhos® PLUS) was not genotoxic and the No Observed Adverse Effect Level (NOAEL) for male and female rats was 1000 mg/kg body weight/day.

Effects of Chrysin and Its Major Conjugated Metabolites Chrysin-7-Sulfate and Chrysin-7-Glucuronide on Cytochrome P450 Enzymes and on OATP, P-gp, BCRP, and MRP2 Transporters.

Chrysin is an abundant flavonoid in nature, and it is also contained by several dietary supplements. Chrysin is highly biotransformed in the body, during which conjugated metabolites chrysin-7-sulfate and chrysin-7-glucuronide are formed. These conjugates appear at considerably higher concentrations in the circulation than the parent compound. Based on previous studies, chrysin can interact with biotransformation enzymes and transporters; however, the interactions of its metabolites have been barely examined. In this in vitro study, the effects of chrysin, chrysin-7-sulfate, and chrysin-7-glucuronide on cytochrome P450 enzymes (2C9, 2C19, 3A4, and 2D6) as well as on organic anion-transporting polypeptides (OATPs; 1A2, 1B1, 1B3, and 2B1) and ATP binding cassette [P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance protein (BCRP)] transporters were investigated. Our observations revealed that chrysin conjugates are strong inhibitors of certain biotransformation enzymes (e.g., CYP2C9) and transporters (e.g., OATP1B1, OATP1B3, OATP2B1, and BCRP) examined. Therefore, the simultaneous administration of chrysin-containing dietary supplements with medications needs to be carefully considered due to the possible development of pharmacokinetic interactions. SIGNIFICANCE STATEMENT: Chrysin-7-sulfate and chrysin-7-glucuronide are the major metabolites of flavonoid chrysin. In this study, we examined the effects of chrysin and its conjugates on cytochrome P450 enzymes and on organic anion-transporting polypeptides and ATP binding cassette transporters (P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2). Our results demonstrate that chrysin and/or its conjugates can significantly inhibit some of these proteins. Since chrysin is also contained by dietary supplements, high intake of chrysin may interrupt the transport and/or the biotransformation of drugs.

Molecular Structure, Binding Affinity, and Biological Activity in the Epigenome.

Development of valid structure-activity relationships (SARs) is a key to the elucidation of pathomechanisms of epigenetic diseases and the development of efficient, new drugs. The present review is based on selected methodologies and applications supplying molecular structure, binding affinity and biological activity data for the development of new SARs. An emphasis is placed on emerging trends and permanent challenges of new discoveries of SARs in the context of proteins as epigenetic drug targets. The review gives a brief overview and classification of the molecular background of epigenetic changes, and surveys both experimental and theoretical approaches in the field. Besides the results of sophisticated, cutting edge techniques such as cryo-electron microscopy, protein crystallography, and isothermal titration calorimetry, examples of frequently used assays and fast screening techniques are also selected. The review features how different experimental methods and theoretical approaches complement each other and result in valid SARs of the epigenome.

Undefeated-Changing the phenamacril scaffold is not enough to beat resistant Fusarium.

Filamentous fungi belonging to the genus Fusarium are notorious plant-pathogens that infect, damage and contaminate a wide variety of important crops. Phenamacril is the first member of a novel class of single-site acting cyanoacrylate fungicides which has proven highly effective against important members of the genus Fusarium. However, the recent emergence of field-resistant strains exhibiting qualitative resistance poses a major obstacle for the continued use of phenamacril. In this study, we synthesized novel cyanoacrylate compounds based on the phenamacril-scaffold to test their growth-inhibitory potential against wild-type Fusarium and phenamacril-resistant strains. Our findings show that most chemical modifications to the phenamacril-scaffold are associated with almost complete loss of fungicidal activity and in vitro inhibition of myosin motor domain ATPase activity.