Nucleotide substitutions at the p.Gly117 and p.Thr180 mutational hot-spots of SKI alter molecular dynamics and may affect cell cycle.

Heterozygous deleterious variants in SKI cause Shprintzen-Goldberg Syndrome, which is mainly characterized by craniofacial features, neurodevelopmental disorder and thoracic aorta dilatations/aneurysms. The encoded protein is a member of the transforming growth factor beta signaling. Paucity of reported studies exploring the SGS molecular pathogenesis hampers disease recognition and clinical interpretation of private variants. Here, the unpublished c.349G>A, p.[Gly117Ser] and the recurrent c.539C>T, p.[Thr180Met] SKI variants were studied combining in silico and in vitro approach. 3D comparative modeling and calculation of the interaction energy predicted that both variants alter the SKI tertiary protein structure and its interactions. Computational data were functionally corroborated by the demonstration of an increase of MAPK phosphorylation levels and alteration of cell cycle in cells expressing the mutant SKI. Our findings confirmed the effects of SKI variants on MAPK and opened the path to study the role of perturbations of the cell cycle in SGS.

TNFRSF13B gene mutation in familial acute myeloid leukemia: A new piece in the complex scenario of hereditary predisposition?

TNFRSF13B mutations are widely associated with common variable immunodeficiency. TNFRSF13B was recently counted among relevant genes associated with childhood-onset of hematological malignancies; nonetheless, its role in acute myeloid leukemia (AML) remains unexplored. We report the study of a family with two cases of AML, sharing a germline TNFRSF13B mutation favoring the formation of a more stable complex with its ligand TNFSF13: a positive regulator of AML-initiating cells. Our data turn the spotlight onto the TNFRSF13B role in AML onset, inserting a new fragment into the complex scenario of a hereditary predisposition to myeloid neoplasms.

Pyruvate dehydrogenase operates as an intramolecular nitroxyl generator during macrophage metabolic reprogramming.

M1 macrophages enter a glycolytic state when endogenous nitric oxide (NO) reprograms mitochondrial metabolism by limiting aconitase 2 and pyruvate dehydrogenase (PDH) activity. Here, we provide evidence that NO targets the PDH complex by using lipoate to generate nitroxyl (HNO). PDH E2-associated lipoate is modified in NO-rich macrophages while the PDH E3 enzyme, also known as dihydrolipoamide dehydrogenase (DLD), is irreversibly inhibited. Mechanistically, we show that lipoate facilitates NO-mediated production of HNO, which interacts with thiols forming irreversible modifications including sulfinamide. In addition, we reveal a macrophage signature of proteins with reduction-resistant modifications, including in DLD, and identify potential HNO targets. Consistently, DLD enzyme is modified in an HNO-dependent manner at Cys477 and Cys484, and molecular modeling and mutagenesis show these modifications impair the formation of DLD homodimers. In conclusion, our work demonstrates that HNO is produced physiologically. Moreover, the production of HNO is dependent on the lipoate-rich PDH complex facilitating irreversible modifications that are critical to NO-dependent metabolic rewiring.

Deconstructing SARS-CoV-2 neutralization: A modular molecular framework for computational design and comparison of antibodies and nanobodies targeting the spike RBD.

Since 2020 the COVID-19 pandemic has led scientists to search for strategies to predict the transmissibility and virulence of new severe acute respiratory syndrome coronavirus 2 variants based on the estimation of the affinity of the spike receptor binding domain (RBD) for the human angiotensin-converting enzyme 2 (ACE2) receptor and/or neutralizing antibodies. In this context, our lab developed a computational pipeline to quickly quantify the free energy of interaction at the spike RBD/ACE2 protein-protein interface, reflecting the incidence trend observed in the transmissibility/virulence of the investigated variants. In this new study, we used our pipeline to estimate the free energy of interaction between the RBD from 10 variants, and 14 antibodies (ab), or 5 nanobodies (nb), highlighting the RBD regions preferentially targeted by the investigated ab/nb. Our structural comparative analysis and interaction energy calculations allowed us to propose the most promising RBD regions to be targeted by future ab/nb to be designed by site-directed mutagenesis of existing high-affinity ab/nb, to increase their affinity for the target RBD region, for preventing spike-RBD/ACE2 interactions and virus entry in host cells. Furthermore, we evaluated the ability of the investigated ab/nb to simultaneously interact with the three RBD located on the surface of the trimeric spike protein, which can alternatively be in up- or down- (all-3-up-, all-3-down-, 1-up-/2-down-, 2-up-/1-down-) conformations.

Targeting mitochondrial impairment for the treatment of cardiovascular diseases: From hypertension to ischemia-reperfusion injury, searching for new pharmacological targets.

Mitochondria and mitochondrial proteins represent a group of promising pharmacological target candidates in the search of new molecular targets and drugs to counteract the onset of hypertension and more in general cardiovascular diseases (CVDs). Indeed, several mitochondrial pathways result impaired in CVDs, showing ATP depletion and ROS production as common traits of cardiac tissue degeneration. Thus, targeting mitochondrial dysfunction in cardiomyocytes can represent a successful strategy to prevent heart failure. In this context, the identification of new pharmacological targets among mitochondrial proteins paves the way for the design of new selective drugs. Thanks to the advances in omics approaches, to a greater availability of mitochondrial crystallized protein structures and to the development of new computational approaches for protein 3D-modelling and drug design, it is now possible to investigate in detail impaired mitochondrial pathways in CVDs. Furthermore, it is possible to design new powerful drugs able to hit the selected pharmacological targets in a highly selective way to rescue mitochondrial dysfunction and prevent cardiac tissue degeneration. The role of mitochondrial dysfunction in the onset of CVDs appears increasingly evident, as reflected by the impairment of proteins involved in lipid peroxidation, mitochondrial dynamics, respiratory chain complexes, and membrane polarization maintenance in CVD patients. Conversely, little is known about proteins responsible for the cross-talk between mitochondria and cytoplasm in cardiomyocytes. Mitochondrial transporters of the SLC25A family, in particular, are responsible for the translocation of nucleotides (e.g., ATP), amino acids (e.g., aspartate, glutamate, ornithine), organic acids (e.g. malate and 2-oxoglutarate), and other cofactors (e.g., inorganic phosphate, NAD+, FAD, carnitine, CoA derivatives) between the mitochondrial and cytosolic compartments. Thus, mitochondrial transporters play a key role in the mitochondria-cytosol cross-talk by leading metabolic pathways such as the malate/aspartate shuttle, the carnitine shuttle, the ATP export from mitochondria, and the regulation of permeability transition pore opening. Since all these pathways are crucial for maintaining healthy cardiomyocytes, mitochondrial carriers emerge as an interesting class of new possible pharmacological targets for CVD treatments.

New Insights Regarding Hemin Inhibition of the Purified Rat Brain 2-Oxoglutarate Carrier and Relationships with Mitochondrial Dysfunction.

A kinetic analysis of the transport assays on the purified rat brain 2-oxoglutarate/malate carrier (OGC) was performed starting from our recent results reporting about a competitive inhibitory behavior of hemin, a physiological porphyrin derivative, on the OGC reconstituted in an active form into proteoliposomes. The newly provided transport data and the elaboration of the kinetic equations show evidence that hemin exerts a mechanism of partially competitive inhibition, coupled with the formation of a ternary complex hemin-carrier substrate, when hemin targets the OGC from the matrix face. A possible interpretation of the provided kinetic analysis, which is supported by computational studies, could indicate the existence of a binding region responsible for the inhibition of the OGC and supposedly involved in the regulation of OGC activity. The proposed regulatory binding site is located on OGC mitochondrial matrix loops, where hemin could establish specific interactions with residues involved in the substrate recognition and/or conformational changes responsible for the translocation of mitochondrial carrier substrates. The regulatory binding site would be placed about 6 Å below the substrate binding site of the OGC, facing the mitochondrial matrix, and would allow the simultaneous binding of hemin and 2-oxoglutarate or malate to different regions of the carrier. Overall, the presented experimental and computational analyses help to shed light on the possible existence of the hemin-carrier substrate ternary complex, confirming the ability of the OGC to bind porphyrin derivatives, and in particular hemin, with possible consequences for the mitochondrial redox state mediated by the malate/aspartate shuttle led by the mitochondrial carriers OGC and AGC.

Proline/Glycine residues of the PG-levels guide conformational changes along the transport cycle in the mitochondrial carnitine/acylcarnitine carrier (SLC25A20).

Mitochondrial carnitine/acylcarnitine carrier (CAC) is a member of the mitochondrial carrier (MC) family and imports acylcarnitine into the mitochondrial matrix in exchange for carnitine, playing a pivotal role in carnitine shuttle, crucial for fatty acid oxidation. The crystallized structure of CAC has not been solved yet, however, the availability of several in vitro/in silico studies, also based on the crystallized structures of the ADP/ATP carrier in the cytosolic-conformation and in the matrix-conformation, has made possible to confirm the hypothesis of the single-binding centered-gated pore mechanism for all the members of the MC family. In addition, our recent bioinformatics analyses allowed quantifying in silico the importance of protein residues of MC substrate binding region, of those involved in the formation of the matrix and cytosolic gates, and of those belonging to the Pro/Gly (PG) levels, proposed to be crucial for the tilting/kinking/bending of the six MC transmembrane helices, funneling the substrate translocation pathway. Here we present a combined in silico/in vitro analysis employed for investigating the role played by a group of 6 proline residues and 6 glycine residues, highly conserved in CAC, belonging to MC PG-levels. Residues of the PG-levels surround the similarly located MC common substrate binding region, and were proposed to lead conformational changes and substrate translocation, following substrate binding. For our analysis, we employed 3D molecular modeling approaches, alanine scanning site-directed mutagenesis and in vitro transport assays. Our analysis reveals that P130 (H3), G268 (H6) and G220 (H5), mutated in alanine, affect severely CAC transport activity (mutant catalytic efficiency lower than 5 % compared to the wild type CAC), most likely due to their major role in triggering CAC conformational changes, following carnitine binding. Notably, P30A (H1) and G121A (H3) CAC mutants, increase the carnitine uptake up to 217 % and 112 %, respectively, compared to the wild type CAC.

3D structures inferred from cDNA clones identify the CD1D-Restricted γδ T cell receptor in dromedaries.

The Camelidae species occupy an important immunological niche within the humoral as well as cell mediated immune response. Although recent studies have highlighted that the somatic hypermutation (SHM) shapes the T cell receptor gamma (TRG) and delta (TRD) repertoire in Camelus dromedarius, it is still unclear how γδ T cells use the TRG/TRD receptors and their respective variable V-GAMMA and V-DELTA domains to recognize antigen in an antibody-like fashion. Here we report about 3D structural analyses of the human and dromedary γδ T cell receptor. First, we have estimated the interaction energies at the interface within the human crystallized paired TRG/TRD chains and quantified interaction energies within the same human TRG/TRD chains in complex with the CD1D, an RPI-MH1-LIKE antigen presenting glycoprotein. Then, we used the human TRG/TRD-CD1D complex as template for the 3D structure of the dromedary TRG/TRD-CD1D complex and for guiding the 3D human/dromedary comparative analysis. The choice of mutated TRG alternatively combined with mutated TRD cDNA clones originating from the spleen of one single dromedary was crucial to quantify the strength of the interactions at the protein-protein interface between the paired C. dromedarius TRG and TRD V-domains and between the C. dromedarius TRG/TRD V-domains and CD1D G-domains. Interacting amino acids located in the V-domain Complementarity Determining Regions (CDR) and Framework Regions (FR) according to the IMGT unique numbering for V-domains were identified. The resulting 3D dromedary TRG V-GAMMA combined with TRD V-DELTA protein complexes allowed to deduce the most stable gamma/delta chains pairings and to propose a candidate CD1D-restricted γδ T cell receptor complex.

Personalized Medicine in Mitochondrial Health and Disease: Molecular Basis of Therapeutic Approaches Based on Nutritional Supplements and Their Analogs.

Mitochondrial diseases (MDs) may result from mutations affecting nuclear or mitochondrial genes, encoding mitochondrial proteins, or non-protein-coding mitochondrial RNA. Despite the great variability of affected genes, in the most severe cases, a neuromuscular and neurodegenerative phenotype is observed, and no specific therapy exists for a complete recovery from the disease. The most used treatments are symptomatic and based on the administration of antioxidant cocktails combined with antiepileptic/antipsychotic drugs and supportive therapy for multiorgan involvement. Nevertheless, the real utility of antioxidant cocktail treatments for patients affected by MDs still needs to be scientifically demonstrated. Unfortunately, clinical trials for antioxidant therapies using α-tocopherol, ascorbate, glutathione, riboflavin, niacin, acetyl-carnitine and coenzyme Q have met a limited success. Indeed, it would be expected that the employed antioxidants can only be effective if they are able to target the specific mechanism, i.e., involving the central and peripheral nervous system, responsible for the clinical manifestations of the disease. Noteworthily, very often the phenotypes characterizing MD patients are associated with mutations in proteins whose function does not depend on specific cofactors. Conversely, the administration of the antioxidant cocktails might determine the suppression of endogenous oxidants resulting in deleterious effects on cell viability and/or toxicity for patients. In order to avoid toxicity effects and before administering the antioxidant therapy, it might be useful to ascertain the blood serum levels of antioxidants and cofactors to be administered in MD patients. It would be also worthwhile to check the localization of mutations affecting proteins whose function should depend (less or more directly) on the cofactors to be administered, for estimating the real need and predicting the success of the proposed cofactor/antioxidant-based therapy.

The Non-Gastric H+/K+ ATPase (ATP12A) Is Expressed in Mammalian Spermatozoa.

H+/K+ ATPase Type 2 is an heteromeric membrane protein involved in cation transmembrane transport and consists of two subunits: a specific α subunit (ATP12A) and a non-specific ß subunit. The aim of this study was to demonstrate the presence and establish the localization of ATP12A in spermatozoa from Bubalus bubalis, Bos taurus and Ovis aries. Immunoblotting revealed, in all three species, a major band (100 kDa) corresponding to the expected molecular mass. The ATP12A immunolocalization pattern showed, consistently in the three species, a strong signal at the acrosome. These results, described here for the first time in spermatozoa, are consistent with those observed for the ß1 subunit of Na+/K+ ATPase, suggesting that the latter may assemble with the α subunit to produce a functional ATP12A dimer in sperm cells. The above scenario appeared to be nicely supported by 3D comparative modeling and interaction energy calculations. The expression of ATP12A during different stages of bovine sperm maturation progressively increased, moving from epididymis to deferent ducts. Based on overall results, we hypothesize that ATP12A may play a role in acrosome reactions. Further studies will be required in order to address the functional role of this target protein in sperm physiology.

Modeling SARS-CoV-2 spike/ACE2 protein-protein interactions for predicting the binding affinity of new spike variants for ACE2, and novel ACE2 structurally related human protein targets, for COVID-19 handling in the 3PM context.

Aims: The rapid spread of new SARS-CoV-2 variants has highlighted the crucial role played in the infection by mutations occurring at the SARS-CoV-2 spike receptor binding domain (RBD) in the interactions with the human ACE2 receptor. In this context, it urgently needs to develop new rapid tools for quickly predicting the affinity of ACE2 for the SARS-CoV-2 spike RBD protein variants to be used with the ongoing SARS-CoV-2 genomic sequencing activities in the clinics, aiming to gain clues about the transmissibility and virulence of new variants, to prevent new outbreaks and to quickly estimate the severity of the disease in the context of the 3PM. Methods: In our study, we used a computational pipeline for calculating the interaction energies at the SARS-CoV-2 spike RBD/ACE2 protein-protein interface for a selected group of characterized infectious variants of concern/interest (VoC/VoI). By using our pipeline, we built 3D comparative models of the SARS-CoV-2 spike RBD/ACE2 protein complexes for the VoC B.1.1.7-United Kingdom (carrying the mutations of concern/interest N501Y, S494P, E484K at the RBD), P.1-Japan/Brazil (RBD mutations: K417T, E484K, N501Y), B.1.351-South Africa (RBD mutations: K417N, E484K, N501Y), B.1.427/B.1.429-California (RBD mutations: L452R), the B.1.141 (RBD mutations: N439K), and the recent B.1.617.1-India (RBD mutations: L452R; E484Q) and the B.1.620 (RBD mutations: S477N; E484K). Then, we used the obtained 3D comparative models of the SARS-CoV-2 spike RBD/ACE2 protein complexes for predicting the interaction energies at the protein-protein interface. Results: Along SARS-CoV-2 mutation database screening and mutation localization analysis, it was ascertained that the most dangerous mutations at VoC/VoI spike proteins are located mainly at three regions of the SARS-CoV-2 spike "boat-shaped" receptor binding motif, on the RBD domain. Notably, the P.1 Japan/Brazil variant present three mutations, K417T, E484K, N501Y, located along the entire receptor binding motif, which apparently determines the highest interaction energy at the SARS-CoV-2 spike RBD/ACE2 protein-protein interface, among those calculated. Conversely, it was also observed that the replacement of a single acidic/hydrophilic residue with a basic residue (E484K or N439K) at the "stern" or "bow" regions, of the boat-shaped receptor binding motif on the RBD, appears to determine an interaction energy with ACE2 receptor higher than that observed with single mutations occurring at the "hull" region or with other multiple mutants. In addition, our pipeline allowed searching for ACE2 structurally related proteins, i.e., THOP1 and NLN, which deserve to be investigated for their possible involvement in interactions with the SARS-CoV-2 spike protein, in those tissues showing a low expression of ACE2, or as a novel receptor for future spike variants. A freely available web-tool for the in silico calculation of the interaction energy at the SARS-CoV-2 spike RBD/ACE2 protein-protein interface, starting from the sequences of the investigated spike and/or ACE2 variants, was made available for the scientific community at: https://www.mitoairm.it/covid19affinities. Conclusion: In the context of the PPPM/3PM, the employment of the described pipeline through the provided webservice, together with the ongoing SARS-CoV-2 genomic sequencing, would help to predict the transmissibility of new variants sequenced from future patients, depending on SARS-CoV-2 genomic sequencing activities and on the specific amino acid replacement and/or on its location on the SARS-CoV-2 spike RBD, to put in play all the possible counteractions for preventing the most deleterious scenarios of new outbreaks, taking into consideration that a greater transmissibility has not to be necessarily related to a more severe manifestation of the disease. Supplementary Information: The online version contains supplementary material available at 10.1007/s13167-021-00267-w.

Zoledronic Acid as a Novel Dual Blocker of KIR6.1/2-SUR2 Subunits of ATP-Sensitive K+ Channels: Role in the Adverse Drug Reactions.

Zoledronic acid (ZOL) is used as a bone-specific antiresorptive drug with antimyeloma effects. Adverse drug reactions (A.D.R.) are associated with ZOL-therapy, whose mechanics are unknown. ZOL is a nitrogen-containing molecule whose structure shows similarities with nucleotides, ligands of ATP-sensitive K+ (KATP) channels. We investigated the action of ZOL by performing in vitro patch-clamp experiments on native KATP channels in murine skeletal muscle fibers, bone cells, and recombinant subunits in cell lines, and by in silico docking the nucleotide site on KIR and SUR, as well as the glibenclamide site. ZOL fully inhibited the KATP currents recorded in excised macro-patches from Extensor digitorum longus (EDL) and Soleus (SOL) muscle fibers with an IC50 of 1.2 ± 1.4 × 10-6 and 2.1 ± 3.7 × 10-10 M, respectively, and the KATP currents recorded in cell-attached patches from primary long bone cells with an IC50 of 1.6 ± 2.8 × 10-10 M. ZOL fully inhibited a whole-cell KATP channel current of recombinant KIR6.1-SUR2B and KIR6.2-SUR2A subunits expressed in HEK293 cells with an IC50 of 3.9 ± 2.7 × 10-10 M and 7.1 ± 3.1 × 10-6 M, respectively. The rank order of potency in inhibiting the KATP currents was: KIR6.1-SUR2B/SOL-KATP/osteoblast-KATP > KIR6.2-SUR2A/EDL-KATP >>> KIR6.2-SUR1 and KIR6.1-SUR1. Docking investigation revealed that the drug binds to the ADP/ATP sites on KIR6.1/2 and SUR2A/B and on the sulfonylureas site showing low binding energy <6 Kcal/mol for the KIR6.1/2-SUR2 subunits vs. the <4 Kcal/mol for the KIR6.2-SUR1. The IC50 of ZOL to inhibit the KIR6.1/2-SUR2A/B channels were correlated with its musculoskeletal and cardiovascular risks. We first showed that ZOL blocks at subnanomolar concentration musculoskeletal KATP channels and cardiac and vascular KIR6.2/1-SUR2 channels.

Protein structure analysis of the interactions between SARS-CoV-2 spike protein and the human ACE2 receptor: from conformational changes to novel neutralizing antibodies.

The recent severe acute respiratory syndrome, known as Coronavirus Disease 2019 (COVID-19) has spread so much rapidly and severely to induce World Health Organization (WHO) to declare a state of emergency over the new coronavirus SARS-CoV-2 pandemic. While several countries have chosen the almost complete lock-down for slowing down SARS-CoV-2 spread, the scientific community is called to respond to the devastating outbreak by identifying new tools for diagnosis and treatment of the dangerous COVID-19. With this aim, we performed an in silico comparative modeling analysis, which allows gaining new insights into the main conformational changes occurring in the SARS-CoV-2 spike protein, at the level of the receptor-binding domain (RBD), along interactions with human cells angiotensin-converting enzyme 2 (ACE2) receptor, that favor human cell invasion. Furthermore, our analysis provides (1) an ideal pipeline to identify already characterized antibodies that might target SARS-CoV-2 spike RBD, aiming to prevent interactions with the human ACE2, and (2) instructions for building new possible neutralizing antibodies, according to chemical/physical space restraints and complementary determining regions (CDR) mutagenesis of the identified existing antibodies. The proposed antibodies show in silico high affinity for SARS-CoV-2 spike RBD and can be used as reference antibodies also for building new high-affinity antibodies against present and future coronaviruses able to invade human cells through interactions of their spike proteins with the human ACE2. More in general, our analysis provides indications for the set-up of the right biological molecular context for investigating spike RBD-ACE2 interactions for the development of new vaccines, diagnostic kits, and other treatments based on the targeting of SARS-CoV-2 spike protein.

Targeting mitochondrial metabolite transporters in Penicillium expansum for reducing patulin production.

There is an increasing need of alternative treatments to control fungal infection and consequent mycotoxin accumulation in harvested fruits and vegetables. Indeed, only few biological targets of antifungal agents have been characterized and can be used for limiting fungal spread from decayed fruits/vegetables to surrounding healthy ones during storage. On this concern, a promising target of new antifungal treatments may be represented by mitochondrial proteins due to some species-specific functions played by mitochondria in fungal morphogenesis, drug resistance and virulence. One of the most studied mycotoxins is patulin produced by several species of Penicillium and Aspergillus genera. Patulin is toxic to many biological systems including bacteria, higher plants and animalia. Although precise biochemical mechanisms of patulin toxicity in humans are not completely clarified, its high presence in fresh and processed apple fruits and other apple-based products makes necessary developing a strategy for limiting its presence/accumulation. Patulin biosynthetic pathway consists of an enzymatic cascade, whose first step is represented by the synthesis of 6-methylsalicylic acid, obtained from the condensation of one acetyl-CoA molecule with three malonyl-CoA molecules. The most abundant acetyl-CoA precursor is represented by citrate produced by mitochondria. In the present investigation we report about the possibility to control patulin production through the inhibition of mitochondrial/peroxisome transporters involved in the export of acetyl-CoA precursors from mitochondria and/or peroxisomes, with specific reference to the predicted P. expansum mitochondrial Ctp1p, DTC, Sfc1p, Oac1p and peroxisomal PXN carriers.

Targeting Penicillium expansum GMC Oxidoreductase with High Affinity Small Molecules for Reducing Patulin Production.

Flavine adenine dinucleotide (FAD) dependent glucose methanol choline oxidoreductase (GMC oxidoreductase) is the terminal key enzyme of the patulin biosynthetic pathway. GMC oxidoreductase catalyzes the oxidative ring closure of (E)-ascladiol to patulin. Currently, no protein involved in the patulin biosynthesis in Penicillium expansum has been experimentally characterized or solved by X-ray diffraction. Consequently, nothing is known about P. expansum GMC oxidoreductase substrate-binding site and mode of action. In the present investigation, a 3D comparative model for P. expansum GMC oxidoreductase has been described. Furthermore, a multistep computational approach was used to identify P. expansum GMC oxidoreductase residues involved in the FAD binding and in substrate recognition. Notably, the obtained 3D comparative model of P. expansum GMC oxidoreductase was used for performing a virtual screening of a chemical/drug library, which allowed to predict new GMC oxidoreductase high affinity ligands to be tested in in vitro/in vivo assays. In vitro assays performed in presence of 6-hydroxycoumarin and meticrane, among the highly affinity predicted binders, confirmed a dose-dependent inhibition (17-81%) of patulin production by 6-hydroxycoumarin (10 µM-1 mM concentration range), whereas the approved drug meticrane inhibited patulin production by 43% already at 10 µM. Furthermore, 6-hydroxycoumarin and meticrane caused a 60 and 41% reduction of patulin production, respectively, in vivo on apples at 100 µg/wound.