Identifying Synergistic Components of Botanical Fungicide Formulations Using Interpretable Graph Neural Networks.

Botanical formulations are promising candidates for developing new biopesticides that can protect crops from pests and diseases while reducing harm to the environment. These biopesticides can be combined with permeation enhancer compounds to boost their efficacy against pests and fungal diseases. However, finding synergistic combinations of these compounds is challenging due to the large and complex chemical space. In this paper, we propose a novel deep learning method that can predict the synergy of botanical products and permeation enhancers based on in vitro assay data. Our method uses a weighted combination of component feature vectors to represent the input mixtures, which enables the model to handle a variable number of components and to interpret the contribution of each component to the synergy. We also employ an ensemble of interpretation methods to provide insights into the underlying mechanisms of synergy. We validate our method by testing the predicted synergistic combinations in wet-lab experiments and show that our method can discover novel and effective biopesticides that would otherwise be difficult to find. Our method is generalizable and applicable to other domains, where predicting mixtures of chemical compounds is important.

Sequestosome-1/p62 is the key intracellular target of innate defense regulator peptide.

Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor alpha-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor alpha-induced p62-protein kinase zeta complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein beta activity, whereas NF-kappaB activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases.

A novel approach for emerging and antibiotic resistant infections: Innate defense regulators as an agnostic therapy.

Innate Defense Regulators (IDRs) are short synthetic peptides that target the host innate immune system via an intracellular adaptor protein which functions at key signaling nodes. In this work, further details of the mechanism of action of IDRs have been discovered. The studies reported here show that the lead clinical IDR, SGX94, has broad-spectrum activity against Gram-negative and Gram-positive bacterial infections caused by intracellular or extracellular bacteria and also complements the actions of standard of care antibiotics. Based on in vivo and primary cell culture studies, this activity is shown to result from the primary action of SGX94 on tissue-resident cells and subsequent secondary signaling to activate myeloid-derived cells, resulting in enhanced bacterial clearance and increased survival. Data from non-clinical and clinical studies also show that SGX94 treatment modulates pro-inflammatory and anti-inflammatory cytokine levels, thereby mitigating the deleterious inflammatory consequences of innate immune activation. Since they act through host pathways to provide both broad-spectrum anti-infective capability as well as control of inflammation, IDRs are unlikely to be impacted by resistance mechanisms and offer potential clinical advantages in the fight against emerging and antibiotic resistant bacterial infections.

Structure-activity relationships for the beta-hairpin cationic antimicrobial peptide polyphemusin I.

The solution structure of polyphemusin I was determined using (1)H-NMR spectroscopy. Polyphemusin I was found to be an amphipathic, beta-hairpin connected by a type I' beta-turn. The 17 low-energy structures aligned very well over the beta-sheet region while both termini were poorly defined due in part to a hinge-like region centred in the molecule about arginine residues 6 and 16. Conversely, a linear analogue, PM1-S, with all cysteines simultaneously replaced with serine was found to be dynamic in nature, and a lack of medium and long-range NOEs indicated that this molecule displayed no favoured conformation. Circular dichroism (CD) spectroscopy confirmed that in solution, 50% trifluoroethanol (TFE) and in the presence of liposomes, PM1-S remained unstructured. The antimicrobial activity of PM1-S was found to be 4- to 16-fold less than that of polyphemusin I and corresponded with a 4-fold reduction in bacterial membrane depolarization. Both peptides were able to associate with lipid bilayers in a similar fashion; however, PM1-S was completely unable to translocate model membranes while polyphemusin I retained this activity. It was concluded that the disulfide-constrained, beta-sheet structure of polyphemusin I is required for maximum antimicrobial activity. Disruption of this structure results in reduced antimicrobial activity and completely abolishes membrane translocation indicating that the linear PM1-S acts through a different antimicrobial mechanism.

Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin.

Daptomycin is a cyclic anionic lipopeptide antibiotic recently approved for the treatment of complicated skin infections (Cubicin). Its function is dependent on calcium (as Ca2+). Circular dichroism spectroscopy indicated that daptomycin experienced two structural transitions: a transition upon interaction of daptomycin with Ca2+, and a further transition upon interaction with Ca2+ and the bacterial acidic phospholipid, phosphatidyl glycerol. The Ca2+-dependent insertion of daptomycin into model membranes promoted mild and more pronounced perturbations as assessed by the increase of lipid flip-flop and membrane leakage, respectively. The NMR structure of daptomycin indicated that Ca2+ induced a conformational change in daptomycin that increased its amphipathicity. These results are consistent with the hypothesis that the association of Ca2+ with daptomycin permits it to interact with bacterial membranes with effects that are similar to those of the cationic antimicrobial peptides.

Membrane binding and permeation by indolicidin analogs studied by a biomimetic lipid/polydiacetylene vesicle assay.

Membrane binding and relative penetration of indolicidin analogs were studied using lipid/polydiacetylene (PDA) chromatic biomimetic membranes. Colorimetric and fluorescence analyses determined that an indolicidin analog with a proline and tryptophan residue substituted with lysines showed more pronounced bilayer surface interactions, while indolicidin and particularly an indolicidin analog in which all prolines were replaced with alanine residues exhibited deeper insertion into the lipid bilayer. The colorimetric data demonstrated that more pronounced blue-red transitions were observed when the chromatic vesicles incorporated lipopolysaccharide (LPS) within the lipid bilayer, indicating that LPS promoted preferred binding and incorporation of the peptides at the lipid/water interface. The fluorescence quenching experiments further confirmed this outcome. The results indicate that the antibacterial activity of indolicidin most likely requires initial binding to the LPS moieties within bacterial membranes, as well as disruption of the bilayer interface. The degree of hemolysis induced by the analogs, on the other hand, correlated to the extent of penetration into the hydrophobic core of the lipid assembly.

Role of membranes in the activities of antimicrobial cationic peptides.

Cationic amphiphilic peptides that are found throughout nature have very broad-spectrum activities against microbes. The initial sites of interaction are with microbial membranes. Although dogma suggests that their lethal action involves disruption of the cytoplasmic membranes, a number of cationic peptides can traverse intact membranes to interact with internal targets.

Interaction and cellular localization of the human host defense peptide LL-37 with lung epithelial cells.

LL-37 is a human cationic host defense peptide that is an essential component of innate immunity. In addition to its modest antimicrobial activity, LL-37 affects the gene expression and behavior of effector cells involved in the innate immune response, although its mode of interaction with eukaryotic cells remains unclear. The interaction of LL-37 with epithelial cells was characterized in tissue culture by using biotinylated LL-37 and confocal microscopy. It was demonstrated that LL-37 was actively taken up into A549 epithelial cells and eventually localized to the perinuclear region. Specific inhibitors were used to demonstrate that the uptake process was not mediated by actin but required elements normally involved in endocytosis and that trafficking to the perinuclear region was dependent on microtubules. By using nonlinear regression analysis, it was revealed that A549 epithelial cells have two receptors for LL-37B, with high and low affinity for LL-37, respectively. These results indicate the mode of interaction of LL-37 with epithelial cells and further our understanding of its role in modulating the innate immune response.

Structure-based design of an indolicidin peptide analogue with increased protease stability.

Indolicidin is an antimicrobial cationic peptide with broad-spectrum activity isolated from bovine neutrophils. An indolicidin analogue CP-11, ILKKWPWWPWRRK-NH(2), with improved activity against Gram-negative bacteria had increased positive charge and amphipathicity while maintaining the short length of the parent molecule. The structure of CP-11 in the presence of dodecylphosphocholine (DPC) micelles was determined using nuclear magnetic resonance spectroscopy. CP-11 was found to be an amphipathic molecule with a U-shaped backbone bringing the N- and C-termini in close proximity. On the basis of this close proximity, a cyclic disulfide-bonded peptide cycloCP-11, ICLKKWPWWPWRRCK-NH(2), was designed to stabilize the lipid-bound structure and to increase protease resistance. The three-dimensional structure of cycloCP-11 was determined under the same conditions as for the linear peptide and was found to be similar to CP-11. Both CP-11 and cycloCP-11 associated with phospholipid membranes in a similar manner as indicated by circular dichroism and fluorescence spectra. The minimal inhibitory concentrations of CP-11 and cycloCP-11 for a range of bacteria differed by no more than 2-fold, and they were nonhemolytic at concentrations up to 256 microg/mL. Cyclization was found to greatly increase protease stability. The half-life of cycloCP-11 in the presence of trypsin was increased by 4.5-fold from 4 to 18 min. More importantly, the antibacterial activity of cycloCP-11, but not that of CP-11, in the presence of trypsin was completely retained up to 90 min since the major degradation product was equally active. A structural comparison of CP-11 and cycloCP-11 revealed that the higher trypsin resistance of cycloCP-11 may be due to the more compact packing of lysine and tryptophan side chains. These findings suggest that cyclization may serve as an important strategy in the rational design of antimicrobial peptides.