Specifically targeting antimicrobial peptides for inhibition of Candidatus Liberibacter asiaticus.

AIMS: Huanglongbing (citrus greening) is a plant disease putatively caused by the unculturable Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), and it has caused severe damage to citrus plantations worldwide. There are no definitive treatments for this disease, and conventional disease control techniques have shown limited efficacy. This work presents an in silico evaluation of using specifically targeting anti-microbial peptides (STAMPs) consisting of a targeting segment and an antimicrobial segment to inhibit citrus greening by inhibiting the BamA protein of CLas, which is an outer membrane protein crucial for bacterial viability. METHODS AND RESULTS: Initially, a set of peptides with a high affinity toward BamA protein were screened and evaluated via molecular docking and molecular dynamics simulations and were verified in vitro via bio-layer interferometry (BLI). In silico studies and BLI experiments indicated that two peptides, HASP2 and HASP3, showed stable binding to BamA. Protein structures for STAMPs were created by fusing known anti-microbial peptides (AMPs) with the selected short peptides. The binding of STAMPs to BamA was assessed using molecular docking and binding energy calculations. The attachment of high-affinity short peptides significantly reduced the free energy of binding for AMPs, suggesting that it would make it easier for the STAMPs to bind to BamA. Efficacy testing in vitro using a closely related CLas surrogate bacterium showed that STAMPs had greater inhibitory activity than AMP alone. CONCLUSIONS: In silico and in vitro results indicate that the STAMPs can inhibit CLas surrogate Rhizobium grahamii more effectively compared to AMPs, suggesting that STAMPs can achieve better inhibition of CLas, potentially via enhancing the site specificity of AMPs.

Identification of Fungicide Combinations for Overcoming Plasmopara viticola and Botrytis cinerea Fungicide Resistance.

Fungal diseases, including downy mildew (caused by Plasmopara viticola) and gray mold (caused by Botrytis cinerea), significantly impact the marketable yield of grapes produced worldwide. Cytochrome b of the mitochondrial respiratory chain of these two fungi is a key target for Quinone outside inhibitor (QoI)-based fungicide development. Since the mode of action (MOA) of QoI fungicides is restricted to a single site, the extensive usage of these fungicides has resulted in fungicide resistance. The use of fungicide combinations with multiple targets is an effective way to counter and slow down the development of fungicide resistance. Due to the high cost of in planta trials, in silico techniques can be used for the rapid screening of potential fungicides. In this study, a combination of in silico simulations that include Schrödinger Glide docking, molecular dynamics, and Molecular Mechanism-Generalized Born Surface Area calculation were used to screen the most potent QoI and non-QoI-based fungicide combinations to wild-type, G143A-mutated, F129L-mutated, and double-mutated versions that had both G143A and F129L mutations of fungal cytochrome b. In silico docking studies indicated that mandestrobin, famoxadone, captan, and thiram have a high affinity toward WT cytochrome b of Botrytis cinerea. Although the QoIs mandestrobin and famoxadone were effective for WT based on in vitro results, they were not broadly effective against G143A-mutated isolates. Famoxadone was only effective against one isolate with G143A-mutated cytochrome b. The non-QoI fungicides thiram and captan were effective against both WT and isolates with G143A-mutated cytochrome b. Follow-up in silico docking and molecular dynamics studies suggested that fungicide combinations consisting of famoxadone, mandestrobin, fenamidone, and thiram should be considered in field testing targeting Plasmopara viticola and Botrytis cinerea fungicide resistance.

In silico and in vitro evaluation of imatinib as an inhibitor for SARS-CoV-2.

The rapid geographic expansion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the infectious agent of Coronavirus Disease 2019 (COVID-19) pandemic, poses an immediate need for potent drugs. Enveloped viruses infect the host cell by cellular membrane fusion, a crucial mechanism required for virus replication. The SARS-CoV-2 spike glycoprotein, due to its primary interaction with the human angiotensin-converting enzyme 2 (ACE2) cell-surface receptor, is considered a potential target for drug development. In this study, around 5,800 molecules were virtually screened using molecular docking. Five molecules were selected for in vitro experiments from those that reported docking scores lower than -6 kcal/mol. Imatinib, a Bcr-Abl tyrosine kinase inhibitor, showed maximum antiviral activity in Vero cells. We further investigated the interaction of imatinib, a compound under clinical trials for the treatment of COVID-19, with SARS-CoV-2 RBD, using in silico methods. Molecular dynamics simulations verified that imatinib interacts with RBD residues that are critical for ACE2 binding. This study also provides significant molecular insights on potential repurposable small-molecule drugs and chemical scaffolds for the development of novel drugs targeting the SARS-CoV-2 spike RBD.Communicated by Ramaswamy H. Sarma.

Evaluation of Candidatus Liberibacter Asiaticus Efflux Pump Inhibition by Antimicrobial Peptides.

Citrus greening, also known as Huanglongbing (HLB), is caused by the unculturable bacterium Candidatus Liberibacter spp. (e.g., CLas), and has caused a devastating decline in citrus production in many areas of the world. As of yet, there are no definitive treatments for controlling the disease. Antimicrobial peptides (AMPs) that have the potential to block secretion-dependent effector proteins at the outer-membrane domains were screened in silico. Predictions of drug-receptor interactions were built using multiple in silico techniques, including molecular docking analysis, molecular dynamics, molecular mechanics generalized Born surface area analysis, and principal component analysis. The efflux pump TolC of the Type 1 secretion system interacted with natural bacteriocin plantaricin JLA-9, blocking the ß barrel. The trajectory-based principal component analysis revealed the possible binding mechanism of the peptides. Furthermore, in vitro assays using two closely related culturable surrogates of CLas (Liberibacter crescens and Rhizobium spp.) showed that Plantaricin JLA-9 and two other screened AMPs inhibited bacterial growth and caused mortality. The findings contribute to designing effective therapies to manage plant diseases associated with Candidatus Liberibacter spp.

Mechanics of controlled release of insulin entrapped in polyacrylic acid gels via variable electrical stimuli.

Controlled release insulin delivery systems possess multiple advantages over conventional ones, including maintaining desired blood glucose levels for prolonged periods and minimizing complications due to insulin overdose. Compared to other controlled-release mechanisms, electro-responsive polymers present the advantages of high controllability and ability to be coupled with microelectronics. This paper reports the possibility of using electro-responsive polyacrylic acid (PAA) and polymethacrylic acid (PMA) hydrogels for controlled delivery of insulin using intermittent electrical signals via matrix deformation. PAA hydrogels showed very good electrical responsivity under both constant and step current inputs, releasing up to 80% of protein at 10 V stimulus, compared to 20% release in the absence of stimulus. Analysis of spatial variation under electrical stimuli suggested that release of protein is a combined effect of deformation of the hydrogel and electrophoresis of protein molecules. Binding interaction analysis revealed that insulin entrapment is largely due to hydrogen bonding between the polymer matrix and insulin, and flooding the matrix with electrical charge likely disrupts the attractive forces that kept protein in place helping the release of the proteins. Understanding the molecular interactions affecting insulin retention and release mechanisms of PAA hydrogels is useful for developing and optimizing hydrogel-based controlled drug release systems.

Electrical Field Reversibly Modulates Enzyme Kinetics of Hexokinase Entrapped in an Electro-Responsive Hydrogel.

In this paper, the potential use of electro-responsive poly(acrylic acid) (PAA) gels as reversible enzyme activity regulators is analyzed. This was evaluated by measuring the glucose conversion by hexokinase embedded PAA hydrogels under external electrical stimuli. Hexokinase physically entrapped within PAA gels showed a significant increase in activity under an electrical stimulus as compared to in the absence of a stimulus. Kinetic studies revealed that the change in reaction rate could be attributed to the change of Vmax under a stimulus, while Km was unaffected by the stimulus, which suggested that the increase in reaction rate under an electrical stimulus was due to increased accessibility of the active site. Optimum stimuli-responsive behavior that resulted in maximum conversion under a stimulus and minimum conversion in the absence of a stimulus was obtained at 5.5 pH and 30 °C. The significant difference between the pH optima for the entrapped enzyme and the pure enzyme can be attributed to the acidic nature of the polymeric matrix. Higher cross-linker concentrations resulted in a reduction of both enzyme release and glucose conversion, and a reasonable trade-off between conversion and release could be obtained at 5% cross-linker concentration. Application of a stepwise electrical stimulus revealed that the entrapped enzymes could sustain responsive properties over multiple cycles of electrical switching. Entrapped hexokinase also showed much better reusability compared to pure hexokinase, a combined result of higher enzyme retention and increased stability. No significant impact of the polymer on the interaction between enzyme and glucose was observed. Thus, this system enables electro-responsive modulation of enzyme activity without any reduction in enzyme activity. The studies revealed that conjugation of electro-responsive polymers to enzymes has the potential to reversibly modulate enzymatic reactions via the application of external electrical stimuli, which is promising for bioprocessing and enzymatic separation applications.