Potency-Enhancing Mutations of Gating Modifier Toxins for the Voltage-Gated Sodium Channel NaV1.7 Can Be Predicted Using Accurate Free-Energy Calculations

Gating modifier toxins (GMTs) isolated from venomous organisms such as Protoxin-II (ProTx-II) and Huwentoxin-IV (HwTx-IV) that inhibit the voltage-gated sodium channel NaV1.7 by binding to its voltage-sensing domain II (VSDII) have been extensively investigated as non-opioid analgesics. However, reliably predicting how a mutation to a GMT will affect its potency for NaV1.7 has been challenging. Here, we hypothesize that structure-based computational methods can be used to predict such changes. We employ free-energy perturbation (FEP), a physics-based simulation method for predicting the relative binding free energy (RBFE) between molecules, and the cryo electron microscopy (cryo-EM) structures of ProTx-II and HwTx-IV bound to VSDII of NaV1.7 to re-predict the relative potencies of forty-seven point mutants of these GMTs for NaV1.7. First, FEP predicted these relative potencies with an overall root mean square error (RMSE) of 1.0 ± 0.1 kcal/mol and an R2 value of 0.66, equivalent to experimental uncertainty and an improvement over the widely used molecular-mechanics/generalized born-surface area (MM-GB/SA) RBFE method that had an RMSE of 3.9 ± 0.8 kcal/mol. Second, inclusion of an explicit membrane model was needed for the GMTs to maintain stable binding poses during the FEP simulations. Third, MM-GB/SA and FEP were used to identify fifteen non-standard tryptophan mutants at ProTx-II[W24] predicted in silico to have a at least a 1 kcal/mol gain in potency. These predicted potency gains are likely due to the displacement of high-energy waters as identified by the WaterMap algorithm for calculating the positions and thermodynamic properties of water molecules in protein binding sites. Our results expand the domain of applicability of FEP and set the stage for its prospective use in biologics drug discovery programs involving GMTs and NaV1.7.

Phylogenetic characteristics of novel Bacillus weihenstephanensis and Pseudomonas sp. to desert locust, Schistocerca gregaria Forskål (Orthoptera: Acrididae)

Thirty bacterial isolates were isolated from the gut contents of diseased/dead locust. Their pathogenicity was tested against 4th instar nymphs of desert locust, Schistocerca gregaria Forskål (Orthoptera: Acrididae). Two isolates, designated DL2 and DL6, out of thirty showed the highest insecticidal activities against locust nymphs in preliminary bracketing. They were bioassayed via leaf dip and per os techniques and toxicity was determined using SAS program. The insecticidal activity of DL6 was more than DL2, whereas LC50’s values of 35 × 106 and 13 × 106 cfu’s/ml were determined for DL2 and DL6, respectively, after 48 h of leaf-dip treatment. However, LD50’s value of 53 × 106 and 26 × 106 cfu’s/ml was determined for DL2 and DL6, respectively, after 24 h of per os treatment. The relative potencies of DL6 to DL2 were (2.6 and 2.03) folds in leaf-dip and per os treatments, respectively. Biochemical characterization was conducted, using GEN III MicroPlate™ Biolog identification system and confirmed with molecular identification via 16S rDNA gene sequencing. Nucleotide sequencing of each was submitted to a gene bank and an accession number was generated for each isolate. Obtained bacterial strains DL2 and DL6 were identified as Bacillus weihenstephanensis (KY630645) and Pseudomonas sp. (KY630649), with a similarity of 100 and 75% to B. weihenstephanensis strain PHCDB9 (NR_024697) and Pseudomonas sp. strain DSM11821 (KF417541), respectively. The tested strains proved their potential to be bio-pesticide agents involved in controlling desert locust nymphs. Graphical abstract

Evaluation of Cannabinoid and Terpenoid Content: Cannabis Flower Compared to Supercritical CO2 Concentrate

A recent cannabis use survey revealed that 60% of cannabis users rely on smelling the flower to select their cannabis. Olfactory indicators in plants include volatile compounds, principally represented by the terpenoid fraction. Currently, medicinal- and adult-use cannabis is marketed in the United States with relatively little differentiation between products other than by a common name, association with a species type, and Δ-9 tetrahydrocannabinol/cannabidiol potency. Because of this practice, how terpenoid compositions may change during an extraction process is widely overlooked. Here we report on a comparative study of terpenoid and cannabinoid potencies of flower and supercritical fluid CO2 (SC-CO2) extract from six cannabis chemovars grown in Washington State. To enable this comparison, we employed a validated high-performance liquid chromatography/diode array detector methodology for quantification of seven cannabinoids and developed an internal gas chromatography-mass spectrometry method for quantification of 42 terpenes. The relative potencies of terpenoids and cannabinoids in flower versus concentrate were significantly different. Cannabinoid potency increased by factors of 3.2 for Δ-9 tetrahydrocannabinol and 4.0 for cannabidiol in concentrates compared to flower. Monoterpenes were lost in the extraction process; a ketone increased by 2.2; an ether by 2.7; monoterpene alcohols by 5.3, 7 and 9.4; and sesquiterpenes by 5.1, 4.2, 7.7, and 8.9. Our results demonstrate that the product of SC-CO2 extraction may have a significantly different chemotypic fingerprint from that of cannabis flower. These results highlight the need for more complete characterization of cannabis and associated products, beyond cannabinoid content, in order to further understand health-related consequences of inhaling or ingesting concentrated forms.