An industry perspective approach and control strategy for implementation of ready-to-use cells in bioassays: survey outcome and recommendations.

The BioPhorum Development Group is an industry collaboration enabling the sharing of common practices for the development of biopharmaceuticals. Bioassays are an important part of an analytical control system. Utilization of ready-to-use cells can increase operational flexibility and improve efficiency by providing frozen cell banks uniform stock while removing challenges associated with maintaining cultured cells. The BioPhorum Development Group-Bioassay workstream conducted an intercompany benchmarking survey and group discussions around the use of ready-to-use cells for bioassays. The results of the collaboration provide alignment on nomenclature, production, qualification and implementation of ready-to-use cells to support the assay life cycle.

A Hexokinase 2 Modulator for Field-Directed Treatment of Experimental Actinic Keratoses.

Overexpression of hexokinase 2, and its binding to VDAC1 on the outer mitochondrial membrane of cancer cells, is key to their metabolic reprogramming to aerobic glycolysis, which enables them to proliferate. We describe Comp-1, an allosteric small molecule that selectively detaches hexokinase 2 from the mitochondria. Detachment of hexokinase 2 reduces glycolysis and triggers apoptosis in cancer cells, without affecting hexokinase 1-expressing normal cells. The anti-cancer activity of Comp-1 was demonstrated in the UVB-damaged skin model in SKH-1 mice. Topical treatment with Comp-1 led to 70% reduction in lesion number and area. This in vivo efficacy was obtained without local skin reactions or other safety findings. Mechanism-related pharmacodynamic markers, including hexokinase 2 and cleaved caspase 3 levels, are affected by Comp-1 treatment in vivo. Good Laboratory Practice toxicology studies in minipigs for 28 days and 13 weeks established no systemic toxicities and minimal dermal reaction for once-daily application of up to 20% and 15% ointment strengths, respectively. Thus, Comp-1 may address a significant unmet medical need for a non-irritating efficacious topical actinic keratosis treatment.

Molding the business end of neurotoxins by diversifying evolution.

A diverse range of organisms utilize neurotoxins that target specific ion channels and modulate their activity. Typically, toxins are clustered into several multigene families, providing an organism with the upper hand in the never-ending predator-prey arms race. Several gene families, including those encoding certain neurotoxins, have been subject to diversifying selection forces, resulting in rapid gene evolution. Here we sought a spatial pattern in the distribution of both diversifying and purifying selection forces common to neurotoxin gene families. Utilizing the mechanistic empirical combination model, we analyzed various toxin families from different phyla affecting various receptors and relying on diverse modes of action. Through this approach, we were able to detect clear correlations between the pharmacological surface of a toxin and rapidly evolving domains, rich in positively selected residues. On the other hand, patches of negatively selected residues were restricted to the nontoxic face of the molecule and most likely help in stabilizing the tertiary structure of the toxin. We thus propose a mutual evolutionary strategy of venomous animals in which adaptive molecular evolution is directed toward the toxin active surface. Furthermore, we propose that the binding domains of unstudied toxins could be readily predicted using evolutionary considerations.

Assembling an arsenal, the scorpion way.

BACKGROUND: For survival, scorpions depend on a wide array of short neurotoxic polypeptides. The venoms of scorpions from the most studied group, the Buthida, are a rich source of small, 23-78 amino acid-long peptides, well packed by either three or four disulfide bridges that affect ion channel function in excitable and non-excitable cells. RESULTS: In this work, by constructing a toxin transcripts data set from the venom gland of the scorpion Buthus occitanus israelis, we were able to follow the evolutionary path leading to mature toxin diversification and suggest a mechanism for leader peptide hyper-conservation. Toxins from each family were more closely related to one another than to toxins from other species, implying that fixation of duplicated genes followed speciation, suggesting early gene conversion events. Upon fixation, the mature toxin-coding domain was subjected to diversifying selection resulting in a significantly higher substitution rate that can be explained solely by diversifying selection. In contrast to the mature peptide, the leader peptide sequence was hyper-conserved and characterized by an atypical sub-neutral synonymous substitution rate. We interpret this as resulting from purifying selection acting on both the peptide and, as reported here for the first time, the DNA sequence, to create a toxin family-specific codon bias. CONCLUSION: We thus propose that scorpion toxin genes were shaped by selective forces acting at three levels, namely (1) diversifying the mature toxin, (2) conserving the leader peptide amino acid sequence and intriguingly, (3) conserving the leader DNA sequences.

Isolation of the first toxin from the scorpion Buthus occitanus israelis showing preference for Shaker potassium channels.

We have purified BoiTx1, the first toxin from the venom of the Israeli scorpion, Buthus occitanus israelis, and studied its activity and genomic organization. BoiTx1 is a 37 amino acid-long peptide contained six conserved cysteines, and is classified as an alpha-KTx3.10 toxin. The pharmacological effects of BoiTx1 were studied on various cloned K(+) channels expressed in Xenopus laevis oocytes. BoiTx1 inhibited currents through Drosophila Shaker channels with an IC(50) value of 3.5+/-0.5nM, yet had much lesser effect on its mammalian orthologs. Thus, BoiTx1 is the first member of the alpha-KTx3 family that preferentially affects insect potassium channels.