"In vivo biosynthesis of N,N-dimethyltryptamine, 5-MeO-N,N-dimethyltryptamine, and bufotenine in E.coli".

N,N-dimethyltryptamine (DMT), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and 5-hydroxy-N,N-dimethyltryptamine (bufotenine) are psychedelic tryptamines found naturally in both plants and animals and have shown clinical potential to help treat mental disorders, such as anxiety and depression. Advances in both metabolic and genetic engineering make it possible to engineer microbes as cell factories to produce DMT and its aforementioned derivatives to meet demand for ongoing clinical study. Here, we present the development of a biosynthetic production pathway for DMT, 5-MeO-DMT, and bufotenine in the model microbe Escherichia coli. Through the application of genetic optimization techniques and process optimization in benchtop fermenters, the in vivo production of DMT in E. coli was observed. DMT production with tryptophan supplementation reached maximum titers of 74.7 ± 10.5 mg/L under fed batch conditions in a 2-L bioreactor. Additionally, we show the first reported case of de novo production of DMT (from glucose) in E. coli at a maximum titer of 14.0 mg/L and report the first example of microbial 5-MeO-DMT and bufotenine production in vivo. This work provides a starting point for further genetic and fermentation optimization studies with the goal to increase methylated tryptamine production metrics to industrially competitive levels.

Evaluation of TrpM and PsiD substrate promiscuity reveals new biocatalytic capabilities.

N-methylated tryptamines, such as the hallucinogenic natural products, psilocybin and N,N-dimethyltryptamine (DMT), are gaining interest from the medical community due to their potential as next generation treatments for mental health disorders. The clinical relevance of these compounds has driven scientists to develop biosynthetic production routes to a number of tryptamine drug candidates, and efforts are ongoing to expand and further develop these biosynthetic capabilities. To that end, we have further characterized the substrate preferences of two enzymes involved in tryptamine biosynthesis: TrpM, a tryptophan N-methyltransferase from Psilocybe serbica, and PsiD, the gateway decarboxylase of the psilocybin biosynthesis pathway. Here, we show that TrpM can N-methylate the non-native amino acid substrate, 4-hydroxytryptophan, a key intermediate in the Escherichia coli-based recombinant psilocybin biosynthesis pathway. However, the ability to incorporate TrpM into a functional psilocybin biosynthesis pathway was thwarted by PsiD's inability to use N,N-dimethyl-4-hydroxytryptophan as substrate, under the culturing conditions tested, despite demonstrating activity on N-methylated and 4-hydroxylated tryptophan derivatives individually. Taken together, this work expands upon the known substrates for TrpM and PsiD, further increasing the diversity of tryptamine biosynthetic products.

Considerations for Polymers Used in Ocular Drug Delivery.

PURPOSE: Age-related eye diseases are becoming more prevalent. A notable increase has been seen in the most common causes including glaucoma, age-related macular degeneration (AMD), and cataract. Current clinical treatments vary from tissue replacement with polymers to topical eye drops and intravitreal injections. Research and development efforts have increased using polymers for sustained release to the eye to overcome treatment challenges, showing promise in improving drug release and delivery, patient experience, and treatment compliance. Polymers provide unique properties that allow for specific engineered devices to provide improved treatment options. Recent work has shown the utilization of synthetic and biopolymer derived biomaterials in various forms, with this review containing a focus on polymers Food and Drug Administration (FDA) approved for ocular use. METHODS: This provides an overview of some prevalent synthetic polymers and biopolymers used in ocular delivery and their benefits, brief discussion of the various types and synthesis methods used, and administration techniques. Polymers approved by the FDA for different applications in the eye are listed and compared to new polymers being explored in the literature. This article summarizes research findings using polymers for ocular drug delivery from various stages: laboratory, preclinical studies, clinical trials, and currently approved. This review also focuses on some of the challenges to bringing these new innovations to the clinic, including limited selection of approved polymers. RESULTS: Polymers help improve drug delivery by increasing solubility, controlling pharmacokinetics, and extending release. Several polymer classes including synthetic, biopolymer, and combinations were discussed along with the benefits and challenges of each class. The ways both polymer synthesis and processing techniques can influence drug release in the eye were discussed. CONCLUSION: The use of biomaterials, specifically polymers, is a well-studied field for drug delivery, and polymers have been used as implants in the eye for over 75 years. Promising new ocular drug delivery systems are emerging using polymers an innovative option for treating ocular diseases because of their tunable properties. This review touches on important considerations and challenges of using polymers for sustained ocular drug delivery with the goal translating research to the clinic.

Probing the mechanisms of inhibition for various inhibitors of metallo-ß-lactamases VIM-2 and NDM-1.

To probe the mechanism of inhibition of several previously-published metallo-ß-lactamase (MBL) inhibitors for the clinically-important MBL Verona integron-encoded metallo-ß-lactamase 2 (VIM-2), equilibrium dialyses with metal analyses, native state electrospray ionization mass spectrometry (ESI-MS), and UV-Vis spectrophotometry were utilized. The mechanisms of inhibition were analyzed for ethylenediaminetetraacetic acid (EDTA); dipicolinic acid (DPA) and DPA analogs 6-(1H-tetrazol-5-yl)picolinic acid (1T5PA) and 4-(3-aminophenyl)pyridine-2,6-dicarboxylic acid (3AP-DPA); thiol-containing compounds, 2,3-dimercaprol, thiorphan, captopril, and tiopronin; and 5-(pyridine-3-sulfonamido)-1,3-thiazole-4-carboxylic acid (ANT-431). UV-Vis spectroscopy and native-state ESI-MS results showed the formation of ternary complexes between VIM-2 and 1T5PA, ANT-431, thiorphan, captopril, and tiopronin, while a metal stripping mechanism was shown with VIM-2 and EDTA and DPA. The same approaches were used to show the formation of a ternary complex between New Delhi Metallo-ß-lactamase (NDM-1) and ANT-431. The studies presented herein show that most of the inhibitors utilize a similar mechanism of inhibition as previously reported for NDM-1. These studies also demonstrate that native mass spectrometry can be used to probe the mechanism of inhibition at lower enzyme/inhibitor concentrations than has previously been achieved.