Novel Psilocin Prodrugs with Altered Pharmacological Properties as Candidate Therapies for Treatment-Resistant Anxiety Disorders.

The psychedelic prodrug psilocybin has shown therapeutic benefits for the treatment of numerous psychiatric conditions. Despite positive clinical end points targeting depression and anxiety, concerns regarding the duration of the psychedelic experience produced by psilocybin, associated with enduring systemic exposure to the active metabolite psilocin, pose a barrier to its therapeutic application. Our objective was to create a novel prodrug of psilocin with similar therapeutic benefits but a reduced duration of psychedelic effects compared with psilocybin. Here, we report the synthesis and functional screening of 28 new chemical entities. Our strategy was to introduce a diversity of cleavable groups at the 4-hydroxy position of the core indole moiety to modulate metabolic processing. We identified several novel prodrugs of psilocin with altered pharmacokinetic profiles and reduced pharmacological exposure compared with psilocybin. These candidate prodrugs have the potential to maintain the long-term benefits of psilocybin therapy while attenuating the duration of psychedelic effects.

A cane toad (Rhinella marina) N-methyltransferase converts primary indolethylamines to tertiary psychedelic amines.

Psychedelic indolethylamines have emerged as potential medicines to treat several psychiatric pathologies. Natural sources of these compounds include 'magic mushrooms' (Psilocybe spp.), plants used to prepare ayahuasca, and toads. The skin and parotid glands of certain toads accumulate a variety of specialized metabolites including toxic guanidine alkaloids, lipophilic alkaloids, poisonous steroids, and hallucinogenic indolethylamines such as DMT, 5-methoxy-DMT, and bufotenin. The occurrence of psychedelics has contributed to the ceremonial use of toads, particularly among Mesoamerican peoples. Yet, the biosynthesis of psychedelic alkaloids has not been elucidated. Herein, we report a novel indolethylamine N-methyltransferase (RmNMT) from cane toad (Rhinella marina). The RmNMT sequence was used to identify a related NMT from the common toad, Bufo bufo. Close homologs from various frog species were inactive, suggesting a role for psychedelic indolethylamine biosynthesis in toads. Enzyme kinetic analyses and comparison with functionally similar enzymes showed that recombinant RmNMT was an effective catalyst and not product inhibited. The substrate promiscuity of RmNMT enabled the bioproduction of a variety of substituted indolethylamines at levels sufficient for purification, pharmacological screening, and metabolic stability assays. Since the therapeutic potential of psychedelics has been linked to activity at serotonergic receptors, we evaluated binding of derivatives at 5-HT1A and 5-HT2A receptors. Primary amines exhibited enhanced affinity at the 5-HT1A receptor compared with tertiary amines. With the exception of 6-substituted derivatives, N,N-dimethylation also protected against catabolism by liver microsomes.

Ume6 Acts as a Stable Platform To Coordinate Repression and Activation of Early Meiosis-Specific Genes in Saccharomyces cerevisiae.

In response to nutrient starvation, the budding yeast Saccharomyces cerevisiae abandons mitotic proliferation and embarks on a differentiation process that leads through meiosis to the formation of haploid spores. This process is driven by cascading waves of meiosis-specific-gene expression. The early meiosis-specific genes are repressed during mitotic proliferation by the DNA-binding protein Ume6 in combination with repressors Rpd3 and Sin3. The expression of meiosis-specific transcription factor Ime1 leads to activation of the early meiosis-specific genes. We investigated the stability and promoter occupancy of Ume6 in sporulating cells and determined that it remains bound to early meiosis-specific gene promoters when those genes are activated. Furthermore, we find that the repressor Rpd3 remains associated with Ume6 after the transactivator Ime1 has joined the complex and that the Gcn5 and Tra1 components of the SAGA complex bind to the promoter of IME2 in an Ime1-dependent fashion to induce transcription of the early meiosis-specific genes. Our investigation supports a model whereby Ume6 provides a platform allowing recruitment of both activating and repressing factors to coordinate the expression of the early meiosis-specific genes in Saccharomyces cerevisiae.

Topical small molecule granzyme B inhibitor improves remodeling in a murine model of impaired burn wound healing.

Granzyme B (GzmB) is a serine protease that has long been thought to function exclusively in lymphocyte-mediated apoptosis. In recent years, this paradigm has been revisited due to the recognition that GzmB accumulates in the extracellular milieu in many autoimmune and chronic inflammatory disorders, and contributes to impaired tissue remodeling due to the cleavage of extracellular matrix proteins. Knockout studies suggest that GzmB-mediated cleavage of decorin (DCN) contributes to impaired collagen fibrillogenesis and remodeling. As DCN is anti-fibrotic and contributes to reduced hypertrophic scarring, GzmB-induced DCN cleavage could play a role in wound healing following burn injury. In the present study, a novel, gel-formulated, first-in-class small-molecule inhibitor of GzmB, VTI-1002, was assessed in a murine model of impaired, diabetic burn wound healing. VTI-1002 exhibited high specificity, potency, and target selectivity. Gel-formulated VTI-1002 was able to penetrate the stratum corneum and was retained in the skin with minimal systemic absorption. Daily topical administration of VTI-1002 gel for 30 days following thermal injury showed significantly accelerated wound closure, increased DCN protein levels, and collagen organization that was translated into significantly increased wound tensile strength compared to controls. Overall, VTI-1002 gel was well-tolerated in vivo and no adverse events were observed. Topical application of VTI-1002 represents a novel therapeutic approach for the treatment of cutaneous burn wounds.

Granzyme B inhibits keratinocyte migration by disrupting epidermal growth factor receptor (EGFR)-mediated signaling.

Chronic non-healing wounds including diabetic, venous, and decubitus skin ulcers are currently lacking effective therapies. Non-healing diabetic ulcers can lead to amputations as progress into a highly chronic state before detection and existing treatments for these wounds often fail. Granzyme B (GzmB) is a serine protease that was, until recently, believed to function exclusively in cytotoxic lymphocyte-mediated apoptosis. However, during excessive or chronic inflammation, GzmB can accumulate in the extracellular milieu, retain its activity, and cleave a number of important extracellular proteins. Epidermal growth factor receptor (EGFR) is a transmembrane receptor involved in cellular processes such as proliferation and migration. EGFR signaling is integral to the wound healing process. The present study investigated the effects of GzmB on keratinocyte cell migration using HaCaT cell line. Using electric cell-substrate impedance sensing and scratch assays, the present study demonstrates that GzmB inhibits keratinocyte migration by interfering with the EGFR pathway. GzmB limited cell transition into a migratory morphology and was found to reduce ligand-induced EGFR phosphorylation. Inhibition of GzmB reversed the aforementioned effects. In summary, data from the present study suggest key role for GzmB in the pathogenesis of impaired wound healing through the impairment of EGFR signaling and cell migration.

Granzyme B Deficiency Protects against Angiotensin II-Induced Cardiac Fibrosis.

Cardiac fibrosis is observed across diverse etiologies of heart failure. Granzyme B (GzmB) is a serine protease involved in cell-mediated cytotoxicity in conjunction with the pore-forming protein, perforin. Recent evidence suggests that GzmB also contributes to matrix remodeling and fibrosis through an extracellular, perforin-independent process. However, the role of GzmB in the onset and progression of cardiac fibrosis remains elusive. The present study investigated the role of GzmB in the pathogenesis of cardiac fibrosis. GzmB was elevated in fibrotic human hearts and in angiotensin II-induced murine cardiac fibrosis. Genetic deficiency of GzmB reduced angiotensin II-induced cardiac hypertrophy and fibrosis, independently of perforin. GzmB deficiency also reduced microhemorrhage, inflammation, and fibroblast accumulation in vivo. In vitro, GzmB cleaved the endothelial junction protein, vascular endothelial (VE)-cadherin, resulting in the disruption of endothelial barrier function. Together, these results suggest a perforin-independent, extracellular role for GzmB in the pathogenesis of cardiac fibrosis.

A comparison of fluorescent DNA binding dyes for flow cytometric analysis of sporulating Saccharomyces cerevisiae.

Flow cytometry is important tool for investigating DNA replication in sporulating Saccharomyces cerevisiae. However, flow cytometry data from maturing spores is often difficult to interpret due to extensive broadening of the fluorescence peaks. This problem is markedly improved by treatment of the spores with potassium hydroxide prior to staining.

Structural elucidation of a PRP8 core domain from the heart of the spliceosome.

The spliceosome is a complex ribonucleoprotein (RNP) particle containing five RNAs and more than 100 associated proteins. One of these proteins, PRP8, has been shown to interact directly with the splice sites and branch region of precursor-mRNAs (pre-mRNAs) and spliceosomal RNAs associated with catalysis of the two steps of splicing. The 1.85-A X-ray structure of the core of PRP8 domain IV, implicated in key spliceosomal interactions, reveals a bipartite structure that includes the presence of an RNase H fold linked to a five-helix assembly. Analysis of mutant yeast alleles and cross-linking results in the context of this structure, coupled with RNA binding studies, suggests that domain IV forms a surface that interacts directly with the RNA structures at the catalytic core of the spliceosome.

The Saccharomyces cerevisiae CLB5 promoter contains two middle sporulation elements (MSEs) that are differentially regulated during sporulation.

The B-type cyclins Clb5 and Clb6 are essential activators of DNA replication during sporulation in Saccharomyces cerevisiae. The expression of CLB5 is maximally induced during the middle phase of sporulation by the transcription factor Ndt80. We have performed an analysis of the CLB5 promoter and have identified two middle sporulation elements (MSEs) that act as binding sites for Ndt80. Although both MSE sequences bind Ndt80 in vitro, they display differential effectiveness in their ability to function as cis-acting regulatory sequences in vivo. Mutation of both MSE sequences in the CLB5 promoter profoundly reduces the induction of CLB5 transcription during the middle phase of sporulation but results in no obvious defect in progression through meiosis and sporulation, implying that the Ndt80-dependent induction of CLB5 is not required for effective DNA replication or chromosome division.

Meiosis-specific regulation of the Saccharomyces cerevisiae S-phase cyclin CLB5 is dependent on MluI cell cycle box (MCB) elements in its promoter but is independent of MCB-binding factor activity.

In proliferating S. cerevisiae, genes whose products function in DNA replication are regulated by the MBF transcription factor composed of Mbp1 and Swi6 that binds to consensus MCB sequences in target promoters. We find that during meiotic development a subset of DNA replication genes exemplified by TMP1 and RNR1 are regulated by Mbp1. Deletion of Mbp1 deregulated TMP1 and RNR1 but did not interfere with premeiotic S-phase, meiotic recombination, or spore formation. Surprisingly, deletion of MBP1 had no effect on the expression of CLB5, which is purportedly controlled by MBF. Extensive analysis of the CLB5 promoter revealed that the gene is largely regulated by elements within a 100-bp fragment containing a cluster of MCB sequences. Surprisingly, induction of the CLB5 promoter requires MCB sequences, but not Mbp1, implying that another MCB-binding factor may exist in cells undergoing meiosis. In addition, full activation of CLB5 during meiosis requires Clb5 activity, suggesting that CLB5 may be regulated by a positive feedback mechanism. We further demonstrate that during meiosis MCBs function as effective transcriptional activators independent of MBP1.