Mining the Metabiome: Identifying Novel Natural Products from Microbial Communities

Microbial-derived natural products provide the foundation for most of the chemotherapeutic arsenal available to contemporary medicine. In the face of a dwindling pipeline of new lead structures identified by traditional culturing techniques and an increasing need for new therapeutics, surveys of microbial biosynthetic diversity across environmental metabiomes have revealed enormous reservoirs of as yet untapped natural products chemistry. In this review, we touch on the historical context of microbial natural product discovery and discuss innovations and technological advances that are facilitating culture-dependent and culture-independent access to new chemistry from environmental microbiomes with the goal of reinvigorating the small molecule therapeutics discovery pipeline. We highlight the successful strategies that have emerged and some of the challenges that must be overcome to enable the development of high-throughput methods for natural product discovery from complex microbial communities

A Prolyl-Isomerase Mediates Dopamine-Dependent Plasticity and Cocaine Motor Sensitization

SummarySynaptic plasticity induced by cocaine and other drugs underlies addiction. Here we elucidate molecular events at synapses that cause this plasticity and the resulting behavioral response to cocaine in mice. In response to D1-dopamine-receptor signaling that is induced by drug administration, the glutamate-receptor protein metabotropic glutamate receptor 5 (mGluR5) is phosphorylated by microtubule-associated protein kinase (MAPK), which we show potentiates Pin1-mediated prolyl-isomerization of mGluR5 in instances where the product of an activity-dependent gene, Homer1a, is present to enable Pin1-mGluR5 interaction. These biochemical events potentiate N-methyl-D-aspartate receptor (NMDAR)-mediated currents that underlie synaptic plasticity and cocaine-evoked motor sensitization as tested in mice with relevant mutations. The findings elucidate how a coincidence of signals from the nucleus and the synapse can render mGluR5 accessible to activation with consequences for drug-induced dopamine responses and point to depotentiation at corticostriatal synapses as a possible therapeutic target for treating addiction

Peptidyl-prolyl Isomerization in the Regulation of Neuronal Signaling Pathways

Peptidyl-prolyl isomerases are a large class of enzymes that catalyze the cis/trans isomerization of prolyl bonds. Initially thought to function solely as chaperones and protein folding catalysts, in recent years a large number of biological substrates for these enzymes had been identified as crucial components in signaling and regulatory networks. However, little remains known about how acceleration of the isomerization rate of a Xaa-Pro bond acts as a regulatory mechanism. In the present work, the mechanisms of the Pin1-mediated regulation of the metabotropic glutamate receptor 5 (mGluR5) signaling and the FKBP mediated gating of TRPC1 channel are examined with respect to the bind-ing and catalytic interactions with the objective of elucidating the molecular mechanisms underlying the physiological function of these receptors. Phosphorylation of the Ser1126-Pro site in mGluR5 and the subsequent interaction of this site with Pin1 have been previously shown to be required for the potentiation of the mGluR5-mediated physiological output involved in the mechanism of neural plasticity. In addition, expression of Homer1a protein was determined to be required in the context of constitutively expressed Homer1c post-synaptic density scaffolding proteins. In Chapter 2 we demonstrate that Homer1a and Homer1c proteins interact with both cis and trans isomers of mGluR5 at the Ser 1126-Pro bond in essentially identical way suggesting that Homer1a does not impart additional functionality on mGluR5. Phosphorylation increases the Homer affinity for mGluR5 ~15-fold but does not significantly affect the cis/trans distribution of pSer1126-Pro bond in the free or Homer-bound mGluR5. Experiments discussed in Chapter 3 directly demonstrate Pin1's ability to efficiently catalyze the isomerization of the pSer 1126-Pro bond. In an unexpected twist, we discovered that the catalytic domain of Pin1 binds single phosphorylated mGluR5 with ∼20-fold grater affinity than the WW domain and that the WW domain is not selective for the trans isomer as had been previously believed. Based on the experimental data we are able to pro-pose a potential model for the function of Pin1 in mGluR5-mediated signaling. In Chapter 4 we provide direct evidence for the FKBP12 and FKBP52 catalysis of the L19-Pro and L644-Pro bonds in the C- and N-terminal domains of the TRPC1 channel believed to be involved in the regulation of the channel gating. Additionally, we identify a previously overlooked FKBP target site in the C-terminal region of TRPC1 catalyzed primarily by FKBP12 and propose that the differential catalysis of the adjacent C-terminal sites by FBP12 and FKBP52 may underlie the different physiological functions of the two isomerases

eSNaPD: A Versatile, Web-Based Bioinformatics Platform for Surveying and Mining Natural Product Biosynthetic Diversity from Metagenomes

SummaryEnvironmental Surveyor of Natural Product Diversity (eSNaPD) is a web-based bioinformatics and data aggregation platform that aids in the discovery of gene clusters encoding both novel natural products and new congeners of medicinally relevant natural products using (meta)genomic sequence data. Using PCR-generated sequence tags, the eSNaPD data-analysis pipeline profiles biosynthetic diversity hidden within (meta)genomes by comparing sequence tags to a reference data set of characterized gene clusters. Sample mapping, molecule discovery, library mapping, and new clade visualization modules facilitate the interrogation of large (meta)genomic sequence data sets for diverse downstream analyses, including, but not limited to, the identification of environments rich in untapped biosynthetic diversity, targeted molecule discovery efforts, and chemical ecology studies. eSNaPD is designed to generate a global atlas of biosynthetic diversity that can facilitate a systematic, sequence-based interrogation of nature’s biosynthetic potential