Biochemical basis for the regulation of biosynthesis of antiparasitics by bacterial hormones.

Diffusible small molecule microbial hormones drastically alter the expression profiles of antibiotics and other drugs in actinobacteria. For example, avenolide (a butenolide) regulates the production of avermectin, derivatives of which are used in the treatment of river blindness and other parasitic diseases. Butenolides and γ-butyrolactones control the production of pharmaceutically important secondary metabolites by binding to TetR family transcriptional repressors. Here, we describe a concise, 22-step synthetic strategy for the production of avenolide. We present crystal structures of the butenolide receptor AvaR1 in isolation and in complex with avenolide, as well as those of AvaR1 bound to an oligonucleotide derived from its operator. Biochemical studies guided by the co-crystal structures enable the identification of 90 new actinobacteria that may be regulated by butenolides, two of which are experimentally verified. These studies provide a foundation for understanding the regulation of microbial secondary metabolite production, which may be exploited for the discovery and production of novel medicines.

Bacteria that dwell in soil known as actinobacteria are the source of many drugs that are used to treat cancer and infectious diseases in humans. In their natural environments actinobacteria produce these drugs, or at least similar compounds, to compete with neighboring microbes for food or to kill their enemies. However, when researchers culture actinobacteria in the laboratory, the bacteria often produce little or none of these compounds. Some actinobacteria produce a compound called avermectin. This compound is closely related to a drug used to treat an infectious disease known as river blindness, which is a common cause of sight loss in people living in West Africa. A bacterial hormone known as avenolide regulates when the bacteria produce avermectin by binding to a receptor known as AvaR1. But the precise details of how this process works remained unclear. To investigate how avenolide binds to AvaR1, Kapoor, Olivares and Nair developed a new strategy to produce large quantities of avenolide in the laboratory from commercially available molecules. A three-dimensional structure of AvaR1 was then generated showing the receptor on its own, bound to avenolide or bound to a short DNA molecule. In the absence of avenolide, AvaR1 sits on DNA. However, binding to avenolide causes AvaR1 to move off the DNA. This revealed how the binding of avenolide changes the receptor protein so that it can be released from DNA to allow the production and release of other small molecule compounds. Further experiments used these structures as guides to identify 90 new species of actinobacteria that may respond to avenolide and other similar bacterial hormones. Understanding how bacterial hormones stimulate actinobacteria to produce avermectin and other compounds will aid efforts to extract new compounds from soil bacteria that have the potential to treat cancer or infectious diseases.

Structure-Guided Analyses of a Key Enzyme Involved in the Biosynthesis of an Antivitamin.

RosB catalyzes the formation of 8-aminoriboflavin 5'-phosphate (AFP), the key intermediate in roseoflavin biosynthesis, from the metabolic precursors riboflavin 5'-phosphate (RP, also known as FMN) and glutamate. The conversion of the aromatic methyl group at position 8 in RP into the aromatic amine in AFP occurs via two intermediates, namely, the aldehyde 8-formyl-RP and the acid 8-carboxy-RP. To gain insights into the mechanism for this chemically challenging transformation, we utilized a structure-based approach to identify active site variants of RosB that stall the reaction at various points along the reaction coordinate. Crystal structures of individual variants in complex with different reaction intermediates, identified via mass spectroscopic analysis, illuminate conformational changes that occur at the active site during multistep conversion. These studies provide a plausible route for the progression of the reaction and a molecular rationale for the mechanism of this unusual biocatalyst.

Integrating Display and Delivery Functionality with a Cell Penetrating Peptide Mimic as a Scaffold for Intracellular Multivalent Multitargeting.

The construction of a multivalent ligand is an effective way to increase affinity and selectivity toward biomolecular targets with multiple-ligand binding sites. Adopting this strategy, we used a known cell-penetrating peptide (CPP) mimic as a scaffold to develop a series of multivalent ligand constructs that bind to the expanded dCTG (CTG(exp)) and rCUG nucleotide repeats (CUG(exp)) known to cause myotonic dystrophy type I (DM1), an incurable neuromuscular disease. By assembling this polyvalent construct, the hydrophobic ligands are solubilized and delivered into cell nuclei, and their enhanced binding affinity leads to the inhibition of ribonuclear foci formation and a reversal of splicing defects, all at low concentrations. Some of the multivalent ligands are shown to inhibit selectively the in vitro transcription of (CTG·CAG)74, to reduce the concentration of the toxic CUG RNA in DM1 model cells, and to show phenotypic improvement in vivo in a Drosophila model of DM1. This strategy may be useful in drug design for other trinucleotide repeat disorders and more broadly for intracellular multivalent targeting.