Mechanistic Probes for the Epimerization Domain of Nonribosomal Peptide Synthetases.

Nonribosomal peptide synthetases (NRPSs) are responsible for the synthesis of a variety of bioactive natural products with clinical and economic significance. Interestingly, these large multimodular enzyme machineries incorporate nonproteinogenic d-amino acids through the use of auxiliary epimerization domains, converting l-amino acids into d-amino acids that impart into the resulting natural products unique bioactivity and resistance to proteases. Due to the large and complex nature of NRPSs, several questions remain unanswered about the mechanism of the catalytic domain reactions. We have investigated the use of mechanism-based crosslinkers to probe the mechanism of an epimerization domain in gramicidin S biosynthesis. In addition, MD simulations were performed, showcasing the possible roles of catalytic residues within the epimerization domain.

Methadone Inpatient and Discharge Prescribing Patterns for Pain at an Academic Health System.

BACKGROUND: Methadone is effective for the treatment of chronic pain, but its unique pharmacology requires additional considerations with dosing and monitoring. OBJECTIVE: The study objective was to evaluate methadone prescribing for pain and subsequent monitoring. METHODS: This retrospective chart review at a single center reviewed patients who received methadone over a two-year period. RESULTS: Of the 100 patients, most were noncancer cases (60%) with unspecified pain (50%). The majority of methadone treatments were initiated by medicine service (37%), followed by burn service (27%), and trauma (10%). Forty-two percent of the patients were being followed by the palliative care team, primarily for the medicine patients (80%). Patients on the burn service had significantly higher oral morphine equivalent (χ2 = 10.6, p = 0.01) and longest length of stay (χ2 = 37.9, p = 0.0001). Patients on medicine service were significantly more likely to have an outpatient discharge plan for methadone starts in the hospital (odds ratio = 3.7, confidence interval: 1.4, 9.7). Only 45% of patients had an electrocardiogram (EKG) checked seven days before methadone start and 37% of those have a measured corrected QT (QTc) of greater than 450 milliseconds. Electrolytes (potassium and magnesium) were not consistently checked and, of those that were evaluated, 15-20% were abnormal. There was an average of 2.6 severe or major drug interactions per patient related to methadone, with the most common being related to concomitant sedatives and other medications that prolonged the QTc. CONCLUSIONS: Prescribing guidelines for methadone would provide a consistent approach for all practitioners involved in using methadone safely and effectively for chronic pain.

Explorations of catalytic domains in non-ribosomal peptide synthetase enzymology.

Many pharmaceuticals on the market today belong to a large class of natural products called nonribosomal peptides (NRPs). Originating from bacteria and fungi, these peptide-based natural products consist not only of the 20 canonical L-amino acids, but also non-proteinogenic amino acids, heterocyclic rings, sugars, and fatty acids, generating tremendous chemical diversity. As a result, these secondary metabolites exhibit a broad array of bioactivity, ranging from antimicrobial to anticancer. The biosynthesis of these complex compounds is carried out by large multimodular megaenzymes called nonribosomal peptide synthetases (NRPSs). Each module is responsible for incorporation of a monomeric unit into the natural product peptide and is composed of individual domains that perform different catalytic reactions. Biochemical and bioinformatic investigations of these enzymes have uncovered the key principles of NRP synthesis, expanding the pharmaceutical potential of their enzymatic processes. Progress has been made in the manipulation of this biosynthetic machinery to develop new chemoenzymatic approaches for synthesizing novel pharmaceutical agents with increased potency. This review focuses on the recent discoveries and breakthroughs in the structural elucidation, molecular mechanism, and chemical biology underlying the discrete domains within NRPSs.

Activity-guided engineering of natural product carrier proteins.

A panel of chimeric carrier proteins was developed and screened for functional activity with essential enzymes involved in carrier protein-mediated biosynthesis. Regions on either side of the recognition helix II within three carrier proteins (CPs) from distinct biosynthetic pathways were swapped in all combinations to generate 24 mutated CPs. This panel of chimeric carrier proteins was tested using two previously established and one novel carrier protein assays. The results suggest a significant contribution from multiple structural units within carrier protein structure, rather than universal recognition helix, is necessary for proper recognition and activity with partner enzymes.

Crosslinking studies of protein-protein interactions in nonribosomal peptide biosynthesis.

Selective protein-protein interactions between nonribosomal peptide synthetase (NRPS) proteins, governed by communication-mediating (COM) domains, are responsible for proper translocation of biosynthetic intermediates to produce the natural product. In this study, we developed a crosslinking assay, utilizing bioorthogonal probes compatible with carrier protein modification, for probing the protein interactions between COM domains of NRPS enzymes. Employing the Huisgen 1,3-dipolar cycloaddition of azides and alkynes, we examined crosslinking of cognate NRPS modules within the tyrocidine pathway and demonstrated the sensitivity of our panel of crosslinking probes toward the selective protein interactions of compatible COM domains. These studies indicate that copper-free crosslinking substrates uniquely offer a diagnostic probe for protein-protein interactions. Likewise, these crosslinking probes serve as ideal chemical tools for structural studies between NRPS modules where functional assays are lacking.

Antibiotic evaluation and in vivo analysis of alkynyl Coenzyme A antimetabolites in Escherichia coli.

Pantothenamides have been the subject of much study as potential inhibitors of CoA and carrier protein dependent biosynthetic pathways. Based on an initial observation of growth inhibition in Escherichia coli by 3, we have synthesized a small panel of pantetheine analogues and re-examined the inhibitory properties of this class of antibiotics with an emphasis on understanding the ability of these compounds to act as substrates of native CoA and carrier protein utilizing biosynthetic pathways. Our findings suggest that a secondary structure-activity relationship is an important factor in the antibiotic activity of these compounds.

Probing the compatibility of type II ketosynthase-carrier protein partners.

Drug discovery often begins with the screening of large compound libraries to identify lead compounds. Recently, the enzymes that are involved in the biosynthesis of natural products have been investigated for their potential to generate new, diverse compound libraries. There have been several approaches toward this end, including altering the substrate specificities of the enzymes involved in natural product biosynthesis and engineering functional communication between enzymes from different biosynthetic pathways. While there exist assays to assess the substrate specificity of enzymes involved in these pathways, there is no simple method for determining whether enzymes from different synthases will function cooperatively to generate the desired product(s). Herein we report a method that provides insight into both substrate specificity and compatibility of protein-protein interactions between the acyl carrier protein (ACP) and ketosynthase (KS) domains involved in fatty acid and polyketide biosynthesis. Our technique uses a one-pot chemoenzymatic method to generate post-translationally modified ACPs that are capable of covalently interacting with KS domains from different biosynthetic systems. The extent of interaction between ACPs and KSs from different systems is easily detected and quantified by a gel-based method. Our results are consistent with previous studies of substrate specificity and ACP-KS binding interactions and provide new insight into unnatural substrate and protein interactions.

An orthogonal purification strategy for isolating crosslinked domains of modular synthases.

Chemo-enzymatic methods for covalently crosslinking carrier proteins with partner enzymes within modular synthases hold promise for elucidating and engineering metabolic pathways. Our efforts to crystallize the ACP-KS complexes of fatty acid synthases have been complicated by difficulties in the purification of the crosslinked complex from the other proteins in the reaction. Here we present a solution that employs an orthogonal purification strategy to achieve the quantity and level of purity necessary for further studies of this complex.