Performing Department
Biochemistry
Non Technical Summary
The introduction of new antibiotics to counter emerging drug resistance will improve human and animal health. We describe the development of potent new analogs of inhibitors of 'old' antibiotics targets as a strategy for protecting animals from diseases and conditions caused by bacterial infections and protecting humans from foods contaminated by pathogenic microorganisms. It is widely recognized that antibiotic resistant bacteria emerge in confined animal feeding operations (particularly drug resistant Campylobacter and Salmonella spp.) can be transferred to humans through food. The Center for Disease Control estimates that there are ~76M cases of foodborne illness each year from viral and bacterial pathogens. The development of new classes of antibiotics--such as those described in this proposal--can relieve some of the pressure currently placed on the dwindling number of effective clinical antibiotics available for treating human infections.
Animal Health Component
25%
Research Effort Categories
Basic
95%
Applied
5%
Developmental
(N/A)
Goals / Objectives
Goals:The major goals of this project are to develop a new class of antibiotics that have broad-spectrum activity against pathogenic bacteria that are food-borne and infect farm animals and humans.Objective 1: Optimize an in silico approach to virtually screen modifications to DNA gyrase inhibitors (the gyramides) to increase their activity and reduce their efflux out of bacteria.Objective 2: Apply the in silico approach to screen a virtual library of gyramide inhibitors for activity; identify lead compounds.Objective 3: Synthesize 50 lead compounds, characterize their structures, and test their activity against a panel of 20 pathogenic bacteria that are relevant to the proposed knowledge areas.Objective 4: Test the activity of the top 5 compounds against recombinant DNA gyrase and measure inhibition and binding constants.
Project Methods
Objectives and hypothesesWe will optimize the potency of the gyramide antibiotics using an iterative cycle of structural biology design, including computational modeling of drug properties, chemical synthesis, and in vivo activity testing. To perform this work, we have formulated the following four-part specific aim.A. Design a virtual library of gyramides. Clinical antibiotics have a spectrum of structural characteristics that are generally considered to be broader than drugs for treating other diseases. O'Shea and Moser recently analyzed the molecular properties of known antibiotics and observed differences that contribute to the activities of small molecules against Gram-positive and Gram-negative bacteria. The information gleaned from these analyses is directly applicable to the design of new gyramide analogs. Although antibiotics active against Gram-positive bacteria are spread over a wide range of molecular weights--with many compounds exceeding 1200 Da--the authors identified a strict upper limit of 600 Da for Gram-negative bacteria. Similarly, the range of distribution coefficients at the physiological pH of human serum is lower for Gram-negative activity and the majority of antibiotics have values between -5 and 2. Antibiotics that are active against Gram-negative bacteria have high average relative polar surface areas (rPSA, which is normalized for total surface area) with values that are typically from 25-60. When the analysis was limited to antibiotics that are active against the Gram-negative human pathogen P. aeruginosa, the physicochemical requirements became very narrow. Not surprisingly, these compounds exhibit a narrow distribution of molecular weight (300-450 Da), cLogD7.4 (-2 to 2), and rPSA. The low and narrow spread of cLogD values for the fluoroquinolones is due to the amine and carboxylic acid groups, which makes them zwitterionic. By comparison, the gyramides exhibit a molecular weight that is moderately close to the fluoroquinolones (ciprofloxacin, 331 Da) and in principle can be modified to exhibit similar cLogD and rPSA properties by "tuning" the basicity of the pyrrolidine nitrogen and the acidity of the sulfonamide. This approach provides an opportunity to optimize the gyramides for bioavailability and antimicrobial activity comparable to ciprofloxacin. This goal is particularly attractive, as we have demonstrated that the gyramides can be used to treat bacteria that are resistant to ciprofloxacin.B. Identify gyramides that satisfy key predictors of biological activity against Gram-negative bacteria. We will optimize the gyramide structure for activity against Gram-negative bacteria by predicting compounds that have molecular properties that closely match successful, clinical antibiotics. We are particularly interested in designing compounds against Salmonella and Campylobacter spp. as these organisms have active drug efflux systems that create challenges for antibiotic uptake, cause widespread infections in both humans and livestock, and are emerging as drug resistant threats, particularly on confined animal feeding operations. We will perform the selection using two strategies: 1) evaluation of mW, cLogD7.4, and rPSA; and 2) application of a permeability model that uses extended connectivity fingerprints and calculated properties from a basis set of 6,500 compounds with known antimicrobial activity. The first strategy will enable a direct comparison of the gyramides to successful clinical antibiotics. The use of mW, cLogD, and rPSA provides an approximate set of guidelines that we can conveniently determine in silico. The second strategy enables us to perform iterative cycles of predicting and evaluating structures for developing and refining a predictive model. The first generation analogs will retain the existing sulfonamide core structure and will consist of pyrrolidine and aryl substituents that confer a range of molecular properties. We will enumerate a virtual library using aromatic aldehydes (2,808) and sulfonyl chlorides (740) that are commercially available en route to designing ~2,000,000 analogs. Both of the selection strategies/models will be applied to this library to generate sets of 2,000 compounds.C. Synthesize two gyramide libraries and assay them in vivo. From the sets of 2,000 compounds, we will select and design two libraries that will each consist of 100 compounds by matrix variation of the two substituents (10xR1 and 10xR2). Variation of matrix elements will ensure maximum synthetic efficiency, as we will synthesize 10 intermediates on a large scale (~1-5 g) and deprotect/sulfonylate in a single step. We will generate a second-generation library from the most active compounds identified by assaying the biological activity of compounds in libraries 1.1 and 1.2 using methods described below. We will select the most active compounds from each library and use them to design a new library that expands on the most effective structural subsets of libraries 1.1 and 1.2. For instance, if heterocyclic sulfonamides have high biological activity, we will make ring substitutions and compare imidazoles, oxazoles, thiazoles, isoxazoles, and other heterocycles. This level of variation is inappropriate for the first libraries, but at this later stage this approach may reveal analogs in which small electronic and structural perturbations enhance activity. Library 2.1 will consist of a focused 5x5 matrix of elements using the most effective compounds from libraries 1.1 and 1.2 as inputs. We will prepare additional 25-membered libraries (2.2 to 2.n) as needed to achieve compounds that display ?M or nM values of the minimum inhibitory concentration (MIC) against bacteria. Preliminary MIC data for gyramides against a relatively small panel of bacteria is summarized in. We will test all of the final library compounds against a panel of Salmonella and Campylobacter spp., including strains isolated from livestock facilities. To obtain and work with these strains, we have developed a relationship with the Wisconsin Veterinary Diagnostics Laboratory, have a portion of our lab cleared for BSL-2 testing, will access a BSL-2 facility within the Department of Biochemistry as needed, and will add these bacterial strains to our current Biosafety Protocol (B00000123).D. Refine compounds based on activity and by modeling into the DNA gyrase X-ray structure. During the synthesis of the two generations of libraries, we anticipate obtaining at least one high-resolution crystal structure of gyramides bound to the GyrBAfus-DNA complex (a recombinant version of a translationally fused Gyr A and B unit, which is not enzymatically function), which will provide a structural basis for the rational design of new analogs. In addition, we envision that the library synthesis will reveal compounds with increased activity against DNA gyrase and high bioactivity in bacteria, which we will use to form complexes with GyrBAfus and dsDNA and to solve the X-ray crystal structure. Based on the structural information provided by these two crystallography studies, we will design new gyramides by altering additional substituents and synthesizing bicyclic analogs. Although synthesis of these new gyramide analogs may require longer routes, the choice of a subset of these analogs should be possible using the conformation of bound analogs determined in the structural biology studies of GyrBAfus.