EditorialINNOVATIVE MEDICINES INITIATIVE

TRANSLOCATION Project: How to Get Good Drugs into Bad Bugs

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Science Translational Medicine  19 Mar 2014:
Vol. 6, Issue 228, pp. 228ed7
DOI: 10.1126/scitranslmed.3008605

Robert A. Stavenger

Mathias Winterhalter

CREDIT: GLAXOSMITHKLINE (R.A.S.); JACOBS UNIVERSITY BREMEN (M.W.)

The scientific literature, lay press, and government bodies all now recognize antimicrobial resistance (AMR) as a major global public health threat (13). Although the need for new antimicrobials is urgent—highlighted by the increasing use of drugs of last resort (such as colistin)—only two new classes of antibiotics have been brought to market in the past 30 years, and many drug developers have left the field. This dearth of new drugs is especially severe for infections caused by Gram-negative bacteria, whose intrinsic penetration barriers make them more difficult to target than their Gram-positive cousins. Leaders in both the industry and government have realized that given the large unmet medical need and the future threat AMR poses to society, a more collaborative approach is needed to improve the likelihood of success in antibacterial R&D (www.cdc.gov/drugresistance/threat-report-2013). The “New Drugs 4 Bad Bugs” (ND4BB) initiative (http://www.nd4bb.eu) is a broad series of public-private partnerships under the umbrella of the Innovative Medicines Initiative (IMI, www.imi.europa.eu) designed to address scientific and financial challenges associated with antibacterial drug discovery and development. Here, we discuss the TRANSLOCATION project, a component of the ND4BB platform specifically aimed at addressing drug penetration into Gram-negative bacteria—that is, getting good drugs into bad bugs (www.imi.europa.eu/content/translocation).

BARRIERS TO THERAPY

Gram-negative bacteria are surrounded by a cellular envelope that comprises outer and inner membranes with distinct properties and provides a potent physical barrier to antibacterial agents. The discovery of new agents to treat drug-resistant Gram-negative infections generally relies on the agent’s ability to penetrate at one or both envelope membranes. Furthermore, even if an agent penetrates these membranes, it can be rapidly transported out of the cell by numerous broadly acting efflux pumps, rendering the agent ineffective. The combination of this intrinsic dual-penetration barrier with the potential for rapid efflux often leads to situations in which compounds with intrinsic activity against intracellular or periplasmic targets display poor antibacterial activity. At present, there are no general reliable methods for measuring these penetration and efflux processes in Gram-negative bacteria, a bottleneck that substantially hinders the ability of scientists to optimize antimicrobial activity in intact bacterial cells. The scope of this deficiency in modern antibacterial drug discovery is too large to be solved by a single group or company because of the complexity of the penetration and efflux processes. The TRANSLOCATION project was created to provide the industrial, biotechnology, and academic communities with renewed optimism as well as tools for the discovery of antibiotics to treat the severe, multidrug-resistant Gram-negative infections that are becoming more and more common across the globe. This large, consortium-based effort spans nine countries, five pharmaceutical companies, five small-medium enterprises, and 14 academic institutions, mostly from across Europe.

An important goal of the TRANSLOCATION project is to optimize in vitro assay conditions so as to better mimic envelope permeability at infection sites. Gram-negative bacteria such as Pseudomonas aeruginosa, a causative agent in many hospital-acquired infections, readily adapt to their environment by remodeling their cell envelopes. This remodeling can take place via differential expression of one or more of the roughly 30 porins (water-filled channels that allow nutrients and other molecules to penetrate this envelope), induction of one or more of ~10 efflux pumps, or modifications to the lipopolysaccharide core of the outer membrane itself. Ultimately, these adaptation processes substantially affect the penetration of antimicrobials. However, current assays for antimicrobial screening in bacterial cells in culture rely on standardized in vitro conditions. Although useful, such conditions may not fully reproduce relevant conditions that pathogens encounter in infected host tissues. As a consequence, antimicrobials with promising activity under standard in vitro conditions might not be as effective in vivo because of insufficient penetration or intracellular residence time in that specific environment. The TRANSLOCATION initiative aims to address this issue.

This work will be complemented by assembling a set of clinical isolates that cover a range of relevant membrane-permeability resistance mechanisms. For this work, researchers will assemble a database in which ~100 clinical strains from P. aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Enterobacter spp. are characterized by various components of their cell envelopes and how those components correlate with antibacterial susceptibility. Such a database will help to rapidly assess the potential spectrum and coverage of new antimicrobial candidates early in the discovery and development process.

In addition, a proteomic shotgun approach will be used to characterize and quantify porin and efflux-pump changes that occur in an in vivo—relative to an in vitro—setting in order to identify the key players in the penetration-efflux continuum. In order to quantify the contribution of these players to drug permeability, a direct and general permeability assay must be developed and validated. Mass spectrometry (MS) is a promising technique for measuring an agent’s ability to permeate the Gram-negative bacterial envelope; the current detection limit of ~20 pmol allows investigators to discriminate the uptake of fewer than 500 antibiotic molecules per bacterial cell, well below the typical minimum inhibitory concentration. Ultimately, this approach might help to characterize the permeability of hundreds to thousands of potential drugs and to quantify antibiotic uptake and efflux kinetics. In the case of fluorescent drugs, uptake can also be studied by using a high-intensity synchrotron light source. For example, the quinolone antibiotic fleroxacin provides a sufficient change in fluorescence to examine its accumulation in single bacterial cells (4).

Detailed information on the rate-limiting steps during antimicrobial permeation can be obtained by combining high-resolution structural information with modeling of the porin-drug interaction and electrophysiology (57). Recent progress in membrane-protein crystallization techniques makes it possible to obtain high-resolution structures from most of the relevant porins and, to a lesser extent, bacterial efflux pumps. Using these high-resolution structures as starting points for all atom modeling allows the identification of a putative permeation pathway and identification of rate-limiting molecular interactions. Although substantial progress has been made, modeling the pathways of antibiotics through these extremely large systems (porins or efflux pumps) requires substantial computation time, and methods to catch rare events must be applied. The validity of pathway modeling can be verified experimentally by using electrophysiology techniques that can detect the interaction of small molecules with single reconstituted porins.

GETTING GOOD DATA INTO A SHARED DATABASE

Another general challenge in drug discovery lies in a lack of mechanisms (and desire) to share data, information, and experience from both failed and successful efforts. Some studies are published in the open literature, but many are not, especially those with negative or inconclusive results. This omission leads to duplication of efforts, lost opportunities for synergy, inefficiencies, and, ultimately, a lack of success in the R&D process. A common remit of the ND4BB projects is to provide a mechanism for an unprecedented sharing of antibacterial R&D information among companies and with the public sector.

The TRANSLOCATION project has been tasked with the creation, management, and population of a ND4BB Information Centre—a data repository that will house not only information from current and future ND4BB projects but also certain legacy antibacterial R&D data, from industry partners in ND4BB, on successes and failures of past efforts. This body of data will be analyzed further by members of the ND4BB community with the goal of answering open questions in antibacterial R&D—for example, what animal efficacy models are most predictive or how to design more efficient and meaningful clinical trials. It is expected that this Information Centre will bring greater transparency and sharing to the field, improvement in efficiency across antibacterial R&D, and, ultimately, more treatment options for patients.

The urgent need for new, effective Gram-negative drugs comes at a time when techniques needed for innovative assays—proteomic analyses, high-sensitivity MS, and computational bioinformatics approaches (8)—are well developed and can provide crucial missing data. Ideally, over the course of the TRANSLOCATION project pieces of the overall penetration-efflux puzzle will form part of a larger understanding of the Gram-negative cell envelope as well as a guide on how to create small molecules that can readily penetrate the bugs’ membranes. This information should move the antibacterial research community away from a “black box” mode of drug discovery and toward more rational approaches, which may enable the delivery of new agents to treat life-threatening infections.

References

  1. Funding: The authors are part of the TRANSLOCATION consortium and have received support from the Innovative Medicines Joint Undertaking under grant agreement 115525, the European Union’s seventh framework program (FP7/2007-2013), and European Federation of Pharmaceutical Industries and Associates companies in-kind contribution.

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