Developing resistance-free antibiotics by targeting quorum sensing
Way back in May 2000, Decision Resources published my short report entitled “New approaches to small-molecule antibacterial drug discovery” as part of its Spectrum Life Sciences series. As might be expected, the report is now out of print.
The report was a brief review of then-novel approaches to antibacterial drug discovery, in the face of the increasing level of antibiotic resistance in pathogenic bacteria. These approaches included genomics and such technologies as high-throughput screening against bacterial-specific targets.
However, the most interesting part of the report was a section on using the study of bacterial physiology to identify targets that are important for the ability of bacteria to cause disease, but are not essential for bacterial proliferation or survival. The hypothesis behind these studies was that it might be possible to develop compounds that prevent these bacteria from causing disease, without selecting for resistant strains of the bacteria.
Antibiotics typically kill or prevent proliferation of bacteria by targeting biomolecules involved in such essential processes as cell wall synthesis, DNA proliferation, or protein synthesis. Treating large populations of bacteria with such agents inevitably selects for a few resistant mutant cells. These proliferate, mutate further, and give rise to antibiotic resistant populations. However, if a therapeutic targets a nonessential pathway that is involved in pathogenesis, resistant populations might not be selected for. That was the hypothesis.
This field of bacterial physiology for drug discovery focused on two related areas–virulence factors and quorum sensing. Virulence factors are not expressed by a strain of pathogenic bacteria in vitro, but are expressed only when the bacteria infect a host. Once expressed, they enable the bacteria to colonize the host and cause disease. Examples of such virulence factors include secretion systems that deliver bacterial effector proteins into host cells. These effector proteins may, for example, kill host cells, inhibit cytokine production or phagocytosis, or may mediate bacterial entry into the host cells.
Quorum sensing is a system by which certain bacteria can monitor their own population density. They accomplish this by secreting specific autoinducer molecules. When the concentration of an autoinducer reaches a critical threshold value (as the result of an increase in bacterial population density), it triggers specific response systems, causing the induction of sets of genes that are only expressed at high population density.
For example, many gram-negative bacteria (e.g., Pseudomonas aeruginosa, Vibrio cholerae, and Escherichia coli) use specific acyl homoserine lactones (AHSLs) as their autoinducers. P. aeruginosa has two quorum sensing systems that use the AHSL autoinducers butyrylhomoserinelactone and 3-oxododecanoylhomoserinelactone, respectively. These systems (operating via specific receptors for the auotoinducers and interacting with each other) control the induction of several genes, some of which are virulence factors. Some of these genes enable the bacteria, when they are at sufficient density, to form biofilms (slimy mats of bacteria and polysaccharide matrix).
P. aeruginosa is an opportunistic pathogen, causing infection in the lungs of people with cystic fibrosis, burn patients, and other hospitalized patients. These infections cause death in over 80% of cystic fibrosis patients. The ability to form biofilms renders the bacteria resistant to antibiotics and to the patient’s own immune system.
Other gram-negative bacteria that form biofilms have been implicated in dental caries, peridontitis, osteomyelitis, and numerous nosocomial infections. Bacterial biofilms can also form on the surface of implanted medical devices, such as catheters and mechanical heart valves, and cause device-related infections.
The gram-positive human pathogen Staphylococcus aureus also has a quorum sensing system. However, it does not use an AHSL as an autoinducer. The S. aureus autoinducers are peptides that contain an unusual thiolactone structure (i.e., a thol ester-linked cyclic structure). The S. aureus quorum sensing system controls the synthesis of virulence factors responsible for the pathogenicity of this organism in vivo. Although specific peptides induce virulence factors in a given strain of S. aureus, there are other specific peptides that inhibit the induction of virulence in strains of the organism other than the one secreting the inhibitory peptides. That finding suggested that researchers should be able to develop specific agents to shut down S. aureus pathogenesis by targeting the quorum sensing system.
Interestingly, quorum sensing-based systems have been used in projects for the International Genetically Engineered Machine (iGEM) competition, an annual undergraduate synthetic biology competition. See the figure above, which was taken from the 2009 Chiba University (Japan) iGEM project. [http://2009.igem.org/Team:Chiba/Project/Signaling-system]
Quorum Sciences and Vertex Pharmaceuticals’ research on quorum sensing
At the time of the writing and publication of our antibacterial drug discovery report, there was a company, Quorum Sciences (Iowa City, IA) that had been established to commercialize the findings of leading researchers on bacterial quorum sensing. As the result of two successive acquisitions in 2000 and 2001, Quorum Sciences passed into the hands of Vertex Pharmaceuticals (Cambridge, MA). In 2006, Vertex researchers and their academic collaborators published a report on the discovery of novel specific inhibitors of the P. aeruginosa quorum sensing system. The last author of this report was quorum sensing pioneer E. Peter Greenberg, formerly of the University of Iowa and chief scientific officer at Quorum Sciences, and from 2005 to the present at the University of Washington School of Medicine. The compounds identified in the 2006 report, discovered via high-throughput screening of a diverse 200,000-compound chemical library, resembled the natural AHSL that binds to the P. aeruginosa quorum sensing receptor LasR. (LasR is a transcription factor that when bound to its specific AHSL, mediates the expression of a set of downstream genes, including those that encode virulence factors.) The researchers concluded that the novel quorum sensing inhibitors might be useful chemical tools, but not drug leads.
In 2010, other academic researchers published a report on the discovery of novel antagonists and agonists of the P. aeruginosa quorum sensing receptor LasR, which were of lower molecular weight and otherwise structurally distinct from the natural P. aeruginosa AHSL. However, these compounds were still deemed to be scaffolds for chemical tools, not drug leads. Nevertheless, the researchers speculated that the compounds “could, with further development, provide a pathway for the design of novel antivirulence agents”. Other researchers are continuing studies aimed at discovery of quorum sensing receptor antagonists, whether synthetic organic molecules or natural products. These involve studies with quorum sensing systems of both gram-positive and gram-negative bacteria.
The 2006 report appears to be the last Vertex publication on quorum sensing. However, Vertex continues to conduct research on antibacterial agents. And the company has a facility in the University of Iowa BioVentures Center (Coralville, IA), which is a continuation of the old Quorum Sciences Iowa facility. As of 2009, Vertex’s Iowa-based team consisted of seven full-time scientists, working on development of antibacterials, and agents to treat hepatitis C and cystic fibrosis, among other areas. The Iowa group participated in the development of Vertex’ now-marketed anti-hepatitis C virus (HCV) agent Incivek (telaprevir).
The May 2012 article “Freezing Time” in The Scientist, and discovery of novel quorum sensing inhibitors
The May 2012 issue of The Scientist contains an article entitled “Freezing Time”, by Vern L Schramm, Ph.D. (Albert Einstein College of Medicine (Bronx, NY). The article focused on design of “transition state analogues”, i.e., compounds with a chemical structure that resembles the transition state of a substrate in an enzyme-catalyzed reaction. Transition state analogs usually act as enzyme inhibitors by blocking the enzyme’s active site. They are exquisitely potent and specific inhibitors, which act at extremely small doses. This makes these compounds potentially attractive as drugs.
A transition state analogue inhibitor that was designed by Dr. Schramm and his colleagues in the early 2000s as an early proof-of-concept molecule is immucillin-H, or forodesine. This is a transition-state analog inhibitor of purine nucleoside phosphorylase. Forodesine is being developed by BioCryst Pharmaceuticals for treatment of relapsed B-cell chronic lymphocytic leukemia, and the results of a Phase 2 trial were published in 2010.
As described in Dr. Schramm’s May 2012 article, his laboratory has been applying their transition-state analogue technology to the field of quorum sensing in bacteria. Instead of targeting the recognition of AHSLs by quorum sensing receptors such as LasR, the researchers targeted the key enzyme in the AHSL biosynthesis pathway in gram-negative bacteria, known as 5′-methylthioadenosine nucleosidase (MTAN). The biosynthetic pathway for the production of AHSLs, including the key role of MTAN, had been elucidated by Dr. Greenberg and his colleagues in the late 1990s.
Dr. Schramm and his colleagues published the results of studies of three transition state analogues that potently inhibited MTANs of gram-negative bacteria. For example, they inhibited the Vibrio cholerae MTAN with dissociation constants of 73, 70, and 208 pM, respectively. They inhibited MTAN in cell of a virulent strain of V. cholerae with IC50 values of 27, 31, and 6 nM respectively, disrupting autoinducer production in a dose-dependent manner without affecting bacterial growth. The compounds were also potent inhibitors of autoinducer production in an enterohemorrhagic strain of Escherichia coli. The transition-state analogues did not inhibit growth in either V. cholerae or E. coli, but one such compound reduced biofilm production by 18% in E. coli and 71% in V. cholerae.
Moreover, the MTAN inhibitors did not appear to select for bacterial resistance in vitro. When V. cholerae bacteria were grown for 26 generations in the presence of a large excess of MTAN inhibitors, subsequent generations of these bacteria were equally sensitive to inhibition by these compounds as bacteria that had not been previously exposed to the inhibitors. These results are consistent with the hypothesis that agents that inhibit targets that are important in the ability of bacteria to cause disease, but are not essential for bacterial proliferation or survival might not select for drug resistance.
As Dr. Schramm said in the May 2012 article in The Scientist, it remains to be seen whether the MTAN-targeting transition-state analogs developed in his laboratory can translate into novel antibiotics that do not select for resistant pathogens. As of March 2009, Dr. Schramm’s team had developed over 20 potent MTAN inhibitors, which will be specific for bacteria and should have no effect on human metabolism. These compounds have been licensed to Pico Pharmaceuticals (Melbourne, Australia), which plans to develop and initiate clinical trials. Dr. Schramm is a Pico Pharmaceuticals co-founder and chairman of its scientific advisory board. Pico claims that one of its quorum sensing inhibitors, designated as PC0208, has demonstrated proof-of-concept in preclinical studies, and now has “pre-IND” status.
Lessons from these studies
Dr. Schramm’s discovery of novel quorum sensing inhibitors was made possible by a strategy that involved a combination of biology-driven drug discovery and sophisticated chemistry technology. The biology-driven drug discovery strategy involved a combination of 1. Building on the quorum sensing studies of Dr. Greenberg and others, and adopting the strategy, as reviewed in our 2000 Spectrum report, of targeting the quorum sensing system in order to discover agents that would have the possibility of not triggering resistance, and 2. Targeting a critical, bacterial-specific pathway enzyme that is upstream of the recognition of AHSLs by quorum sensing receptors (the usual target of most researchers in this area). This enzyme, MTAN, has a key role in the biosynthesis of AHSLs.
The sophisticated chemical technology employed by Dr. Schramm and his colleagues was of course the transition state analogue technology developed in his own laboratory. Combined with the biology-driven strategy described in the last paragraph, Dr. Schramm’s approach has succeeded in the discovery of compounds that are potential drug candidates, while approaches based on high-throughput screening for AHSL antagonists have so far failed to produce any such compounds. Dr. Scharamm’s laboratory has also obtained evidence that treatment with their compounds should not result in the selection of resistant strains of pathogenic bacteria.
It is possible that other chemistry approaches might be successfully employed to discover quorum sensing inhibitors, both for gram-negative bacteria and gram-positive organisms such as S. aureus.
As we have discussed in numerous articles on this blog, biology-driven drug discovery strategies, often coupled with innovative approaches to chemistry (in the case of small-molecule drug discovery) are applicable to very many different targets involved in a whole range of human diseases. (Biology-driven drug discovery has also been central to discovery and development of many successful large-molecule drugs.) The quorum sensing case study in this article is a simple, understandable, and elegant example of such a strategy.
In addition to the scientific, clinical, and medical aspects of antibacterial drug discovery, the other major issue is the business of antibacterial discovery and development. The economics of drug discovery and development have shifted pharmaceutical industry investment away from the development of drugs targeting short course therapies for acute diseases (such as antibacterials) and towards long-term treatment of chronic conditions. At the same time, discovery of novel antibacterials has gotten more difficult. As a result, during the 2000-2010 period, such companies as Wyeth, Aventis, Eli Lilly, GlaxoSmithKline, Bristol-Myers Squibb, Abbott Laboratories, Proctor & Gamble, and Merck have either deprioritized anti-bacterial R&D or left the field altogether. Meanwhile, antibiotic resistance, which was a problem in 2000, has become an even greater problem in 2012, in some cases reaching crisis proportions [e.g, methicillin resistant S. aureus (MRSA) that is also resistant to the drug of last resort, vancomycin].
As a result of these economic, scientific, and medical challenges, a €223.7 consortium of five pharmaceutical companies and leading academics, called NewDrugs4BagBugs (ND4BB) was launched in Europe in May 2012. The program is envisioned to involve a three-stage approach – to improve the understanding of antimicrobial resistance, to design and implement efficient clinical trials, and finally, to take novel drug candidates through clinical development.
And at least one venture capitalist has observed that biotechs that specialize in antibacterial drug development (as well as those that specialize in other areas that have been deemphasized by Big Pharmas) have provided “contrarian opportunities” in biotech venture. According to a June 2 2012 article by Bruce Booth of Atlas Venture published in Forbes, what has been deprioritized by some (or several) Big Pharmas, are likely be re-prioritized by others several years later. Such antibacterial drug developers as Calixa, Cerexa, Novexel, Neutec, Paratek, Pennisula, Protez, and Vicuron have produced some of the best returns in biotech venture capital from merger/acquisition exits. These biotechs included companies that were built around compounds outlicensed from Big Pharma, and others that conducted new research on novel targets, especially for MRSA and other resistant bacteria. By taking advantage of a strategic depriorization in Pharma, these biotechs and their venture backers were able to create considerable value in the past decade out of antibacterial drug development.
Meanwhile, antibiotic specialist Cubist Pharmaceuticals (Lexington, MA) remains an independent, and profitable, biotech company that is continuing to conduct R&D, including on discovery and development of agents to treat pathogens that are resistant to current antibiotics. It has expanded into development and marketing of peripheral mu-opioid receptor antagonists (including via acquisition of Adolor in 2011), and has recently expanded its R&D facilities.
Can Pico Pharmaceuticals (which has oncology programs in addition to antibacterials) experience similar success?
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