Research

Infectious Disease

Jump to: Vancapticin | Mode of action of antibiotics | Dengue | Antifungal | Anaerobes | Gram positives |Gram negatives |

 

Vancapticin

Bacterial resistance to antibiotics is an increasingly serious threat to the ability to routinely treat microbial infections, and is resulting in increased hospitalisation and mortality. Two Gram positive bacteria, Enterococcus faecium and Staphylococcus aureus, are members of the ‘ESKAPE’ family of pathogen ‘superbugs’ that have developed partial or complete resistance to multiple antibiotics [1]. Other Gram positive strains, such as Streptococcus pneumonia and Clostridium difficile, are also responsible for many harmful infections. The glycopeptide antibiotics, predominantly vancomycin (approved in 1958), have come to be relied upon as a drug of ‘last resort’ for life-threatening Gram positive infections that do not respond to other antibiotics. However, VISA/GISA (Vancomycin/Glycopeptide Intermediate resistant Staphylococcus aureus) and VRE (Vancomycin Resistant Enterococci) strains have emerged. In addition to the long-standing existing clinical glycopeptide antibiotics vancomycin and teicoplanin, several new glycopeptide antibiotics have either recently been approved (Telavancin, FDA approved 2009) or are in late stage clinical trials (Oritavancin, Dalbavancin).

While it is well recognized that there is an urgent need to develop new antibiotics that are effective against resistant bacteria, attempts to identify novel classes of compounds have been remarkably unproductive, with almost a 30 year gap before the clinical introduction of two new types of systemic antibiotics in 2000 (the oxazolidinones, linezolid FDA approved 2000) and 2003 (the lipopeptides, daptomycin FDA approved 2003) [2]. Furthermore, structural or mechanistic novelty does not guarantee immunity from resistance, with strains resistant to linezolid identified prior to FDA approval. Modifying existing antibiotics to overcome resistance mechanisms presents an opportunity to rationally develop effective new drugs more rapidly than screening for new structures.

We have developed a series of potent glycopeptide antibiotics by chemical modification of the out-of-patent antibiotic vancomycin, attaching membrane-selective elements that target the drug to bacterial membranes in preference to those of human cells. In addition these motifs improve binding to the bacterial cell wall precursor Lipid II. The latest generation of compounds are 10- to 100-fold more active than other glycopeptides against the drug-resistant bacteria MRSA and VRE, with no measurable haemolytic or cytotoxic activity and >90% stability in human plasma for over 6 hours. Preliminary pharmacokinetic studies in mice indicate an extended half life compared to vancomycin, with the potential for once a day dosing. The group’s efforts are focused on conducting further structure-activity-relationship studies and optimising physicochemical properties, with the goal of producing a preclinical drug candidate. Mode of action studies and structural characterisation (x-ray, NMR, molecular modelling) are also in progress.

 

[1] Boucher, H.W.; et. al., Clin. Infect. Dis. 2009, 48, 1.

[2] Hamad, B., Nat. Rev. Drug Discov. 2010, 9, 675.

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Mode of action of antibiotics

The mode of action of antibiotics, a term used to describe exactly how they kill bacteria, is commonly separated into four categories:

  1. Cell wall (e.g. penicillins, cephalosporins, glycopeptides, daptomycin)
  2. Protein synthesis (e.g. macrolides, aminoglycosides, oxazolindones, tetracyclines, pleuromutilin)
  3. DNA replication (e.g. quinolone, novobiocin)
  4. Folate synthesis inhibition (e.g. trimethoprim, sulfonamides)

Determining the mode of action of synthesised antibiotics is an integral part of our antibiotic drug discovery strategy. We believe that a thorough understanding of mode of action is important information that can be combined with biological activity data to design better drugs. These data are also very important when selecting candidate compounds for further development.

Many of our established mode of action studies have been based around bacterial cell wall inhibiting antibiotics. For example, a radioligand assay is used to probe bacterial cell wall biosynthetic steps that are inhibited after antibiotic treatment with 14C UDP-GlcNAc / -MurNAc-pentapeptide soluble precursors.

Other assays are used to measure antibiotic effect on bacterial, human cell line and erythrocyte membranes. Examples include the fluorescent measurement of the release of membrane associated dye diSC3-(5), which correlates with membrane disruption in bacterial cells and erythrocytes, and measurement of incorporating of DNA staining dye SYTOX Green into bacteria. We have also developed surface plasmon resonance (SPR) methods that use reconstituted lipid extracts derived from human erythrocytes, liver and kidney cells versus those derived from sensitive and drug-resistant bacterial strains to measure compound membrane binding. Antagonism (ligand competition) assays (e.g. obtaining MICs of vancomycin derivatives in the presence of Ac2-Lys-DAla-DAlaand Ac2-Lys-DAla-DLac) are also undertaken to gain insights into the relative activity and specificity of antibiotics.

Whole genome sequencing of antibiotic resistant bacterial strains obtained from clinical isolates, by direct selection and serial passage studies are used to probe the origin and evolution of resistance on a molecular level.

We use various forms of microscopy to examine the antibiotic effects on bacteria, cells and erythrocytes. For example, we have observed the direct effect of an antibiotic on erythrocytes using a wide field microscope, image specifically designed fluorescent derivatives to observe antibiotic localisation and used a Single Electron Microscope (SEM) to observe bacterial damage caused by antibiotic treatment.

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Dengue

Dengue is a significant public health threat, with estimates of 50 to 100 million cases per year and around 3 billion people at risk of infection [1]. There have now been epidemics reported in over 100 countries [2]. Importantly, the global burden from dengue infections is likely to be much higher than current prevalence data suggests with only 10% of symptomatic cases believed to be reported [3].

Dengue is a febrile illness caused by the dengue virus, a group of single stranded RNA viruses. The four serotypes of dengue are transmitted to humans primarily via Aedesaegypti mosquitoes. Infection can result in a broad spectrum of disease syndromes ranging from an asymptomatic or mild infection, classical dengue fever (DF), to the potentially fatal dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) [4]. There are currently no vaccines or effective therapeutics available and early and accurate detection is imperative to assist with the clinical management of symptoms.The ongoing challenge for dengue diagnosis, and in particular low cost serological assays, is the need for sensitivity towards all four related, but antigenically distinct, serotypes whilst at the same time limiting unwanted cross-reactivity to other members of the flaviviridae family that co-circulate in dengue endemic regions, such as Japanese Encephalitis, and Yellow Fever.

 

The Cooper group is currently involved in a number of different dengue related projects including the development of reagents for improved diagnosis and surveillance, and screening of anti-viral therapeutics. We are actively engaged and collaborating with experts and key opinion leaders from academia, industry and clinical research.

We are working with Prof. Paul Young and Prof. Bostjan Kobe at the School or Chemistry and Molecular Biosciences (UQ) as well as Prof. Che at the Southern Medical University (Guangzhou, China) to better understand the structural and molecular biology of the interaction between the virus and host cells, such as the role of domain III of the virus envelope protein in cell fusion. We are also collaborating with A/Prof Stephen Mahler’s group at AIBN in the development of high affinity reagents against the non-structural protein-1 (NS1), an important early marker of infection.We currently have an ARC linkage grant with Alere, a multinational diagnostics company with a local operation that specialises in rapid tests, to develop best-in-class diagnostics. We are also undertaking a comprehensive bioinformatic analysis of published dengue sequences and whole genome sequencing on present-day clinical isolates to aid in the identification of highly specific antigens for immunassay and serology testing platforms.

[1] Kyle, J. L.; Harris E., Annu. Rev. Microbiol. 2008, 62, 71.

[2] Teles, F. R. R.; Prazeres, D. M. F.; Lima-Filho, J. L., Rev. Med. Virol. 2005, 15, 287.

[3] World Health Organisation., “Strengthening Implementation of the Global Strategy for Dengue Fever/Dengue Haemorrhagic Fever Prevention and Control” 2000

[4] Mairuhu, A. T. A.; Wagenaar, J.; Brandjes, D. P. M.; Gorp, E. C. M., Eur. J. Clin. Microbiol. Infect. Dis. 2004, 23, 425.

 

Antifungal

Opportunistic pathogenic fungi such as Cryptococcus neoformans, Candida Albicans and Aspergillus Fumigatus pose an increasing threat to human health. The mortality rate for fungal infections is now comparable to that for tuberculosis and malaria.(1) Fungal infections are particularly common for immunocompromised individuals typically HIV/AIDS patients and persons undergoing immunosuppressive treatment such as transplant and chemotherapy.(2) One of the most problematic fungal infections is C. neoformans which enters the host through inhalation of the spores.(3) The fungus can disseminate from the lungs, through the bloodstream, to the central nervous system to cause meningoencephalitis - invariably fatal if untreated. The most recent data from the CDC estimates the cryptococcal meningitis mortality rate in developed nations to be around 12%. By contrast, sub-Saharan Africa has the greatest incidence of disease with approximately 1 million cases per year and mortality rate of around 60%.(4)


Fungal infections are very difficult to treat(1) primarily due to the similarities between fungal and human physiology. Pharmacologic management of cryptococcal meningoencephalitis in developed nations usually involves induction therapy with amphotericin B and flucytosine, followed by consolidation therapy with fluconazole for as long as 12 months. Immunodeficient individuals who survive the initial infection are often given lifelong antifungal therapy and frequently undergo relapse, although the incidence decreases for patients undergoing immune reconstitution therapy. Antifungal therapies are few in number, costly with high toxicity and notoriously variable efficacy across the spectrum of human fungal pathogens. Furthermore fungal strains are now emerging which are resistant to currently used therapeutics. It is alarming to note that antifungal therapies have not altered significantly in over a decade and, in many cases, are proving to be inadequate.(5,6)


We and have identified a number of exciting molecules active against common pathogenic fungi from high throughput screening of small molecules and natural products. We are using structure based drug discovery and medicinal chemistry to fully investigate the therapeutic potential of these promising highly novel molecules in our pipeline, actively working towards antifungal agents of the future.


REFERENCES

1. Xie, J. L., Polvi, E. J., Shekhar-Guturja, T., and Cowen, L. E. (2014) Elucidating drug resistance in human fungal pathogens. Future microbiology 9, 523-542
2. Perfect, J. R. (2014) Cryptococcosis: a model for the understanding of infectious diseases. The Journal of clinical investigation 124, 1893-1895
3. Sabiiti, W., and May, R. C. (2012) Mechanisms of infection by the human fungal pathogen Cryptococcus neoformans. Future microbiology 7, 1297-1313
4. CDC Centers for Disease Control and Prevention (2014). Cryptococcus Statistics. http://www.cdc.gov/fungal/diseases/cryptococcosis-neoformans/statistics.html: Accesssed: 13th October 2014.
5. Park, B. J., Wannemuehler, K. A., Marston, B. J., Govender, N., Pappas, P. G., and Chiller, T. M. (2009) Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23, 525-530
6. Pfaller, M. (2010) Drug resistance in Cryptococcus: Epidemiology and molecular mechanisms. in Cryptococcus: From human pathogen to model yeast (Heitman, J., Kozel, T. R., Kwon-Chung, K. J., Perfect, J. R., and Casadevall, A. eds.), ASM Press, Washington, DC. pp 203-216

 

Anaerobe

An urgent threat to human health is gut infections caused by the anaerobic bacterial pathogen Clostridium difficile. (1) This Gram-positive bacteria causes life-threatening diarrhoea and inflammation to the gut which costs society over 1 billion dollars annually.1 People with an imbalance in their healthy gut microbiota, such that arises from prior antibiotic use, are susceptible to infection. Clostridium difficile infection affects all age groups, including the children and the elderly. Unfortunately there are only three main antibiotic treatments for C. difficile infections and two of these, metronidazole and vancomycin, result in recurrence of infection in up to 30% of patients. (2) Part of the problem in controlling C. difficile infection is the formation of dormant and hardy bacterial spores that can persist in the environment for a long time. These spores are the vehicle for infection transmission between individuals in hospitals and the community. (3) The Cooper Group is tackling this infectious pathogen by developing next generation antibiotic treatments.

We are investigating the action of vancapticins, lipoglycopeptide antibiotics designed and developed by the Cooper Group, against C. difficile. This work is currently supported by an NHMRC grant of $725 000 (2014-2016). We also design, synthesise and evaluate novel nitro-heterocyclic antibiotics for antimicrobial activity against C. difficile as well as other pathogenic anaerobic organisms.

We are also developing compounds that are better at preventing C. difficile spores from forming and have minimal impact on the healthy resident anaerobic gut microbiota, with the goal of reducing the rate of C. difficile infection recurrence.

The small molecule organic synthesis and peptide synthesis that underpins the medicinal chemistry program on anaerobic organisms is supported by the world-class chemistry facilities and expertise within the Cooper group and IMB. The Cooper group is equipped with a Coy anaerobic chamber for growing C. difficile and the gut microbiota associated with good health under strict anaerobic conditions (no oxygen). Robotic liquid handlers facilitate the microbiological evaluation of compounds, developed in-house and by external collaborators, in a high throughput fashion.

References:

1. Antibiotic Resistance Threats in the United States, 2013; Centers for Disease Control and Prevention, 2013.
2. Johnson, S.; Louie, T. J.; Gerding, D. N.; Cornely, O. A.; Chasan-Taber, S.; Fitts, D.; Gelone, S. P.; Broom, C.; Davidson, D. M.; Polymer Alternative for CDI Treatment (PACT) investigators. Vancomycin, Metronidazole, or Tolevamer for Clostridium difficile Infection: Results from Two Multinational, Randomized, Controlled Trials. Clinical Infectious Diseases 2014, 59, 345–354.
3. Eyre, D. W.; Cule, M. L.; Wilson, D. J.; Griffiths, D.; Vaughan, A.; O'Connor, L.; Ip, C. L. C.; Golubchik, T.; Batty, E. M.; Finney, J. M.; Wyllie, D. H.; Didelot, X.; Piazza, P.; Bowden, R.; Dingle, K. E.; Harding, R. M.; Crook, D. W.; Wilcox, M. H.; Peto, T. E. A.; Walker, A. S. Diverse Sources of C. difficile Infection Identified on Whole-Genome Sequencing. N Engl J Med 2013, 369, 1195–1205.

 

Gram positives

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Gram-positives
The rise of drug-resistant ‘superbugs’ is threatening global health to the point that we risk a catastrophic return to the pre-antibiotic era unless radical action is taken to discover new antibiotics. The 2013 US-CDC report1 on antibiotic resistance conservatively estimates that 2 million illnesses are caused by antibiotic resistant infections each year in the US, with 1.6 million attributable to Gram-positive bacteria. Over 90% of these infections arise from 3 organisms alone: methicillin resistant Staphylococcus aureus (MRSA; 80,000 infections and 11,000 deaths), multi-drug resistant Streptococcus pneumoniae (1.2 million infections and 7,000 deaths) and Clostridium difficile (250,000 infections and 14,000 deaths). This greatly exceeds both infection and mortality attributed to Gram-negative bacteria. Despite this disproportionate Gram-positive associated morbidity, most major companies have either abandoned antibiotic research or are focusing predominantly on Gram-negative therapies. MRSA and MDR S. pneumoniae are classed as ‘serious threats’ in the 2013 US CDC report, whereas C. difficile is an urgent threat that requires immediate and aggressive attention.

Vancapticin
Despite the emergence of glycopeptide insensitivity and resistance triggered through years of widespread use of glycopeptide antibiotics, vancomycin remains the mainstay treatment for MRSA infections.2 Although total resistance to vancomycin in S. aureus is still rare (VRSA), the increased incidence of infections caused by MRSA strains that display subtle reductions in susceptibility has dramatically increased (hVISA); such infections correlate with treatment failure and increased mortality.3 S. aureus strains of this nature arise in the hospital setting during failed prolonged glycopeptide therapy, particularly for patients with high bacterial load infections (e.g. endocarditis, osteomyelitis/septic arthritis, deep abscesses, infection of prosthetic devices) and/or history of prior vancomycin exposure. Vancomycin, which is dosed intravenously, has relatively poor pharmacokinetic properties, including a short half-life and poor tissue penetrating properties. The inability of vancomycin to fully penetrate tissues harbouring deep seated infections may promote selection of resistance due to inadequate drug concentrations reaching target bacteria. It is therefore of great concern that vancomycin, the current mainstay treatment for MRSA infections, is becoming less effective in the clinic. The urgent development of new therapies is therefore needed to maintain our arsenal against Gram-positive pathogens.4 In stark contrast to vancomycin, which enjoyed decades of resistance-free use, alternative therapies like daptomycin, linezolid, telavancin, third-generation cephalosporins (e.g. ceftaroline) and the glycylcycline tigecycline antibiotic classes have led to resistance soon after their clinical introduction. Newer therapies have gained recent regulatory approval (ceftobiprole, tedizolid, dalbavancin and oritavancin) which will improve the anti-MRSA landscape; however, based on the previous decade of resistance evolution, it is highly likely that resistance to these therapies will also develop.
In order to restore the activity of vancomycin toward MRSA and MDR S. pneumoniae, we have developed a novel strategy not found in any other Gram-positive antibiotic. By covalently appending a short peptidic motif onto vancomycin, we are able to selectively target it to bacterial cell membranes, in the process restoring and improving the in vitro and in vivo activity of vancomycin toward MRSA and MDR S. pneumoniae. This is achieved through a greater avidity for lipid II (the bacterial target of glycopeptide antibiotics) versus vancomycin, leading to cell death through potent inhibition of peptidoglycan synthesis and membrane disruption.
High potency therapies with good safety profiles and convenient dosing regimens have the potential to facilitate earlier patient discharge and reduce costs associated with hospitalisation.  Our latest generation glycopeptides (vancapticins) are up to 66-fold more potent than comparator compounds against MRSA, and require one-fortieth of the dose of vancomycin to achieve a similar therapeutic outcome in mouse MRSA infection models. With an improved safety profile over current standard of care, and pharmacokinetic properties consistent with once-daily dosing, the vancapticin glycopeptide antibiotics are well positioned in the competitive Gram-positive space to provide a treatment option targeting parenteral antibacterial treatment in an outpatient setting.
The vancapticin program is well advanced, with several lead optimisation candidates having been identified and scrutinised in multiple in vitro and in vivo assays. The group’s efforts are currently focused on optimising the physicochemical properties of the lead optimisation series toward candidate selection for IND (investigation new drug) enabling studies.

 

Clostridium difficile

Clostridium difficile (C. difficile) is a Gram-positive spore forming anaerobic bacterium that causes life-threatening diarrhea. Infection by this organism has become an enormous economic and social burden in the last ten years. Epidemic strains have spread throughout hospitals, while community acquired infections and other sources ensure a constant inoculation of spores into hospitals.
C. difficile infections (CDI) are common amongst hospitalised or recently hospitalised patients who have received antibiotic treatment, and deaths related to this organism increased 400% between 2000 and 2007, in part due to the emergence and epidemic spread of a novel strain of the bacteria. C. difficile spores are an ideal vehicle for transmission between patients because they persist in the environment for long periods and are resistant to heat and typical disinfectants.


The first line treatment for C. difficile infection is the antibiotic metronidazole for mild to moderate infection, or oral vancomycin for moderate to severe infection. Both of these drugs have been used to treat CDI since the late 1970s, but in 20-30% of cases do not effectively treat the infection or prevent relapsing infection. Although in vitro resistance to antibiotics used for the treatment of CDI has been noted, it is not a significant problem at this time. The macrolide antibiotic fidaxomicin is the first new drug on the market to treat C. difficile and has been available since 2011. Due to its narrow spectrum of activity, it offers improvements on relapse rates by reducing collateral damage to the resident gut microbiota, but is not 100% effective. Thus, decreasing response rates and high recurrence rates with current frontline drugs highlights the need for the continued development of new antibacterial agents.


The group has taken two approaches toward the development of new CDI therapies, based on the 5-nitroimidazole and glycopeptide antimicrobial classes. In the first approach, we are developing next generation metronidazole analogues through chemical modification of its core structure. We have identified several novel compounds with broad spectrum activity against C. difficile, and the anaerobic parasites G. lamblia (causing the parasitic disease giardiasis) and E. histolytica (causing the gastrointestinal infection amoebiasis). Work is on-going in this area.


The second approach is aimed at expanding the utility of our vancapticin compounds from a parenteral treatment targeting MRSA and MDR S. pneumoniae to a oral treatment selectively targeting CDI. Given the current landscape of vancomycin-resistance in the clinic, alternatives to vancomycin for treating CDI are highly sought after. We are currently screening our diverse library of novel vancapticin compounds against C. difficile.

 

 

References

1. U.S. Department of Health and Human Services (Centres for Disease Control and Prevention); http://www.cdc.gov/drugresistance/threat-report-2013/ (accessed on 12 September 2014).
2. Butler, M. S.; Hansford, K. A.; Blaskovich, M. A.; Halai, R.; Cooper, M. A., Glycopeptide antibiotics: Back to the future. The Journal of Antibiotics 2014. doi: 10.1038/ja.2014.111.
3. Howden, B. P.; Davies, J. K.; Johnson, P. D.; Stinear, T. P.; Grayson, M. L., Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and heterogeneous vancomycin-intermediate strains: resistance mechanisms, laboratory detection, and clinical implications. Clinical Microbiology Reviews 2010, 23 (1), 99-139.
4.  Butler, M. S.; Blaskovich, M. A.; Cooper, M. A., Antibiotics in the clinical pipeline in 2013. The Journal of Antibiotics 2013, 66 (10), 571-91.

 

Gram negatives

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When Danish bacteriologist Hans Christian Gram published a stain method for distinguishing bacteria in 1884, the hidden world of bacteria was reveled to human kind. The difference between Gram-negative and Gram-positive bacteria can be seen in the Gram staining method of bacterial differentiation. The Gram-negative bacteria don’t retain a blue dye (crystal violet) but are stained pink or red by use of a counterstain.

Some authors have called the rise of the Gram-negative resistant bacteria “the coming of a new Red Plague”, making a reference to the Bubonic plague, or the “Black Death” which killed more than a third of Europe’s population from 1346 to 1351, and the “White Plague” (tuberculosis) that became an epidemic in Europe throughout the 19th century (1).
In the clinic, common human Gram-negative pathogens are usually Escherichia, Klebsiella, Enterobacter, Proteus and Pseudomonas species. These organisms cause infections such as urinary tract infections, peritonitis, biliary tract infection, hospital-acquired pneumonia, and less common but more serious infections such as liver abscess and neonatal meningitis, among others. While antimicrobial agents were initially highly successful in treating these infections, their unregulated use in both humans and animals has seen rates of antimicrobial resistance rise alarmingly, especially in the developing world.
The World Health Organization (WHO) has identified antimicrobial resistance as one of the three most important problems for human health. Some authors have summarized this phenomenon with the word 'ESKAPE', to include the most frequent MDR microorganisms: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.
The Gram-negative bacteria are bounded by two membranous structures (Fig. 2.1). The inner membrane (IM) structure, also called the plasma membrane, is a phospholipid bilayer, while the outer membrane (OM) consists of proteins, including porins, receptors, and an asymmetric distribution of lipopolysaccharide (LPS) and lipids (3).
gram negative.tiff
At the Cooper group we are committed to the development of new antibiotics to address the serious lack of new alternatives in the market to fight the “super bugs”. We have several synthetic approaches in house focusing mainly in OM disruption. Some of our most successful leads are based on cyclopeptide structures.

1. Med J Aust 2013; 198 (5): 243-244.
2. S. N. Chatterjee and K. Chaudhuri, Outer Membrane Vesicles of Bacteria, SpringerBriefs in Microbiology, DOI: 10.1007/978-3-642-30526-9_2.
3. Crit Care. 2011; 15(2): 215.