Drugs and diagnostics for superbugs, viruses and cancer

Superbugs and Inflammation: halting their advance

Every week in Australia 170 people lose their life to bacterial infection. Drug-resistant bacteria are now taking lives at an astonishing rate, and too few scientists are tackling the problem. An epidemic is coming, and we are unprepared.

Diseases like breast cancer and prostate cancer have the world in pursuit, but bacterial sepsis is killing more people every week than prostate, breast cancer and car accidents combined.

Bacteria have been evolving, and some have become so advanced that they can now evade antibiotics altogether, leaving us defenseless to fight them. They are aptly named ‘superbugs’.

Professor Matt Cooper is part of a small contingent of scientists in the world working to prevent the impending crisis.

Research overview

The Cooper Group is discovering new ways to detect, rapidly and accurately diagnose and treat bacterial infections. Most importantly they are designing and developing a new line of defense: antibiotics that are active against drug-resistant bacteria.

To that end, Professor Cooper founded CO-ADD, the Community for Open Antimicrobial Drug Discovery.

“CO-ADD is crowdsourcing compounds from chemists across the globe to uncover diverse compounds with antimicrobial activity,” said Professor Cooper. 

The group is also addressing inflammation, which lies at the heart of many, if not most diseases. Within the immune system, complexes inside our immune cells called inflammasomes generate signals that activate and maintain inflammation. The Cooper Group created a drug that can block these signals – halting inflammation.

The Cooper Group are particularly interested in diseases of the brain such as Parkinson’s and Alzheimer’s. They are also exploring the relationship between gut biota and inflammation in the development of diseases like asthma, chronic obstructive pulmonary disease, diabetes, and cancer.

Research projects

Jump to:  Infectious disease  |  Inflammation  |  Drug discovery

 

Infectious disease

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.

 

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.

 

Antifungal

Opportunistic pathogenic fungi such as Cryptococcus neoformansCandida 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 201459, 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 2013369, 1195–1205.

 

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

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 EscherichiaKlebsiellaEnterobacterProteus 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).
 
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.

 

Inflammation

The complement system and inflammation

The complement system is a very important part of the innate immune system that helps us to combat invading pathogens. Activation of the complement cascade is a tightly regulated and well-maintained process, however when dysregulated it has been implicated in a number of inflammatory and autoimmune disorders. The research in our lab focuses predominantly on the C5a and C3a complement proteins, both key inflammatory mediators. C3a binds to C3aR and C5a binds to either C5a1 and C5a2.

These receptors we are researching belong to a particular class of receptors involved in inflammation called GPCRs, or G protein coupled receptors. GPCRs account for up 30% of the targets of drugs currently on the market and are of interest to Big pharma, academia and biotech. They are commonly referred to as 7TM proteins as they are composed of seven helices that span the membrane. They are implicated in many disease types, due to their widespread expression in a number of different cell types, particularly immune cells. We are interested in GPCRs and the pathology of native and synthetic ligands for these receptors that play a central role in host defense [1].  Excessive complement receptor activation and dysregulation has been linked to a number of different disease types, including rheumatoid arthritis [2], sepsis [3] and Alzheimer’s [4]. Whilst much effort (20 years+) has been put into the development of potent antagonists against this receptor, there are no marketed antagonists to date.

Our group has attempted to understand the complex relationship between the two different GPCRs of C5a, demonstrating that C5a2 is not just a ‘dud’ receptor, as previously believed. Our findings have given us a foundation to pursue novel therapeutics that specifically act via C5a2 and not C5a1.

Key publications

  • Klos, A., Wende, E., Wareham, K.J., and Monk, P. N. International Union of Basic and Clinical Pharmacology. LXXXVII. Complement Peptide C5a, C4a, and C3a Receptors. Pharmacol Rev (2013), 65, 500-543
  • Schofield, Z.V, Woodruff, T.M., Halai, R., Wu, M.C.L., Cooper, M. A. Neutrophils-a key component of ischemic reperfusion injury.(2013), Shock, 40, 463-70
  • Croker, D. E., Halai R., Fairlie, D. P., Cooper M.A. C5a, but not C5a-des Arg, induces upregulation of heteromer formation between complement C5a receptors C5aR and C5L2. (2013) Immunol Cell Biol, 91, 625-633
  • Wu, M.C.L, Brennan, F.H, Lynch, J.P., Mantovani, S., Phipps, S., Wetsel, R.A., Ruitenberg, M.J., Taylor, S.M., Woodruff, T.M. The receptor for complement component C3a mediates protection from intestinal ischemia-reperfusion injuries by inhibiting neutrophil mobilization (2013) PNAS, 110, 9439-9444
  • Reid, R.C., Yau, M., Singh, R., Hamidon, J.K., Reed, A.N., Chu, P., Suen, J.Y., Stoermer, M.J., Blakeney, J.S., Lim, J., Faber, J.M., & Fairlie, D.P. Downsizing a human inflammatory protein to a small molecule with equal potency and functionality (2013), Nat. Commun. 4, 1-9

References

[1] Monk, P. N.; Scola, A. M.; Madala, P.; Fairlie, D. P., Br. J. Pharmacol. 2007, 152, 429.

[2] Jose, P. J.; Moss, I. K.; Maini, R. N.; Williams, T. J., Ann. Rheum. Dis1990, 49, 747.

[3] Czermak, B. J.; Sarma, V.; Pierson, C. L.; Warner, R. L.; Huber-Lang, M.; Bless, N. M.; Schmal, H.; Friedl, H. P.; Ward, P. A., Nat. Med. 1999, 5, 788

[4] Velazquez, P.; Cribbs, D. H.; Poulos, T. L.; and Tenner, A. J., Nat. Med. 1997, 3, 77.

[5] Gilchrist, A., Trends Pharmacol. Sci. 2007, 28, 431.

[6] Urban, J. D.; Clarke, W. P.; von Zastrow, M.; Nichols, D. E.; Kobilka, B.; Weinstein, H.; Javitch, J. A.; Roth, B. L.; Christopoulos, A.; Sexton, P. M.; Miller, K. J.; Spedding, M.; Mailman, R. B., J. Pharmacol. Exp. Ther2007, 320, 1.

[7] Michel, M. C.; Alewijnse, A. E., Mol. Pharmacol. 2007, 72, 1097.

[8] Hutchinson, D. S.; Chernogubova, E.; Sato, M.; Summers, R. J.; Bengtsson, T., Naunyn-Schmiedeberg's Arch. Pharmacol2006, 373, 158.

[9] Sato, M.; Horinouchi, T.; Hutchinson, D. S.; Evans, B. A.; Summers, R. J., Mol. Pharmacol. 2007, 72, 1359.

 

Intestinal reperfusion injury and inflammation

Inflammation is the body’s response to injury and or infection and provides a necessary function for protection and repair. Inflammation is typically considered a mechanism of innate immunity which is a closely regulated system. However, in rare cases, such as trauma and severe infection, acute inflammation can become aberrant causing ischemia reperfusion injury (IRI). 
IRI is a common, damaging and untreatable event that can lead to multiple organ failure (MOF) and death (1). The excessive injury seen in IRI is caused by an exacerbated inflammatory response mediated by neutrophils (2). Current therapeutic options provide palliative care at best, with significant un-met medical need. Thus it is still a research imperative to investigate the mechanisms and find treatments to prevent IRI.  

Our group is investigating the role of the innate immune system with a particular interest for neutrophils and the G protein coupled receptor (GPCR) free fatty acid receptor 2 (FFA2). FFA2, the endogenous ligand for short chain fatty acids(3), is highly expressed on neutrophils. Our twofold approach is exploring the innate mechanisms of IRI in vivo and ex vivo using wild type and knockout models as well as primary cells. Whereas our in vitro work is receptor focused and aims to reveal discreet signalling pathways using FRET, BRET, FLIPR and label free technologies for the purpose of designing optimum therapeutics.

1.        Widgerow AD: Ischemia-Reperfusion Injury: Influencing the Microcirculatory and Cellular Environment. Annals of plastic surgery 00(00): 1–8, 2012 Dec 13. 
2.        Schofield ZV, Woodruff TM, Halai R, Wu MC-L, Cooper MA: Neutrophils-a key component of ischemia-reperfusion injury. Shock (Augusta, Ga) 40(6): 463–70, 2013 Dec. 
3.        Le Poul E, Loison C, Struyf S, Springael J-Y, Lannoy V, Decobecq M-E, et al.: Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation. The Journal of biological chemistry 278(28): 25481–9, 2003 Jul 11.

 

Drug discovery process

Drug Discovery

Modern drug discovery seeks to identify novel small molecules that effectively and selectively modulate a disease related function. In the past, nature provided the majority of such novel molecules, but in the last 30 years synthetic compounds have become the basis of compound screening sets. As a consequence, the size of such libraries is typically in the order of thousands to millions of molecules, which requires highly efficient screening technologies and efficient analysis and decision-making methodologies.

Such screening-based drug discovery approaches can start from an identified target, in which case a target-based screening assay needs to be developed, which usually involves the testing of small molecules against a purified protein. Recent advances in screening technologies have led to the (re)emergence of cell- or organism-based phenotypic assays, in which the effect of small molecules are investigated in the cellular context, rather than on isolated components. Depending on disease and target context both approaches have their advantages and disadvantages.


Target Validation

For target-based screening approaches Target Validation is an important step in the drug discovery and development process. Target validation investigates which type of molecules are able to modulate the target and if the target modulation has an effect on the desired phenotype or disease state. Target druggability is usually linked to the likelihood to find a small molecule with high affinity binding to a given protein [1]. However, the concept can be expanded to the likelihood of a high affinity binder actually reaching the target in the cell, and to the likelihood of a high affinity binder to be specific enough to have the desired activity without displaying adverse effects.

The Cooper Group applies several predictive tools to evaluate the druggability of a target, including structural binding pocket evaluation combined with bioinformatics evaluation of how conserved or variable the potential pocket is between isoforms of the target, within the normal population of the host, and throughout the evolution. The Cooper Group is equipped with a range of in silico methodologies and technologies, including in-house developed workflows. 

The Cooper Group also uses its medicinal chemistry capability to develop tool compounds which can be used to investigate Target Validation in various cell-based and in vivo studies. Tool compounds usually constitute drug candidates with limited potential, either due to limited IP protection or limited application in humans. Despite their reduced potential as final drug candidate they represent a valuable tool for early drug discovery, early target validation and proof-of-concept studies. We are currently using such tool compounds to investigate the druggability and target validation of developmental transcription factors in the treatment of solid tumours, and intra cellular and membrane-bound receptors for the modulation of inflammatory responses.

 

Target Identification and Mode of Action

For phenotypic screening approaches Target Identification and Mode-of-Action (MoA) studies are important for the validation and progression of drug leads. Detailed information on the molecular and structural biology on how an active compound modulates a phenotype is crucial to the design any lead molecules with improved activity and improved ADME/Tox properties. Several approaches are usually applied for target identification, such as direct biochemical methods, genetic interaction methods and computational inference methods [2]. Direct methods involve labelling of protein, ligand/inhibitor or both, and detection or separation of the labelled molecules. Genetic manipulations involve the modulation of presumed targets, ether as knock-outs or inactive mutations, and investigate changes in the sensitivity of the small molecules. Computational approaches use pattern recognition or similarity algorithm to infer possible target by similarity to known targets and/or known bioactives.  

The Cooper Group uses its medicinal chemistry capability to develop labelled small molecule probes, by adding immune-histological or fluorescence tags to known active molecules. A large range of molecular probes have been developed for antimicrobial targets [3], as well as anti-inflammation targets. The Cooper Group also uses regularly genetic methods to generate mutants, as well as transposon-directed insertion site sequencing (TraDIS) methods [4] to investigate resistance mechanisms of bacteria against anti-microbial. In addition, the Cooper Group applies a range of computational methods for protein and chemical similarity analysis [5].


Lead Discovery

The outcome of any screening based drug discovery process depends largely on the selection of the set or library of small molecules going into a screening campaign. Libraries are either selected to represent diversity, exploring chemical and structural variations, or are focused, exploring the structure-activity relationship around hits. However, as most compound library becoming more and more hydrophobic [5], the selection also requires to consider physico-chemical properties of the compounds not only to aim for good oral-bioavailability (or drug-likeness), but also to minimize screening errors due to low solubility or promiscuous binding.

The Cooper Group applies several in silico methods, for optimal selection of screening libraries, including chemo-informatic selection of diverse library with desirable properties and virtual screening for focused libraries [6, 7]. The Cooper Group has access to a wide range of commercial and open-source software, including an extended database of compounds, commercial and bioactives, curated and maintained specifically for our drug discovery projects.

 

Lead Optimization

The next step in the drug discovery process is the optimization of hits from the screening into lead candidates. Chemical modifications are applied to the hit structures to improve their efficacy while reducing any toxicity or adverse effects the small molecules might have. Various methods are applied to guide the chemical modifications, using structure-activity relationship analysis from a set of active ligands, using structure information of the target to improve activity, or applying medicinal chemistry guidelines to improve pharmaco-kinetic properties such as stability, half-life and distribution.

The Cooper Group uses chemo-informatic and machine learning tools to build predictive model for structure-activity relationship, which are applied for the selection of screening libraries, as well as optimization of the hit structure for improved activity. The Cooper Group uses its in-house databases of compounds and bioactivity data, which the group maintains and curates. The Cooper Group also has access to the in-house protein crystallisation facilities which are used for co-crystallisation experiment of the protein with the active small molecules. These facilities are compensated with high field NMR facilities and molecular dynamic simulations to investigate the dynamic behaviour of the ligand-target interaction, including interaction of small molecules with membranes of various species (i.e. bacterial vs. mammalian).

The medicinal chemistry facilities in Cooper Group are able to take the concept of the design and implement the chemical modifications not only for discovery but also for in vivo studies (i.e. larger scale). The Cooper Group has several antibacterial leads currently in animal studies.  

Key publications
1.            Cheng, A.C., et al., Structure-based maximal affinity model predicts small-molecule druggability. Nat Biotechnol, 2007. 25(1): p. 71-5.
2.            Schenone, M., et al., Target identification and mechanism of action in chemical biology and drug discovery. Nat Chem Biol, 2013. 9(4): p. 232-40.
3.            Phetsang, W., et al., An azido-oxazolidinone antibiotic for live bacterial cell imaging and generation of antibiotic variants. Bioorganic & medicinal chemistry, 2014. 22(16): p. 4490-8.
4.            van Opijnen, T. and A. Camilli, Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol, 2013. 11(7): p. 435-42.
5.            Zuegg, J. and M.A. Cooper, Drug-likeness and increased hydrophobicity of commercially available compound libraries for drug screening. Curr Top Med Chem, 2012. 12(14): p. 1500-13.
6.            Karoli, T., et al., Identification of antitubercular benzothiazinone compounds by ligand-based design. J Med Chem, 2012. 55(17): p. 7940-4.
7.            Karoli, T., et al., Structure aided design of chimeric antibiotics. Bioorg Med Chem Lett, 2012. 22(7): p. 2428-33.

 

Drug discovery research

Drug induced kidney injury

Nephrotoxicity, or damage to the kidney, is a side effect of many marketed drugs, with 19-25% of acute renal failures caused, in part, by drug exposure [1]. Many of these drugs are antibiotics, and include a number of classes that are the focus of drug discovery projects within the group. In the search for improved versions of existing drugs, and for the development of novel therapies, it would be very helpful to be able to screen for potential nephrotoxicity. Unfortunately, the gold standard for preclinical compound testing is kidney histopathology from animal studies, a low throughput and expensive procedure that requires sacrifice of the animal. In vivo monitoring of serum creatinine (SCr) or blood urea nitrogen (BUN) levels provides an alternative readout to kidney biopsies, but sensitivity and correlation to injury is poor. There has been an intensive effort in recent years to identify in vivo biomarkers that can be used to selectively monitor kidney damage [2-8]. In particular, proteins such as NGAL (Neutrophil gelatinase-associated lipocalin) [9] and Kim-1 (Kidney injury molecule-1) [7, 10] have been highlighted as proteins with much more relevance to early detection of kidney injury than traditional serum creatinine levels.

For drug discovery screening, a cell-based assay is much more useful than an in vivo assay, as potential liabilities can be assessed at a much earlier timepoint, and structure-activity relationships can be explored at a reasonable cost. There have been numerous research reports on the use of cellular assays systems to detect nephrotoxicity. However, the scope of these studies has been restricted by either the types of cells employed, the toxicity readouts assessed, or the drugs applied to the cells. Furthermore, the possible in vitro utility of many of the potential new in vivo biomarkers requires investigation.

We are undertaking a comprehensive research program using a matrix of cell types, reference compounds, and readouts to identify in vitro cellular assays that can be used to predict nephrotoxicity. We will investigate multiple human kidney-related cell types and compare the results to those seen in non-renal cells, allowing for the differentiation of general cytotoxicity from nephrotoxicity. We will test a range of known nephrotoxic and cytotoxic reference compounds. The cellular effects will be monitored using general cytotoxicity assays, biomarker-based assays, high content screening using fluorescent labelled markers, and label-free assays using both optical and electrical impedence biosensors. The results will provide us with an ability to counterscreen the group’s antibiotic drug discovery efforts for nephrotoxic side effects. In the future we hope to expand this effort to all drugs as a tool for the scientific and pharmaceutical research communities.

Figure 1. (a) Biomarkers of kidney injury and (b) Drugs that elicit site-specific toxicity [1]

[1] Bonventre, J.V.; et al., Nat. Biotechnol2010, 28, 436.

[2] Ozer, J.S.; et al., Nat. Biotechnol. 2010, 28, 486.

[3] Mattes, W.B.; et al., Nat. Biotechnol2010, 28, 432.

[4] Sistare, F.D.; et al., Nat. Biotechnol. 2010, 28, 446.

[5] Dieterle, F.; et al., Nat. Biotechnol2010, 28, 455.

[6] Dieterle, F.; et al., Nat. Biotechnol. 2010, 28, 463.

[7] Hoffmann, D.; et al., Toxicol. Sci. 2010, 116, 8.

[8] Yu, Y.; et al., Nat. Biotechnol2010, 28, 470

[9] Paragas, N.; et al., Nat. Med. 2011, 17, 216.

[10] Vaidya, V.S.; et al., Nat. Biotechnol2010, 28, 478.

 

Drug-membrane interaction

All living cells are surrounded by one or more membranes. These membranes, composed of lipids and proteins, play important roles in cell survival and function. Drug design generally focuses on the interactions between ligands and their receptor or enzyme targets, and largely ignores the role played by cell membranes, particularly for membrane-based protein targets. However, knowledge of drug-membrane interactions is essential for understanding a drug’s biodistribution, activity, selectivity and toxicity.

The development of analytical tools for the study of drug-membrane interactions of increasing interest to scientists. Methods such as high-performance liquid chromatography (HPLC), fluorescence techniques and NMR are commonly used. We are undertaking several projects using surface plasmon resonance (SPR), cell impedance and resonant waveguide photonics; all label-free techniques, to investigate the interactions between novel antibiotics and cell membranes [1-5]. These provide an ideal model of cell membrane that can be varied to determine the binding affinity and kinetics of drugs on different membrane types. The results not only provide valuable information for drug design and development, but also contribute to investigations into the mode of action of novel antibiotics and cancer therapeutics.

 

[1] Cooper, M. A.; Williams, D. H., Chem. Biol. 1999, 6, 891.

[2] Cooper, M.A., Label-free biosensors : techniques and applications. 2009 Cambridge University Press: Cambridge.

[3] Chia, C. S.; Gong, Y.; Bowie, J. H.; Zuegg, J.; Cooper, M. A., Biopolymers2010, doi: 10.1022/bip.21438.

[4] Nussio, M. R.; Sykes, M. J.; Miners, J. O.; Shapter, J. G., ChemMedChem. 2007, 2, 366.

[5] Cooper, M.A., J. Mol. Recognit. 2004, 17, 286.

 

Drug-drug interactions

With the general population now often taking more than one drug at the same time, adverse effects of drug-drug interactions are becoming very prominent. Knowing if there are side effects and changes in pharmacokinetics of a drug as a result of taking two different drugs at one time, is critical for drug efficacy. Pharmocokinetic interaction between drugs arise thereby if one drug changes the absorption, distribution, metabolism, or excretion of another drug, changing the concentration of active drugs in the body, in some case above their maximal tolerable dose.

Several systems are involved in changing the pharmacokinetic properties of a drug, including Cytochrome P450 and residence time on serum albumin. Inhibition of the CYP450 of one drug can change the metabolism of a second drug, whereas competitive binding to albumin affects the concentration and half-life of drugs in the blood. This research project is focusing on the development of fast, high throughput screening methods to investigate drug-drug interactions.

 

Chemoinformatics

Chemoinformatic methods, generally described as computer and informational techniques in the field of chemistry, are nowadays essential tools in the discovery and development of new active compounds. In combination with databases such as chEMBL or PubChem, they provide essential information about existing compounds and compounds classes. But more importantly, using various statistical and predictive modeling tools Chemoinformatic methods are able to provide prediction on on- and off-target activity/selectivity and biochemical and pharmacokinetic properties (ADME-Tox)

The research project aims to develop and implement a comprehensive toolset and database for chemoinformatic. The main focus of the project will be the integration of molecular modelling (structural biology) and bioinformatic (molecular biology) methods with the chemoinformatic (chemistry) toolset.

For example, drug-target networks combine the sequence similarity between targets with the chemical similarity of their targets, to establish a wider relationship between structures and targets than would have been possible using only a single similarity relationship. Similarly, integrating structure docking and target homology procedures into the chemoinformatic toolsets will enhance its functionality.

Research training opportunities

Research title: Drug discovery and diagnostics

Summary of research interests: We believe that we can more effectively treat people by improving the way we understand and diagnose disease. Our research is aimed at discovering new ways of diagnosing and treating viral and bacterial infections, as well as diseases associated with chronic inflammation such as asthma, chronic obstructive pulmonary disease, type 2 diabetes and cancer. We have a major focus on the design and development of novel antibiotics active against drug-resistant bacteria, also known as ‘superbugs’.

Traineeships, honours and PhD projects include

  • Antibacterial and antifungal medicinal chemistry
  • Small molecule inhibitors of inflammation
  • Antibiotic mode of action studies
  • Chemoinformatics, microbiology and nanotechnology for diagnostics.

Contact: Professor Matt Cooper

+61 7 3346 2044
m.cooper@uq.edu.au


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Featured publications

  • Mridha, Auvro R., Wree, Alexander, Robertson, Avril A. B., Yeh, Matthew M., Johnson, Casey D., Van Rooyen, Derrick M., Haczeyni, Fahrettin, Teoh, Narci C. -H., Savard, Christopher, Ioannou, George N., Masters, Seth L., Schroder, Kate, Cooper, Matthew A., Feldstein, Ariel E. and Farrell, Geoffrey C. (2017) NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice. Journal of Hepatology, 66 5: 1037-1046. doi:10.1016/j.jhep.2017.01.022

  • Blaskovich, Mark A. T., Butler, Mark S. and Cooper, Matthew A. (2017) Polishing the tarnished silver bullet: the quest for new antibiotics. Antimicrobial Resistance, 61 1: 103-114. doi:10.1042/EBC20160077

  • Chitty, Jessica L., Tatzenko, Tayla L. , Williams, Simon J., Koh, Y. Q. Andre E., Corfield, Elizabeth C., Butler, Mark S., Robertson, Avril A. B., Cooper, Matthew A., Kappler, Ulrike, Kobe, Bostjan and Fraser, James A. (2017) GMP synthase is required for virulence factor production and infection by cryptococcus neoformans. Journal of Biological Chemistry, 292 7: 3049-3059. doi:10.1074/jbc.M116.767533

View all Publications

Engagement and impact

Professor Cooper is affiliated with the Pfizer Australia Stakeholder Faculty, he is a Fellow of the Queensland Academy of Arts and Sciences, a Fellow of the Royal Society of Medicine, Member of the Royal Australian Society of Chemistry, the Australian Society for Infectious Diseases and the Australian Society for Antimicrobials.

He is also highly engaged with the community. He has recently been involved in the following conferences:

Professor Cooper is also pro-active in his engagement with the media. He has recently featured in an ABC Catalyst special episode on Antibiotic Resistance, and an episode of  Science over Coffee – Antibiotics and the Emerging Superbug Threat.  

He has consulted with Private Equity Investment, Pharmaceutical, Biotechnology and Diagnostics companies, was Managing Director of Cambridge Medical Innovations (part of Alere Inc.) and CSO of Akubio Ltd. He is an inventor and driver of several antibiotic drug discovery programmes with lead compounds pre-clinical.

Professor Cooper was founder of the Community for Open Antimicrobial Drug Discovery (CO-ADD), which is not for profit initiative. CO-ADD screens comounds for antimicrobial activity for academics for free to help researchers worldwide find new, diverse compounds to combat drug resistant infections.

Professor Cooper is also CEO of Inflazome Ltd., a company that is developing drugs to address clinical unmet needs in inflammatory disease by targeting the inflammasome. 

His group have gained a deeper understanding of the role of gut biota, which are the bacteria that live in our digestive system, and how this affects inflammation in the development of diseases such as asthma, chronic obstructive pulmonary disease, diabetes and cancer. This basic research helps us to develop new methods to diagnose and more effectively treat patients affected by these complex and deadly diseases. 

Professor Cooper is a co-founder and Director of Defensin Therapeutics, a company developing novel molecules that modulate our microbiome (the bugs in our guts), to treat disease.

Partners and collaborators

Through the Community for Open Antimicrobial Drug Discovery (CO-ADD) Professor Cooper is collaborating with 200 academic group collaborators from 38 countries. Strategic partners and funders include the Wellcome Trust, The University of Queensland and the Institute for Molecular Bioscience. Project partners include the School of Chemistry and Molecular Biosciences at the University of Queensland, Compounds Australia, ChEMBL, and QFAB.

Professor Cooper is in partnership with more than 30 laboratories worldwide, designing and developing new molecules to target the interface between infection and our immune system’s response that leads to acute and chronic inflammatory disease. 

The research group has received major awards from the Wellcome Trust, Bill & Melinda Gates Foundation, Michael J Fox Foundation, the NIH and the NHMRC.
 

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Prof Matt Cooper

Professor Matthew Cooper

Director, IMB Centre for Superbug Solutions

Director, Community for Open Antimicrobial Drug Discovery

  +61 7 3346 2044  
  m.cooper@uq.edu.au
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    Institute for Molecular Bioscience

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