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August 2013 Archive

Posted by Chris on August 15th, 2013  ⟩  0 comments

Aminoglycosides are one of the largest and most common classes of antibiotics we use on a daily basis. The founding member of this group, Streptomycin, was discovered early in the golden age of antibiotics and was the first antibiotic found to be efficacious in treating tuberculosis. In the decades that followed, many new aminoglycosides were discovered or synthetically developed, including: kanamycin, spectinomycin, amikacin, apramycin and hygromycin. These drugs have been a bulwark of our antibiotic armament over the years due to their effectiveness against both gram positive and gram negative bacteria, their synergism with other antibiotic classes and their generally low cost. But as with all antibiotics, organisms have been developing resistance to them as fast as we have been able to develop new variants.

There are three main methods by which organisms have gained resistance to aminoglycosides: decreased intracellular concentration due to active efflux of the antibiotic (as with lung isolates of P. aeruginosa), targeted modifications of genes like rpsL or rrs which change their protein structure slightly, but significantly, and most commonly by enzymatic modification by aminoglycoside-modifying enzymes (AMEs). AMEs change the structure of the aminoglycoside which then prevents their binding to the ribosome. One of the ways AMEs does this is through APHs (nucleoside triphosphate-dependent phosphotransferases). There are over 100 different AMEs described in literature, and 40 crystal structures of 8 different APH enzymes have been analyzed, demonstrating the diversity and variability of this class of enzymes.

APH Structure

The primary, historical method of circumventing AME-mediated resistance has been in the creation of steric hindrance. In this process, the inclusion of precisely placed side chains such as AHB ((S)-4-amino-2-hydroxybutyrate) offer resistance to the actions of many AMEs, a process that has worked effectively in creating new antibiotics like amikacin from kanamycin. Even more recently, Plazomicin has been synthesized by the addition of an AHB group (to position 1 of the 2-deoxystreptamine) and a hydroxyethyl group (to position 6’) to the sisomicin molecule. The effect is an aminoglycoside which appears to be resistant to all but one aminoglycoside resistance enzymes (the exception was AAC(2’)) (Armstrong and Miller, 2010)! There have also been a few others which have shown some efficacy against resistant strains, but in the greater war, that’s not a lot of reserve options. And AMEs are certain to coevolve equally fast with these new antibiotics.

An ideal APH would provide resistance against all members of the AME family. However, that might not be feasible as we learn more about the structures of the different subfamilies of APH enzymes. Researching more specific APH inhibitors might be more useful, but would likely prove less profitable. Considering our inability to produce a lot of next-generation aminoglycosides, however, perhaps specific APH inhibitors are a better choice after all if we want to slow down the impending ARA (Antibiotic Resistance Apocalypse). You can find a more complete review of this subject online from Kun Shi and associates from McGill University.

 
 

Shi, K., Caldwell, S. J., Fong, D. H., & Berghuis, A. M. Prospects for circumventing aminoglycoside kinase mediated antibiotic resistance. Frontiers in Cellular and Infection Microbiology, 3, 22.

Armstrong, E. S., and Miller, G.H. (2010). Combating evolution with intelligent design: the neoglycoside ACHN-490. Curr.
Opin. Microbiol. 13, 565–573.

Category Code: 79101

Posted by Chris on August 22nd, 2013  ⟩  0 comments

Autophagy is a very complicated process when it comes to cancer. In general, autophagy isn’t complicated at all. It is a highly conserved process in which damaged or long-lived proteins and organelles can be removed from cells. Functioning through lysosomal machinery, this process ensures cellular survival during starvation by maintaining cellular energy levels. Think of it as a waste removal/renewable energy source system; bad or unnecessary stuff gets broken down in order to sustain the rest of the cell. This happens in nearly all cells and it works wonderfully.

In cancer, however, the role of autophagy is a bit murkier. Sometimes, autophagy is working with the doctors in the suppression of tumor cells; isolating damaged organelles, allowing cell differentiation or promoting cell death of the cancerous cells. But other times, autophagy is working against the doctors; allowing stressed tumor cells to undergo dormancy and resistance to chemotherapeutic drugs. Such a dichotomy in roles can be very frustrating to the doctors and ultimately deadly to a patient if the therapy is compromised due to the lack of effective autophagy or if the cancer comes back due to effective autophagy. Knowing which cancers use autophagy for survival and which cancers are susceptible to autophagy is key to corrective treatment.

None of this is particularly new, however. The role of autophagy in cancer cells is quite known (there is a dedicated Autophagy Journal, after all). There are also a number of clinical studies on various cancer types which are looking into the use of anti-autophagy drugs in conjunction with chemotherapy drugs. Autophagy-inhibitory drugs can come in one of two types: there are the early-stage inhibitors which can target class III P13K and interfere with its membrane recruitment, and late-stage inhibitors (such as chloroquine, hydrochloroquine and monensin) which prevent the acidification of lysosomes (Yang, 2011). Below is a table of various ongoing (c. 2011) clinical trials utilizing some of these late-stage, autophagy inhibitors.

Autophagy Clinical trials

More recently, a group in South Korea headed by Cheol Hyeon Kim investigated the effect of monensin on two anticancer drugs (erlotinib and rapamycin) in the treatment of lung cancer cells. Erlotinib is an Epidermal Growth Factor Receptor (EGFR) inhibitor which binds to the ATP binding site on the EGFR, which prevents the formation of an EGFR homodimer and its ensuing signaling cascade. Autophagy pathwayRapamycin is a macrocyclic triene, antibacterial drug which also has immunosuppressant and anticancer activities. The major target of rapamycin in mammalian cells is the mTOR pathway, which is a major regulator of autophagy, and which is downstream of the P13K-AKT pathway.

Kim’s group saw an increase in apoptosis in the cells treated with both the anticancer drug as well as nanomolar concentrations of monensin, with a 40% reduction in cells compared to the control and around ~20% reduction compared to the anticancer drug alone (P <0.01). Perhaps this isn’t really that surprisingly, after all. Hydrochloroquine in conjunction with erlotinib is already in a phase 2 trials for lung cancer (see table above). But their published record further assists in the elucidation of how autophagy functions in the realm of cancer cell tumorigenesis and ultimately, that is most important thing right now.

 
 

Choi, H. S., Jeong, E. H., Lee, T. G., Kim, S. Y., Kim, H. R., & Kim, C. H. (2013). Autophagy Inhibition with Monensin Enhances Cell Cycle Arrest and Apoptosis Induced by mTOR or Epidermal Growth Factor Receptor Inhibitors in Lung Cancer Cells. Tuberculosis and Respiratory Diseases, 75(1), 9-17.

Yang, Z. J., Chee, C. E., Huang, S., & Sinicrope, F. A. (2011). The role of autophagy in cancer: therapeutic implications. Molecular cancer therapeutics, 10(9), 1533-1541.

Category Code: 88221 79101

Posted by Chris on August 26th, 2013  ⟩  0 comments

A few weeks ago, we had a blog post about the issues we’re facing due to our overuse of specific antibiotics, namely Cephalosporins. The overuse of these and similar antibiotic compounds has resulted in selecting for a whole new breed of multi-drug resistant bacteria, which is a serious threat to our collective well-being.  So now that we have this problem, what do we do about it? For many researchers, the answer continues to be the discovery and classification of novel antibiotic compounds. Just this month there have been two papers published that describe two new antibiotics that are showing effectiveness against major health issues caused by drug resistant organisms, mainly tuberculosis and methicillin-resistant Staphylococcus aureus (MRSA) infections.

The tuberculosis drug, code named Q203, was found in a survey of over one hundred thousand different chemical compounds that a team of researchers were testing for potential effectiveness.   The researchers isolated around 100 potential compounds, where Q203 stood out for its ability to kill drug resistant strains. With a few chemical tweaks, the researchers were able to test the drug on mice, and found that it was tolerated at high doses, and more effective than the current tuberculosis drug isoniazid. Q203 is part of a larger group of chemical compounds called imidazopyridine amides, or IPA’s and there have been other studies done on similar IPA’s, but none have shown the same effectiveness on drug resistant TB. The researchers found that Q203 works by targeting the cytochrome b1 complex in the bacteria, and inhibits the energy transduction system in the cells, inhibiting their growth.  Other researchers working on similar compounds hope that the knowledge they gain from Q203 will lead to a whole new class of IPA antibiotic agents that are easy and inexpensive to manufacture. You can find the paper in its entirety here at Nature Medicine.

While the tuberculosis drug Q203 was found by surveying thousands of different previously classified chemical compounds, the antibiotic effective against MRSA was found through a different method entirely. Researchers at the Scripps Institution of Oceanography, La Jolla recently published a paper in the journal Angewandte Chemie, describing a new antibiotic they found being produced by Streptomyces sp. they had isolated from ocean sediment off the coast of Santa Barbara, CA.  They named the drug Anthracimycin, for its ability to inhibit Bacillus anthracis, which causes anthrax, and MRSA. Anthracimycin is different from the antibiotics produced by other strains of Streptomyces, and has the potential to work on a wide variety of additional pathogens.  The full paper can be found here.

Unfortunately though, finding novel antibiotics like the two just described are extremely rare, and involve a lot of research effort. It can take many years just to isolate the specific compound, and it still has to go through a number of rigorous studies to determine its effectiveness and any potential side effects it may have.  This process can take many years, and sometimes the smallest issue with one of the preliminary studies can prevent a drug from getting FDA approval. Also some scientists argue that developing and using new antibiotics will just perpetuate the microbial arms race, and we’ll be in the same situation again in a matter of years. While we will still need to figure out this issue for the long term, for the short term at least we have a few new promising candidates that may help humanity battle off serious infection. At least for a little while.

Sources:

Kevin Pethe, Pablo Bifani, Jichan Jang, Sunhee Kang, et al. 2013. Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis. Nature Medicine;  doi:10.1038/nm.3262

Kyoung Hwa Jang et al. 2013. Anthracimycin, a Potent Anthrax Antibiotic from a Marine-Derived Actinomycete. Angewandte Chemie, vol. 52, pages 7822–7824; doi: 10.1002/anie.201302749

Category Code: 79102 88221 79101

Posted by Chris on August 29th, 2013  ⟩  0 comments

All of our naturally derived antibiotics come from the ground, or more specifically the organisms that live in the ground. It’s estimated that there are more organisms living in just one square inch of soil than there are humans in the entire world. And we know and understand so very little of what thrives at that level. The bacteria that exist on this plane of existence have been fighting for millions of years, far beyond our own petty rivalries. Their weapons of choice are a variety of antibiotic chemicals, battling to suppress rival bacteria and an almost equal variety of activatable genes that work to rebuff any counter measures. Even in the smallest physical parts of our world, it seems that it really is a jungle out there.

With our widespread overuse of antibiotics, both in humans as well as for our livestock, and the corresponding resistance to the majority of our antibiotics, there is a tremendous push to better understand where the resistance is accurately coming from. Over the last 10 years, a great deal of effort has gone into researching the bacteria resistome where it all began: in the soil. There have been many papers (See Antibiotic Fall from Grace) suggesting that an excess of antibiotics spill out into the ground we live around, which then drive the development of resistant bacteria, which then complete the cycle back into our system. These papers state that we are creating our own worst nightmares and are nearly helpless to prevent it. As further proof of this effect, Gautam Dantas’ lab in Washington University of St. Louis has published several papers finding exact matches between soil bacteria antibiotic-resistant genes which are identical to ones found in clinically resistant bacteria! The findings of one of his lab’s research were published in Science last year. Suddenly, it’s not just a suggestion anymore.

Fiona Walsh seems to have taken a slightly different stand, however. While still focusing on the soil ecosystem and the soil bacterial resistome, she sees a less direct relationship between the clinically resistant genes and the naturally occurring ones in the soil. She began by testing soil samples both from agricultural and urban locations (close to human activity) as well as more pristine locations (with minimal human impact). Walsh was looking to establish the levels of culturable resistant bacteria and identify the different mechanisms of resistance were most often utilized. What she found was over 80% resistance in over 400 isolates to 16-23 of the antibiotic drugs which were screened…even in the pristine locations! There was no relationship at all between the variety of resistance and the level of human activity. Instead, Walsh found a number of bacteria which were able to utilize the efflux capabilities of surrounding resistant bacteria to assume their own resistance. There was also little correlation in her study between the intrinsic resistances of soil bacteria to clinical bacteria. And contrary to Dantas’ findings, Walsh did not find any ESBL (Extended Spectrium β-Lactamases) or quinolone resistant genes in the bacteria she tested (caveat: Walsh primarily tested in Switzerland, while Dantas’ work was across the US).

Enzyme Inhibition Assay - Walsh 2013

Ultimately though, everyone agrees that soil bacteria are an important reservoir for resistance mechanisms and that more work needs to be done to understand the myriad mechanisms at play. This research is crucial in our own tiny war against the multitude of bacteria that plague us and academic groups like Dantas’ and Walsh are on the forefront in discovering the rules of this war and using those rules to better treat the diseases caused by those pathogens.

 
 

Walsh, F., & Duffy, B. (2013). The Culturable Soil Antibiotic Resistome: A Community of Multi-Drug Resistant Bacteria. PloS one, 8(6), e65567.

Forsberg, K. J., Reyes, A., Wang, B., Selleck, E. M., Sommer, M. O., & Dantas, G. (2012). The shared antibiotic resistome of soil bacteria and human pathogens. Science, 337(6098), 1107-1111.

Category Code: 79105 79102 88221 79101
 

Posted by Chris on August 8th, 2013  ⟩  0 comments

It’s extremely well known that our antibiotics are failing us. Alexander Fleming noticed it from the very beginning. Improper use of antibacterial agents leads to improved bacterial resistance to the agent. Throughout the decades since Fleming’s discovery of penicillin, we have watched as diseases have slowly (or quickly, in some cases) developed resistance to one antibiotic after another. The speed with which they are developing resistance has nearly surpassed the rate with which we can develop new antibiotics. And our newest antibiotics are losing their efficacy faster than their predecessors. It’s a viscous cause and effect cycle which we seem destined to lose.

As I searched for interesting research on Cephalosporins this week, I was reintroduced to this scary fact from several recent articles discussing the plight of increased resistance throughout the world to some of our most potent antibitics. First, I found a letter from Peter Collignon and colleagues to the CDC journal, Emerging Infectious Diseases, about the over-use of third generation cephalosporins in poultry through Europe. Collignon cited an ever rising tide of resistant E. coli (G3CREC) across Europe and that G3CREC-related illnesses have already been associated with over 900 hospital bed-days needed to treat the resistant infections and even resulted in 21 deaths in the Netherlands! The strangest part is that most countries have a “prudent use” policy in place concerning medical delivery of many antibiotics. At the same time, however, farm and veterinary implementation of these policies are lagging far behind. With the high rate of gene similarity between the G3CREC in humans and poultry in samples taken throughout Europe, it’s easy to see why the practice of large scale inoculations is so dangerous.

In another part of the world, Bhasker Thakuria and Kingshuk Lahon continued the cry for better “prudent use” polices in developing nations like India. There, it is cost prohibitive to do a full biological screening of every infection or illness. Through the lack of antibiotic susceptibility resources and the lack of guidelines for more common diseases, there is also a prevalence in using state-of-the-art antibacterial agents, such as later generation cephalosporins, to treat every patient’s illness, regardless of their specific needs. Thakuria and Lahon issued a call to improve the empirical use of drugs like cephalosporins before they become completely obsolete in our war against disease.

Finally, there was an article written by Douglas Call and associates detailing the possibility that increased antibiotic resistance might be related not just to our over-use of the drugs, but the downstream destinations of those drugs. Call found that metabolites of third generation cephalosporins, such as ceftiofur, excreted from cattle can still persist and remain biologically active for up to 3 days at room temperature (or several weeks at 4°C). And since 70% of ceftiofur is excreted through urine within 24 hours of injection, there is a high rate of probability that it will affect bacteria in the surrounding soil. The eventual resistant bacteria reenters the cattle through their environment, resulting in further resistance down the chain.

Our irresponsible use of antibiotics have created a pothole in the road to healthier living. Our continued irresponsible use will only serve to widen and deepen the hole we’ve made, eventually leading to the complete ineffectuality of our “antibiotic age” and the resurgence of some of mankind’s greatest diseases. In truth, perhaps it is not so much that our antibiotics are failing us, but that we are failing our antibiotics.

 
 

Collignon, P., Aarestrup, F., Irwin, R., and McEwen, S. (2013) C. D. C. Human Deaths and Third-Generation Cephalosporin use in Poultry, Europe (letter). Emerging Infectious Diseases, 19 (8), 1339.

Thakuria, B., & Lahon, K. (2013). The Beta Lactam Antibiotics as an Empirical Therapy in a Developing Country: An Update on Their Current Status and Recommendations to Counter the Resistance against Them. Journal of Clinical and Diagnostic Research, 7(6), 1207-1214.

Call, D. R., Matthews, L., Subbiah, M., & Liu, J. (2013). Do antibiotic residues in soils play a role in amplification and transmission of antibiotic resistant bacteria in cattle populations?. Frontiers in microbiology, 4 (193).

Category Code: 79102