Unraveling The Resistance Of Antibiotics!

Stories seem to emerge daily regarding the threat to the human race regarding the rise in resistance of antibiotics toward common diseases.  One side of the spectrum, the pro-antibiotic sector are dispersing antibiotics like candy to farm animals and patients without caution.  Whereas on the other side of the spectrum, there is a growing community of researchers, advocates, and concerned citizens -- yelling at the top of their voices to stop administering antibiotics needlessly.  With both sides of the spectrum known, the following question emerges naturally:



Where is scientific research at on the issue?



Are advancements in discovery being made to deal with the potential threat?



The short answer is that the discovery process takes time and is complicated.  Which is no answer at all.  Whereas the long term solution involves research being done.  As I explained in a previous post on drug discovery, research advances are arduous and take time.  Recently, though, progress has been reported in the scientific community and worth giving a "shout out" about.  Below is the short post regarding the advance.

Antibiotic Resistance?

Yes, whenever I hear about antibiotic resistance, I stop and pause for a moment of scare.  Then I think about the progress that is being made (hopefully).  I cannot have myself worry too much about the issue since I do not perform research directly toward a solution.  Although, I can support students who are biochemistry undergraduates and graduate students while educating them on the need and importance of such research.  Couple that with a proper training on the scientific instrument needed to perform the research and my job ends there.



Sounds scary right?



Well, not all is held in limbo with regard to antibiotic resistance.



First, what is antibiotic resistance?  In order to understand the issue, what is the problem?



Here is an excerpt taken from the 'Wikipedia' page for "Antibiotic Resistance" is shown below:

Antimicrobial resistance (AMR) is the ability of a microbe to resist the effects of medication previously used to treat them.[2][3][4] This broader term also covers antibiotic resistance, which applies to bacteria and antibiotics.[3] Resistance arises through one of three ways: natural resistance in certain types of bacteria; genetic mutation; or by one species acquiring resistance from another.[5] Resistance can appear spontaneously because of random mutations; or more commonly following gradual buildup over time, and because of misuse of antibiotics or antimicrobials.[6] Resistant microbes are increasingly difficult to treat, requiring alternative medications or higher doses—which may be more costly or more toxic. Microbes resistant to multiple antimicrobials are called multidrug resistant (MDR); or sometimes superbugs.[7] Antimicrobial resistance is on the rise with millions of deaths every year.[8] A few infections are now completely untreatable because of resistance. All classes of microbes develop resistance (fungi, antifungal resistance; viruses, antiviral resistance; protozoa, antiprotozoal resistance; bacteria, antibiotic resistance).

Antibiotics should only be used when needed as prescribed by health professionals.[9] The prescriber should closely adhere to the five rights of drug administration: the right patient, the right drug, the right dose, the right route, and the right time.[10] Narrow-spectrum antibiotics are preferred over broad-spectrum antibiotics when possible, as effectively and accurately targeting specific organisms is less likely to cause resistance.[11] Cultures should be taken before treatment when indicated and treatment potentially changed based on the susceptibility report.[12][13] For people who take these medications at home, education about proper use is essential. Health care providers can minimize spread of resistant infections by use of proper sanitation: including handwashing and disinfecting between patients; and should encourage the same of the patient, visitors, and family members.[12]

Rising drug resistance can be attributed to three causes use of antibiotics: in the human population; in the animal population; and spread of resistant strains between human or non-human sources.[6] Antibiotics increase selective pressure in bacterial populations, causing vulnerable bacteria to die—this increases the percentage of resistant bacteria which continue growing. With resistance to antibiotics becoming more common there is greater need for alternative treatments. Calls for new antibiotic therapies have been issued, but new drug-development is becoming rarer.[14] There are multiple national and international monitoring programs for drug-resistant threats. Examples of drug-resistant bacteria included in this program are: methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant S. aureus (VRSA), extended spectrum beta-lactamase (ESBL), vancomycin-resistant Enterococcus (VRE), multidrug-resistant A. baumannii (MRAB).[15]

A World Health Organization (WHO) report released April 2014 stated, "this serious threat is no longer a prediction for the future, it is happening right now in every region of the world and has the potential to affect anyone, of any age, in any country. Antibiotic resistance—when bacteria change so antibiotics no longer work in people who need them to treat infections—is now a major threat to public health."[16] Increasing public calls for global collective action to address the threat include proposals for international treaties on antimicrobial resistance.[17] Worldwide antibiotic resistance is not fully mapped, but poorer countries with weak healthcare systems are more affected.[9] According to the Centers for Disease Control and Prevention: "Each year in the United States, at least 2 million people become infected with bacteria that are resistant to antibiotics and at least 23,000 people die each year as a direct result of these infections." [18]

Is that a comprehensive definition?



Instead, why not start with a simple pictorial representation of what antimicrobial resistance is.  In a previous post on the need for greater science communication, Dr. Tyler Dewitt gave a great explanation of the modes of how virus's and bacteria invade a host organism like the human body.  I suggest taking a look at the post.  Once the invader (virus or bacteria) enters the system, the invader takes hold (control) of your system.  The control allows the invader to make several copies of itself to proliferate and grow to become a problem (onset of disease).



What can be done about such a state?



One methodology that has become increasingly common if the invader is a microbe or bacteria is to administer antibiotics which wipe out the infection.  Shown below is a picture taken from the 'Wikipedia' page again for clarity to illustrate the point of action of antibiotics:

Source:By NIAID – NIH

The image above is very simple to understand the action of an antibiotic.

Advances In Antibiotic Resistance

Recently, there has been advances in research surrounding antibiotic resistance that may speed up the ability to deal with the looming threat.  In an article from the website 'Sciencedaily.com' titled "Mystery of bacteria's antibiotic resistance unravelled" new developments have been made in unraveling the mode of disabling the effect of antibiotics by researchers.  Here is an excerpt discussing the advancement:

One of the mechanisms leading to rifampicin's resistance is the action of the enzyme Rifampicin monooxygenase.

Pablo Sobrado, a professor of biochemistry in the College of Agriculture and Life Sciences, and his team used a special technique called X-ray crystallography to describe the structure of this enzyme. They also reported the biochemical studies that allow them to determine the mechanisms by which the enzyme deactivates this important antibiotic.

The results were published in the Journal of Biological Chemistry and PLOS One, respectively.

"In collaboration with Professor Jack Tanner at the University of Missouri and his postdoc, Dr. Li-Kai Liu, we have solved the structure of the enzyme bound to the antibiotic," said Sobrado, who is affiliated with the Fralin Life Science Institute and the Virginia Tech Center for Drug Discovery. "The work by Heba, a visiting graduate student from Egypt, has provided detailed information about the mechanism of action and about the family of enzymes that this enzyme belongs to. This is all-important for drug design."

 Before I make a few comments on the success of the discovery in the pipeline to a marketable drug or treatment, I would like to add another excerpt from the same article highlighting the importance of the antibiotic rifampicin is to an array of diseases:

Rifampicin, also known as Rifampin, has been used to treat bacterial infections for more than 40 years. It works by preventing the bacteria from making RNA, a step necessary for growth.

The enzyme, Rifampicin monooxygenase, is a flavoenzyme -- a family of enzymes that catalyze chemical reactions that are essential for microbial survival. These latest findings represent the first detailed biochemical characterization of a flavoenzyme involved in antibiotic resistance, according to the authors.

Tuberculosis, leprosy, and Legionnaire's disease are infections caused by different species of bacteria. While treatable, the diseases pose a threat to children, the elderly, people in developing countries without access to adequate health care, and people with compromised immune systems.

As you can see, the ability of the antibiotic rifampicin to knock out an array of important diseases cannot be overstated.  Therefore, any advancement in understanding modes of action or in this case 'inaction' are critical to drug designers for the future.  At this point, you might be wondering what the structure of rifampicin looks like?  Shown below is the chemical structure of rifampicin take from 'Wikipedia':

Rifampicin

Source of image: By Vaccinationist - Rifampicin on PubChem

With the discovery of the mechanism by which Rifampicin Monooxygenase deactivates rifampicin's ability to act as an antibiotic, should we all throw our hands up and celebrate?



The discovery of the deactivation mechanism of rifampicin is a major step for drug makers in producing new lines of antibiotics in the future.  As I mentioned in a previous blog on drug discovery, the flow of patentable drug includes discoveries made at the university level.  This discovery certainly qualifies as one -- certainly.  Although, more studies will have to be followed up in order to realize the discovery into a better antibiotic in the future.



I should mention one major point of contention about the discovery of the mechanism.  The spectroscopic technique that was used was x-ray crystallography.   X-ray crystallography as a technique just celebrated it't 100th year since the discovery of the technique.  Here is an excerpt from the 'Wikipedia' page describing the technique of 'x-ray crystallography':

X-ray crystallography is a tool used for identifying the atomic and molecular structure of a crystal, in which the crystalline atoms cause a beam of incident X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder and various other information.

Since many materials can form crystals—such as salts, metals, minerals, semiconductors, as well as various inorganic, organic and biological molecules—X-ray crystallography has been fundamental in the development of many scientific fields. In its first decades of use, this method determined the size of atoms, the lengths and types of chemical bonds, and the atomic-scale differences among various materials, especially minerals and alloys. The method also revealed the structure and function of many biological molecules, including vitamins, drugs, proteins and nucleic acids such as DNA. X-ray crystallography is still the chief method for characterizing the atomic structure of new materials and in discerning materials that appear similar by other experiments. X-ray crystal structures can also account for unusual electronic or elastic properties of a material, shed light on chemical interactions and processes, or serve as the basis for designing pharmaceuticals against diseases.

The spectroscopic technique is very powerful and is commonly used in a wide range of areas of research for structural determination.  One drawback is the constraint of having to grow a crystal -- a rather large crystal to subject the x-rays to in order to obtain a diffraction pattern.



Why does this matter?



One commonly held belief among spectroscopists is that x-ray crystallography is extremely useful in a range of areas as a first step or a confirmation step.  The constraint of having to grow a crystal is also a large point of contention regarding the usefulness of the information obtained by the diffraction pattern.  Why?



The reason is centered around the fact that processes in the body (i.e., at physiological conditions) are performed in a 'liquid-state' rather than a 'crystalline-state' (i.e., solid state).  Scientists argue about the true degree of accuracy of a structure obtained by x-ray crystallography rather than say a structure obtained by nuclear magnetic resonance (NMR) in the liquid state.  The structure obtained by NMR is believed to be more representative of the actual conditions (liquid state, pH, temperature, etc.).



Nevertheless, the discovery above is extremely important.  The realization of a site of deactivation for rifampicin monooxygenase can now be further explored and compared to other antibiotics.  Scientists in industry and academia (university settings) will use incorporate this mechanism into their current understanding and models to produce a better antibiotic.



Further, understanding 'antimicrobial resistance' is a hot topic.  Just today, the 'Los Angeles Times' published an editorial discussing two bills that are hitting legislature for consideration.  Lets hope the combined efforts of all of these actions, leads to fewer cases of deaths in hospitals along with safer and better antibiotics.

Conclusion...

Should we be celebrating?


The advancement discussed above is cause to celebrate momentarily. Although, as I mentioned in the previous post regarding the drug discovery process, the path is long and arduous. Advancements such as these improve the ability of researchers to add another piece to the puzzle. Given more information, further advances can be pushed even further. Of course, that goes without being said (i.e., thank you captain obvious). Research is a long process and needs a lot of funding and time to test and retest procedures to make sure that scientists get the process right -- to eliminate the problem the first time around. Adjustments often have to be made due to inefficiencies of a given treatment or terrible side effects. Although, with a better understanding of the mode of action and inaction, drug manufacturers create more accurate drugs and research is one step further in understanding how nature operates to take control over our immune systems or subject us to terrible diseases.



Last but not least, the overall importance of writing a post like this is to convey the excitement and importance of such research. To demystify the meaning of "antibiotic resistance" or "antimicrobial resistance." Raising awareness of the magnitude of the issue will hopefully rally support on part of the public (your support) to elevate the need for funding and research into such issues. Do your part. Advocate for science and educate yourself on the successes and challenges (failures, obstacles). Give us some feedback.


Until next time, have a wonderful day!

How Do Chemists Discover New Drugs? A Brief Introduction!

How do chemists discover new drugs?  Obviously, in the laboratory!  Is that all one can say about the process?  Certainly not.  There is a process by which discovery happens.  The process may vary depending on which laboratory a chemist works in.  Although, the process does not vary so greatly as to eliminate a general procedure or process a drug takes from laboratory to the marketplace.  In the blog below, I introduce the general process by which drug discovery proceeds.  I want to highlight the word "introduction" since depending on your level of understanding, the process can be described in different ways.

Drug Discovery - General Route

I recently stumbled upon a video made by the 'National Institutes of Allergy and Infectious Diseases' (NIAID) titled "How A Drug Becomes A Drug" which I will show below in a moment.  Before I emphasize the importance of viewing the short video (less than 4 minutes), I want to introduce the agency NIAID -- which is a sub-agency of the 'National Institutes of Health'.  Here is an excerpt describing the organization taken from the "Wikipedia" page for the "NIAID" below:

The National Institute of Allergy and Infectious Diseases (NIAID) is one of the 27 institutes and centers that make up the National Institutes of Health (NIH), an agency of the United States Department of Health and Human Services (HHS). NIAID's mission is to conduct basic and applied research to better understand, treat, and prevent infectious, immunologic, and allergic diseases.[1]

NIAID has "intramural" (in-house) laboratories in Maryland and Montana, and funds research conducted by scientists at institutions in the United States and throughout the world. NIAID also works closely with partners in academia, industry, government, and non-governmental organizations in multifaceted and multidisciplinary efforts to address emerging health challenges such as the pandemic H1N1/09 virus.

The three main mission areas can be summarized from the "Wikipedia" page as follows:

Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS)

The goals in this area are finding a cure for HIV-infected individuals; developing preventive strategies, including vaccines and treatment as prevention; developing therapeutic strategies for preventing and treating co-infections such as TB and hepatitis C in HIV-infected individuals; and addressing the long-term consequences of HIV treatment.

Biodefense and Emerging Infectious Diseases (BioD)

The goals of this mission area are to better understand how these deliberately emerging (i.e., intentionally caused) and naturally emerging infectious agents cause disease and how the immune system responds to them.

Infectious and Immunologic Diseases (IID)

The goal of this mission area is to understand how aberrant responses of the immune system play a critical role in the development of immune-related disorders such as asthma, allergies, autoimmune diseases, and transplant rejection. This research helps improve the understanding of how the immune system functions when it is healthy or unhealthy and provides the basis for development of new diagnostic tools and interventions for immune-related diseases.

The above mission covers every disease known and unknown.  The National Institutes of Health is a huge organization made up of sub-agencies like the NIAID to divide up the mission.  As such, the NIAID oversees the funding of drug research to a large extent in order to understand how infectious disease compromise the immune system -- the body at large.  Additionally, the NIAID is interested in how drug discovery overcomes infectious diseases that have invaded our body.  This includes the research behind the disease at the academic level.



I mentioned above a short video to highlight the general process of drug discovery from the academic level up all the way through to the consumer level -- i.e. the pharmacy.  Here is the video below -- which is worth watching:

In the video above, the research is said to start at the basic science level at the university.  This is true to an extent.  Basic research into disease function and origin typically starts at the university level.  Although, I would add that a fair amount of research is done at the industry level too by large drug companies.  That research is typically targeted at a specific disease in which the pathway of progression or origin is known.  I will explain more about the last sentence shortly.



The drug companies take the research done at the academic (university) level and carry the "small molecule" or "drug target" out to an actual therapeutic that is sold on the shelf of the pharmacy.



Why is this important to know?



Periodically, in the popular news, stories emerge about the over pricing of medication by companies like Turing pharmaceuticals (outrageous pricing) which cause wonder as to why such high prices exist for a given medication.  These instances (of over pricing) are minimal compared to the price point needed to make a profit and move onto research more efficient drugs.  The point is that research at the companies take time and money along with infrastructure.



The overall benefit of such research could be realized through an "open-access" network of drug targets and therapeutics (proprietary information at the moment) to which other researchers could access at their leisure.  Arguments for such a system is that the funding has been provided by a government agency.  Whereas arguments against such a system is loss of proprietary information.  Tough call.  Sorry for the divergence.



The goal of research is to find effective therapeutics (drugs) that treat a large part of the population.  Side effects come about as a result of non-target delivery.  The drug misses the target of intent or hits additional targets and causes extra problems.  This is where the concept of "personalized medicine" comes in and will be discussed in future blog posts.  For now, lets focus on designing drugs for a certain disease.

Drug Design 101

In order to design a drug to treat a certain disease or ailment, the pathology of the disease needs to be known.  The origin of the disease needs to be known.  How did the disease originate in the body?



Is the disease the result of a mutation in the genetic make-up of the person?


Is there a mutation in the DNA of the patient which causes a downstream mutation in the production of proteins?


Is the protein distorted in shape, contour which affects function?  



Is the disease caused by an external agent (i.e. virus or bacteria)?



These problems can plague researchers success greatly for years.  Luckily, over time, drug companies have built up libraries of "molecules" that serve as "messengers" or "therapeutics" that can hit a specific target that is involved in the process of the disease.  Here is an excerpt from the "Wikipedia" page for "drug design" which I think will help you understand the process at the research level in either the university or industry setting:

Drug design, often referred to as rational drug design or simply rational design, is the inventive process of finding new medications based on the knowledge of a biological target.[1] The drug is most commonly an organic small molecule that activates or inhibits the function of a biomolecule such as a protein, which in turn results in a therapeutic benefit to the patient. In the most basic sense, drug design involves the design of molecules that are complementary in shape and charge to the biomolecular target with which they interact and therefore will bind to it. Drug design frequently but not necessarily relies on computer modeling techniques.[2] This type of modeling is sometimes referred to as computer-aided drug design. Finally, drug design that relies on the knowledge of the three-dimensional structure of the biomolecular target is known as structure-based drug design.[2] In addition to small molecules, biopharmaceuticals and especially therapeutic antibodies are an increasingly important class of drugs and computational methods for improving the affinity, selectivity, and stability of these protein-based therapeutics have also been developed.[3]

The phrase "drug design" is to some extent a misnomer. A more accurate term is ligand design (i.e., design of a molecule that will bind tightly to its target).[4] Although design techniques for prediction of binding affinity are reasonably successful, there are many other properties, such as bioavailability, metabolic half-life, side effects, etc., that first must be optimized before a ligand can become a safe and efficacious drug. These other characteristics are often difficult to predict with rational design techniques. Nevertheless, due to high attrition rates, especially during clinical phases of drug development, more attention is being focused early in the drug design process on selecting candidate drugs whose physicochemical properties are predicted to result in fewer complications during development and hence more likely to lead to an approved, marketed drug.[5] Furthermore, in vitro experiments complemented with computation methods are increasingly used in early drug discovery to select compounds with more favorable ADME (absorption, distribution, metabolism, and excretion) and toxicological profiles.[6]

The drug designer is looking for a "biological target" that is involved in either the origin or progression of the disease.  As mentioned above, there are two popular processes: computer-aided drug based design and structure-based drug design.  Both involve information on the biological target of interest.



What might a biological target look like?



A biological target can vary in definition depending on the nature of the disease.  For instance, if the disease involves the distortion or mutation of a protein, then the surface of the protein would be considered the biological target.  Specifically, the site of interest for a drug to interact with is referred to as the "active site."  Here is an image take from the "Wikipedia" page for "Active Site" to help assist the reader in what that might look like:

Source: Thomas Shafee - Own work, CC 

As you can see, the protein appears to be like a "blob" in the image above.  The reason for that to emphasize the "binding site" or "catalytic site" not the overall structure which is of little concern to the drug designer.  Remember that proteins are made up of amino acids which in turn form large "macromolecules" some of which are referred to as Proteins.  I wrote an earlier blog about oligosaacharides are made up of simple sugars.



Starting from the picture above, now, the video below might make sense to watch before we proceed with our discussion of drug design.  The video is titled "A Basic Introduction to Drugs, Drug Targets, and Molecular Interactions" and is just over 4 minutes long -- and definitely worth watching.

The video above is more technical than some readers might want to view in order to understand the process.  Therefore, we should back off a little on the "technical side" and focus on the "development" side of drug development -- from a simplistic standpoint.



Are you ready to understand drug design from a simple standpoint?



Alright, here we go!



In order to do so, I decided to borrow a few slides from a recent webinar offered online by the American Chemistry Society.  The webinar was titled "Crystallography As A Drug Design And Delivery Tool" and was given by Dr. Vincent Stoll of AbbVie -- where he serves as the Director of Structural Biology.



One of the examples that Dr. Stoll used to talk about drug design was binding to the transmembrane molecule B-Cell-Lymphoma-Extra-Large or bcl-xl in the mitochondria.  In his talk, he focused on a few binding sites shown in the slide below with a picture:

Specifically, in this case the company wanted to design a drug candidate that would "mimic" the peptide Bak binding.  Shown to the right on the slide are the sites or "active sites" that the peptide Bak bind to on the transmembrane molecule bcl-xl.  In order to find a drug that will mimic the binding of the peptide, the drug will have to have the ability to bind to multiple sites on the transmembrane molecule.



Fortunately, over time, large drug companies have built up a data base or library of 'molecules' that will bind to similar or exact sites.  In the slide below, I show a yellow surface with two molecules hovering above the surface -- slightly bound -- taken from Dr. Stoll's talk:

There is a lot of information on the slide shown above.  Let me walk you through the relevant information for drug design 101.  First, I mentioned that each drug company kept a library or database with a bunch of 'fragments' that are intended to hit specific targets on biological surfaces.  These biological surfaces can be viewed as the picture shown to the right in the slide.  They might be a protein surface, or another biological surface of interest to drug manufacturers.



In the case above, the two molecules shown on the yellow surface -- one is a brown color while the other is a green color.  The different color is to illustrate that the molecules are fragments designed to hit a specific type of target or active site on a biological surface.  In this case, the biological surface is the transmembrane molecule bcl-xl.

 

Once the fragments have been identified that will occupy and hit the desired targets or active sites, then the challenge is to link the fragments together by chemistry.  This step in of itself is often challenging and does not guarantee that the newly formed molecule (of two fragments with a linker molecule) will work.  Therefore, in the picture above, there are possible linker molecules that exist within the pharmaceutical database that have been shown to work in other cases.



After linking the two fragments together, the next step is to verify by spectroscopy that the total or linked molecule worked.



How is this accomplished?



In the lab, the substrate or biological surface will have a drop of the linked molecule injected onto the surface.  Then the surface which should have the drug bound to the active sites will be investigated using a spectroscopic technique like Nuclear Magnetic Resonance Spectroscopy.  Upon confirmation, a number will be reported as shown on the slide that indicates the binding affinity of the molecule onto the surface:

In the slide above, there are a couple of numbers reported that make sense to drug designers but probably not the reader -- you.  Do not worry.  Over time (through other blog posts) you will come to understand their meaning.  What is important to understand at this point is that after linking molecular fragments together, an experiment occurs to understand if the drug or linked molecule is as effective as the fragments are alone.



Furthermore, the pharmaceutical company might understand the chemistry of the active site to a large extent and further modify the linked molecules to make a more "potent" drug or linked molecule.  On the slide below, I show from Dr. Stoll's talk such a modification:

Again, the overall take home message is that the molecular modification done to the linked molecule has some effect.



Is that effect better or worse?



Can there be a further modification to the linked molecule or now drug to enhance the ability to mimic the peptide binding?



Who knows.  That is why research is continuously pushed forward and costs money to find out.

Conclusion...

In the above paragraphs, my intention was to introduce briefly the process of drug design.  As we speak though, changes are being made to parts of the process.  Outsourcing of linker molecules is occurring as are mergers and acquisitions of large companies by even larger pharmaceutical companies.  Which potentially means that the shared database or libraries of available drug targets is growing.  The process is dynamic but slow at the same time.



Discovering the mechanisms of disease and cures as a result is the dream of every drug designer.  Progress is unfortunately slowed down by the trial and error process.  Research takes time and money to complete.  Furthermore, improvements to existing drugs take time.  I will leave you with another short video about the progression of the medical research field:






Until next time, have a great day!!!!