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An Interview with Professor Manuel Varela: Sulfonamides – and their Cousins

Oct 12, 2018 by

Michael F. Shaughnessy

Image result for Gerhard Domagk
Dr. Gerhard Johannes Paul Domagk

1) Professor Varela, there is always some talk about something called sulfonamides. I am guessing it is related to Sulphur.

The sulfonamide antimicrobials are named as such because this collection of chemicals contain a sulfur (Sulphur) atom, which is a prominent element in their overall structures and is necessary for their antimicrobial activity.  The sulfonamide group of antimicrobial agents are commonly known as the sulfa drugs. These sulfur-containing chemical agents have been used on a clinical basis for almost a century in order to effectively treat a variety of serious bacterial infections. These sulfa drugs are still clinically used in modern times.

The discovery by Dr. Gerhard Domagk of the first clinically important sulfonamide-based agent, Prontosil, with its antibacterial activity, represented a fulfillment of sorts towards a largely concerted effort to find a magic bullet, an effort first started by Prof. Paul Ehrlich near the beginning of the 20th century. Dr. Ehrlich had long sought to find a chemical agent, a magic bullet, which could be used to selectively inhibit or kill certain microbial pathogens in a human patient while presenting little to no serious side effects upon the patient. While Dr. Ehrlich’s discovery of the first magic bullet candidate, Salvarsan, was of limited success, it did inspire others, like Dr. Domagk, to pursue their own searches for perhaps more successful magic bullets.  Dr. Domagk’s discovery of the sulfur-containing Prontosil appears to be the first clearly successful magic bullet, starting what is considered now to be the modern field of antimicrobial chemotherapy of bacterial infections.

2) Who was and what was his involvement in this?

Dr. Gerhard Johannes Paul Domagk, a biochemist and bacteriologist, was born in a small town called Lagow (now Poland), in the province of Brandenburg Marches, Prussia, now Germany, on the 30th day of October, in the year1895.  Young Gerhard attended elementary school at Sommerfeld (now considered Lubsko, Poland).  He attended university at Kiel, where his education was interrupted by World War I (the Great War) and was wounded in 1915 and then served in the Medical Corp at his grenadier regiment for the duration of the war.  After finishing his duty in the war, in 1918, he returned to the University of Kiel, studying the discipline of medicine and taking his medical degree in 1921. At Kiel, he taught and became an assistant to Profs. Max Burger and Felix Hoppe Seyler.

In 1923, Dr. Domagk moved to the University of Greifswald, in the department of pathology and anatomy. Then, in 1925, Dr. Domagk relocated to the University of Münster, eventually becoming a professor there.  At about this time, Dr. Domagk married Gertrud Strübe, and together the couple had four children: one girl and three boys.  In the years between 1927 and 1929, Dr. Domagk participated in the performance of research at a prominent dye-making institution called I.G. Farbenindustrie (I.G. Farben), Bayer, at Wuppertal-Elberfeld.  In 1927, Dr. Domagk became director of research in the area of pathology and of bacteriology at I.G. Farben, where he remained for the duration of his scientific career. 

In the year 1939, just prior to the start of World War II, Dr. Domagk was awarded the Nobel Prize in the areas of physiology or medicine for his role in the discovery of Prontosil.  However, because Hitler and his Nazi party officials had been upset with the earlier bestowment of the Nobel Prize for peace to a German dissident, Carl von Ossietzky, in 1935, Dr. Domagk was forced to decline the offer from Stockholm.  It is said that Dr. Domagk was able to receive the Nobel after the war, in 1947, but without the monetary reward that is traditionally bequeathed to Nobel Laureates.  In 1964, on the 24th day of April, Dr. Domagk passed away at the age of 68. 

In 1932, Dr. Domagk discovered the antimicrobial nature of the Prontosil, a red dye produced by chemists Fritz Meitsch and Joseph Klarer who combined sulfonamide with a red azo chemical dye.  At first, the agent, called then Prontosil Rubrum, did not appear to inhibit or kill bacteria in Petri dishes, in vitro. Nevertheless, the agent showed good antimicrobial activity against bacterial infection in laboratory test animals, in vivo. In particular, the Prontosil was effective against the dreaded pathogens of the genera Staphylococcus and Streptococcus when these bacteria were used to artificially infect laboratory mice.  This finding strongly suggested that, in the living host, Prontosil was converted by host metabolism into an active form, which was, in turn, able to inhibit or kill the infecting bacteria.

Later studies by Jacques and Theresa Tréfouël working in the laboratory of Daniel Bovet showed that the Prontosil, an inactive form of the agent, was metabolized in vivo and converted to the active form, called sulfanilamide, making it antimicrobial inside the body. 

3) Prontosil seems to be somehow related to sulfonamides- or am I incorrect in this?

No. You are correct. You see, Prontosil, a red dye, is the first important member of the sulfonamides class of antimicrobial chemotherapeutics to be discovered.  The Prontosil chemical is actually an inactive precursor that is metabolized by patients into an active principle, called sulfanilamide, a structure that has potent antimicrobial activity against a variety of bacteria, including both Gram-positive and Gram-negative bacterial, as well as certain acid-fast bacteria. That is, the active structure of the sulfonamides is sulfanilamide, and it represents a modern class of anti-bacterial agents, many of which are still used today.  If some of these sulfonamides are provided in particular combinations with each other, these combinations will then enhance their therapeutic natures against certain bacterial aliments.

In modern times, the sulfanilamide-containing class of sulfonamide antimicrobials are further grouped into a larger type of chemotherapeutics, called antimetabolites. These so-called antimetabolites kill or inhibit the growth of certain bacteria by preventing the synthesis of precursor metabolites such as nucleic acids, building blocks which in turn are necessary for the synthesis of larger important molecules, like DNA and RNA. The bacteria that are targets of the antimetabolites cannot grow and live without their own DNA or RNA. Thus, the bacteria will be prevented from causing pathogenic damage to an individual. 

4) We often hear about families of drugs – that seem to be similar, yet have distinctly different actions. Can you explain?

There are indeed several important families of antimicrobial drugs.  Each of these distinctive drug families have their own unique sets of mechanisms for accomplishing their antimicrobial activities upon their bacterial targets. A summary of these antimicrobial mechanisms follows here.

First, the antimetabolites, represented in this case by the sulfanilamide-containing sulfa drugs (sulfonamides), constitute one of these types of antimicrobial drug classes. In general, antimetabolites function by inhibiting enzymes needed to make the precursor molecules that are further necessary to make certain building blocks, such as nucleic acids.  Without these required nucleic acids, various bacteria may not be able to undergo DNA or RNA production. Thus, the bacteria will die or cease to grow further.

In particular, the sulfanilamides (e.g., sulfamethoxazole, a synthetic agent) serve as structural analogs of the metabolic precursor called para­­-aminobenzoic acid (PABA), getting in the way of the PABA, preventing it from binding to its enzyme and making it terribly difficult for a bacterium to make a folic acid called dihydrofolic acid from the PABA substrate. Another metabolic step that is prevented by the antimetabolites (e.g., trimethoprim) involves the production of another folic acid called tetrahydrofolic acid from the dihydrofolic acid. Frequently, a combination of two sulfanilamides (e.g., trimethoprim-sulfamethoxazole) is used clinically, with good or even enhanced antibacterial activities.

Without these folic acids, bacteria cannot, therefore, make purine or pyrimidine nucleotides, both metabolic precursors of which are most certainly necessary for bacterial production of DNA and RNA. Bacteria that lack these important molecules will be unable to grow and mount an infection. The antimetabolites harbor elements of the magic bullet properties because humans apparently lack the folic acid producing machinery targets. 

Another mechanism of antimicrobial action is referred to as prevention of bacterial cell wall synthesis.  In this mechanism, certain antimicrobial agents like the β-lactams (e.g., the penicillins, the cephalosporins, or the carbapenems), the glycopeptides (e.g., vancomycin), the lipopeptides (e.g., daptomycin), the polypeptides (e.g., bacitracin), and many others, inhibit the cell wall-making machinery of the bacteria. 

As a result, the normally protective bacterial cell walls are either not built or are structurally weak. If the bacteria lose the ability to form cell walls, they will die. On the other hand, bacteria that form weak cell walls may no longer be as strong, structurally speaking, possibly making the bacteria burst from their own internal cellular metabolic activities. The end-result, of course, is that the bacteria, targets of the cell wall synthesis inhibitors, explode, in a bacterial process known as lysis. We humans lack many of the bacteria-based cell wall components in our own cells, resulting in a selective effect upon the bacteria without lysing human cells or other animal cells. It is another realization of the magic bullet effect.

A third antimicrobial mechanism is called protein synthesis inhibition. In this antimicrobial mechanism, the bacterial cell target resides in the cytoplasm, where an important giant macromolecule called the ribosome works.

The ribosomes function by making protein; as such, they are considered to be protein factories. Important classes of antimicrobials that fall into this category include the aminoglycosides (e.g., streptomycin), the tetracyclines, the oxazolidinones (e.g., linezolid), the chloramphenicols, the macrolides (e.g., erythromycin), the lincosamides (e.g., clindamycin), the glycylcyclines (e.g., tigecycline), or the streptogramins (e.g., quinupristin-dalfopristin), to name a few. Protein synthesis inhibitors such as these, and others, act on the ribosomes as their prime target, preventing the production of needed bacterial protein.

Without protein, bacteria simply cannot make many of the types of cellular molecules that are necessary for their lives.  Bacteria that lack their protein may not be able, for example, to undergo metabolism, acquire and process biological energy, produce cellular machinery for growing, or to produce their genetic hereditary material. Bacteria without their proteins, thus, cannot accomplish many of the functions they need to multiply; and they can stop growing or even die, as a result. The magic bullet concept hold here, too, in that humans and bacteria have distinctively shaped ribosomes, different enough for the protein synthesis disruptors to affect bacterial ribosomes with minimal interruption to human ribosomes.

A fourth antimicrobial mechanism of action is referred to as prevention of nucleic acid synthesis. In this case, antimicrobials directly interfere with the ability of bacteria to synthesize DNA (a process called DNA replication) or with the ability to synthesize RNA (called transcription).  Without bacterial DNA or RNA, these microbes cannot send their genetic information into the next generation or express their proteins. Bacteria that are affected in this way will fail to thrive, perhaps evening dying. In any case, the bacteria may be unable to cause an infectious illness. One type of antimicrobial agents that affect DNA replication include the quinolones, another group of compounds that were discovered by Dr. Domagk. 

The quinolones, and their chemical derivatives called the fluoroquinolones, inhibit DNA gryase, which is needed for DNA synthesis.  Another type of nucleic acid synthesis inhibitors include those which prevent transcription, the process of producing RNA. An important class of molecules that affect transcription includes the RNA polymerase inhibitors such as rifampin or one of its derivatives called rifabutin.

Again, a comparison of the nucleic acid synthesis machinery possessed by humans versus bacteria indicates that these two systems are sufficiently different on a structural level such that nucleic acid synthesis inhibitors primarily affect the bacteria more so than the humans who have an infectious disease.

An antimicrobial mechanism of action that could affect humans and bacteria more similarly is referred to as disruption of the plasma or cytoplasmic membrane.  In this case, the antimicrobial targets are sufficiently similar and affect both humans and bacteria, producing more toxicity (side effects) in human hosts of bacterial infection. 

Thus, these membrane disruptors are often used topically, on the outside skin surfaces of humans. These membrane disruptions are produced by making membranes leak, allowing molecules to move across them. 

The main membrane disrupting mechanism works by insertion of porous proteins, called cyclic polypeptides such as the polymyxins B and E (e.g., colistin), into the membranes.  Such porous membrane leakage prevents the containment of important biomolecules inside the cells, causing such cells to lyse, as well. Such antimicrobial agents can cause serious internal toxicity if taken internally.

Certain superbug bacteria, such as Acinetobacter baumannii or Pseudomonas aeruginosa, are pathogens that are highly virulent, and one of the chief reasons is because colistin, one of the main drugs to which these bacteria were susceptible to, may no longer work as effectively as they used to on these newer superbugs.

5) And we hear about “second generations” of drugs, where subtle differences are made and changes implemented. How does this come about?

After a new antimicrobial agent has been discovered and shown to be effective against infectious disease-causing microbes, almost invariably the new drug shows less antimicrobial activity with time. Frequently, the target microbes can mutate or acquire some antimicrobial resistance mechanism that reduces the effectiveness of the first generation agents. The new bacterial variants may not succumb to the first generation antimicrobial agents as their predecessors had.  Thus, the antimicrobial agent can be modified chemically, producing a second generation of variant antimicrobials. Often, these second-generation compounds can inhibit the growth or kill the bacterial microbes more effectively than their previous parental counterparts can do so.

The cycle may repeat itself in certain cases. The second generation of drugs that no longer work as effectively to treat infections are themselves changed yet again into a third generation of drugs. These newer third-generation drugs then work well, at first, to inhibit bacterial growth or even kill the bacteria outright. Then, as is usually the case, the third generation encounters newly drug-resistant bacteria. Thus, the third-generation of drugs will be chemically modified yet again to produce now a fourth generation of drugs.

The cephalosporin class of antimicrobials represents a good example of this repeating cycle.  As of this writing there are currently five generations of modified cephalosporins. Each new generation is frequently more effective than the previous generation. It is anticipated that the repeating cycle of competing generations of antimicrobial agents versus microbial variants may occur indefinitely.

6) Gerhard Domagk- what were some of his other discoveries? Moreover, what else is he known for?

In addition to playing a key role in the discovery, in 1932, of the sulfonamides, Dr. Domagk was apparently key in the important discovery of the newer quaternary ammonium compounds (QACs, pronounced “quacks” or sometimes “quots”), in 1935.  These more recent QAC agents served to provide reasonably good disinfection activity against a wide spectrum of bacteria located on various hospital surfaces and on medical instruments, etc. In modern times, certain key bacterial pathogens have developed various antimicrobial resistance mechanisms that render the QAC less effective in killing them.

Dr. Domagk was also key to the discovery, in 1946, involving a new class of anti-tuberculosis agents, called thiosemicarbazones, such as aminothiazole or aminothiodiazole. While these thiosemicarbazone-based agents were shown by others to have profound toxicity, or failed to work in the clinical setting, Dr. Domagk made the important suggestion that the thiosemicarbazones be combined with another new class of anti-tuberculosis agents, in particular, one called isoniazid.  The thiosemicarbazone-isoniazid combination therapy was, thus, shown to be an effective treatment against tuberculosis, enhancing the effect of the isoniazid.

7) What have I neglected to ask about sulfonamides?

The story is told that during the time when Dr. Domagk was actively studying his famous Prontosil, his only daughter, Hildegard, then a six-year old, was stricken with a severe case of septicemia by the Streptococcus bacteria, Dr. Domagk administered the Prontosil, saving her from amputation of her infected arm, and, importantly, saving her life.  It is also recorded that young Hildegarde acquired a lasting reddish stain on her skin due to the red-dye nature of the Prontosil.

Apparently, according to some sources, Dr. Domagk’s Prontosil saved other prominent individuals from the dreaded septicemia.  A one Winston Churchill comes to mind.  President Franklin D. Roosevelt also comes to mind. 

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