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Posts Tagged ‘mechanism of action’


C. botulinum toxin activity

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

The botulinum toxin as a therapeutic agent: molecular and pharmacological insights

Roshan Kukreja,1 Bal Ram Singh2
Dove  8 Dec 2015; Volume 2015: 5: 173—183
DOI http://dx.doi.org/10.2147/RRBC.S60432

 

Botulinum neurotoxins (BoNTs), the most potent toxins known to mankind, are metalloproteases that act on nerve–muscle junctions to block exocytosis through a very specific and exclusive endopeptidase activity against soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of presynaptic vesicle fusion machinery. This very ability of the toxins to produce flaccid muscle paralysis through chemical denervation has been put to good use, and these potentially lethal toxins have been licensed to treat an ever expanding list of medical disorders and more popularly in the field of esthetic medicine. In most cases, therapeutic BoNT preparations are high-molecular-weight protein complexes consisting of BoNT, complexing proteins, and excipients. There is at least one isolated BoNT, which is free of complexing proteins in the market (Xeomin®). Each commercially available BoNT formulation is unique, differing mainly in molecular size and composition of complexing proteins, biological activity, and antigenicity. BoNT serotype A is marketed as Botox®, Dysport®, and Xeomin®, while BoNT type B is commercially available as Myobloc®. Nerve terminal intoxication by BoNTs is completely reversible, and the duration of therapeutic effects of BoNTs varies for different serotypes. Depending on the target tissue, BoNTs can block the cholinergic neuromuscular or cholinergic autonomic innervation of exocrine glands and smooth muscles. Therapeutic BoNTs exhibit a high safety and very limited adverse effects profile. Despite their established efficacy, the greatest concern with the use of therapeutic BoNTs is their propensity to elicit immunogenic reactions that might render the patient unresponsive to subsequent treatments, particularly in chronic conditions that might lead to long-term treatment and frequent injections.

Therapeutic botulinum toxins: introduction and historical background

Botulinum neurotoxins (BoNTs) produced by anaerobic, spore-forming bacteria of the genusClostridium are the most toxic proteins known with mouse lethal dose 50% (LD50) values in the range of 0.1–1 ng/kg.1 They are solely responsible for the pathophysiology of botulism, a severe neurological disease characterized by flaccid muscle paralysis, resulting from BoNT-mediated blockage of acetylcholine release at the nerve–muscle junctions.2 BoNTs constitute a family of seven structurally similar but antigenically distinct proteins (types A–G) produced by different strains ofClostridium botulinum. BoNT serotypes share a high degree of sequence homology, but they differ in their toxicity and molecular site of action.2

Botulism was first identified in the early 19th century by Justinus Kerner, a German doctor and poet, when he linked deaths from food intoxication with a poison found in smoked sausages.3 He had even speculated about a variety of potential therapeutic uses of botulinum toxin for movement disorders, hypersecretion of body fluids, ulcers, etc.4 The scientific parameters of the disease were uncovered in 1897 by Emile van Ermengem, who successfully isolated the bacterium and named it Bacillus botulinus,5,6 which was renamed C. botulinum in later years.

In 1928, Snipe and Sommer at the University of California isolated BoNT as a stable acid precipitate for the first time,7 following which, standardized preparations of BoNT and maintenance of rigorous safety standards for its therapeutic use were achieved by Edward J Schantz, Carl Lammana, and colleagues from the Department of Microbiology and Toxicology at the University of Wisconsin, Madison.810 The first documented use of BoNT for the treatment of disease was in the 1970s, approximately 150 years after Kerner’s initial observations about the potential use of BoNT as a therapeutic, when Dr Alan Scott, an ophthalmologist, used local injection of minute doses of BoNT to selectively inactivate muscle spasticity in strabismus in monkeys.11 Following the success of a series of clinical studies on humans suffering from strabismus,12 the Food and drug Administration (FDA) in 1989 approved the use of BoNT/A (BOTOX®), manufactured by Allergan pharmaceuticals, for the treatment of strabismus, blepharospasm, and hemifacial spasm. Since then the very lethal botulinum toxins, botulinum types A and B, have been extensively used for the treatment of a myriad of dystonic and nondystonic movement disorders and a host of other medical conditions, including axillary hyperhidrosis, spasticity, tremors, and pain management. The high efficacy of BoNT/A coupled with a good safety profile has prompted its empirical use in a variety of ophthalmological, urological, gastrointestinal, secretory, and dermatological disorders.13 Incredibly, the list of conditions treated with botulinum toxin is expanding at a brisk rate.

The potential use of BoNT/A in esthetics was first demonstrated in 1987 based on the observation that facial wrinkles were diminished on treatment with BoNT/A for blepharospasm.14 Dynamic facial lines and wrinkles are caused by patterns of repetitive muscle contractions or facial expressions. Injection with BoNT temporarily paralyzes the nerve impulses responsible for muscle contraction, resulting in flattened facial skin and improved cosmetic appearance.15 This effect, although temporary, is extremely popular with patients, has a low incidence of side effects, making the use of BoNT/A the most common cosmetic procedure worldwide for facial enhancement.16 Botulinum toxin injections have revolutionized the nonsurgical approach to rejuvenation of an aging face and are now widely used for several esthetic procedures, including treatment of glabella frown lines, forehead furrows, and periorbital wrinkles.17

Molecular structure of BoNTs

BoNT is produced as a single polypeptide chain with a molecular mass of ~150 kDa that displays low intrinsic activity. This precursor protein is subsequently cleaved by bacterial proteases at an exposed protein-sensitive loop generating a fully active neurotoxin, composed of a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC). The HC and LC remain linked by both noncovalent protein–protein interactions and a conserved interchain disulfide bridge, called the belt, which extends from the HC and wraps around the LC.2 During intoxication process, the interchain bridge is reduced, and this is a necessary prerequisite for the intracellular action of the toxins.18 The three-dimensional structures of BoNTs reveal that they are folded into three distinct domains that are functionally related to their cell intoxication mechanism. The N-terminal domain is the 50 kDa LC, which is a Zn2+-dependent endoprotease. The 100 kDa HC consists of a N-terminal translocation domain and a C-terminal receptor-binding domain2 (Figure 1).

https://www.dovepress.com/cr_data/article_fulltext/s60000/60432/img/fig1.jpg

Figure 1 Schematic representation of different domains of BoNT/A.
Note: The heavy chain receptor binding domain is marked in red, green is the heavy chain translocation domain, and the light chain catalytic domain is colored blue.
Abbreviation: BoNT/A, botulinum neurotoxin type A.

BoNTs are secreted from C. botulinum in the form of multimeric complexes, with a set of nontoxic proteins coded for by genes adjacent to the neurotoxin gene.19 These protein complexes range in size from 300 kDa to 900 kDa. These large protein complexes consist of the 150 kDa neurotoxin moiety and the set of complexing proteins that are made of a nontoxic-nonhemagglutinin protein (or neurotoxin binding protein [NBP]) and several hemagglutinin proteins. These are known as neurotoxin-associated proteins (NAPs) and also as complexing or accessory proteins. Stabilized through noncovalent interactions, NAPs account for ~70% of the total mass.20

The nontoxic NAPs are believed to protect the neurotoxin from degradation during its passage through the low pH environment of the gastrointestinal tract.21 They are also known to assist BoNT translocation across the intestinal mucosal layer.22,23 The association of NAPs with the toxin is pH dependent, and at physiological pH, this complex is reported to rapidly dissociate allowing release of the neurotoxin in the blood stream.24,25 When used for therapeutic purposes, where BoNT/is not delivered orally, the role of these accessory proteins in protection against gastric pH extremes and proteases and in transport across the intestinal epithelium is not clear and not relevant to clinical efficiency.

Mechanism of action of BoNTs

When therapeutic BoNT preparation is injected into the target tissue, it acts as a metalloproteinase that enters peripheral cholinergic nerve terminals and cleaves proteins that are crucial components of the neuroexocytosis apparatus, causing a persistent but reversible inhibition of neurotransmitter release. The exact molecular mechanism of BoNT action still remains to be completely understood but existing experimental evidence suggests that BoNT intoxication occurs through a multistep process involving each of the functional domains of the toxin.26 These steps include binding of the neurotoxin to specific receptors at the presynaptic nerve terminal, internalization of the toxin into the nerve cell and its translocation across the endosomal membrane, and intracellular endoprotease activity against proteins crucial for neurotransmitter release.

BoNTs have a high affinity and specificity for their target cells and use two different coreceptors for binding at the neuronal cell surface. Binding of BoNTs to the neuromuscular junction involves a tight association between its receptor-binding HC domain and complex polysialogangliosides, particularly GT1b and GD1b that are known to be enriched in neurons.27,28 Upon binding to the gangliosides, the membrane-bound ganglioside–toxin complex moves to reach the toxin-specific receptor. Different BoNT serotypes bind to different protein receptors. SV2 (isoforms A–C), a synaptic vesicle glycoprotein, has been identified as a receptor for BoNT/A and BoNT/E.29,30 Synaptotagmin, a synaptic vesicle protein, has been identified as the receptor for BoNT types B and G.31,32

Following binding to neuronal cell surface receptors, BoNT is internalized into cellular compartments by receptor-mediated endocytosis.1 After BoNTs are incorporated within the early endosomes, the acidic environment of the endocytotic vesicles is believed to induce a conformational change in the neurotoxin structure. The HC is inserted into the synaptic vesicle membrane forming a transmembrane protein-conducting channel that translocates the LC into the cytosol.33

Upon internalization into the neuronal cytosol, BoNTs exert their toxic effect by virtue of the metalloprotease activity of the LC, which specifically cleave one of three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that are integral to vesicular trafficking and neurotransmitter release.2 The specific SNARE protein targeted and the site of hydrolytic cleavage vary among the seven BoNT serotypes. BoNT serotypes A and E specifically cleave SNAP-25 at a unique peptide bond. BoNT serotypes B, D, F, and G hydrolyze VAMP/synaptobrevin, at different single peptide bonds, and BoNT/C cleaves both syntaxin and SNAP-252 (Figure 2).

https://www.dovepress.com/cr_data/article_fulltext/s60000/60432/img/fig2small.jpg

Figure 2 Schematic model of mode of action of botulinum neurotoxins.
Notes: (A) Synaptic vesicles containing neurotransmitters dock and fuse with the plasma membrane through interaction of the SNARE proteins (Synaptobrevin, SNAP-25, and Syntaxin). (B) Botulinum neurotoxin binds to the presynaptic membrane through gangliosides and a protein receptor followed by internalization into the endosomes via endocytosis. Following this, the light chain is translocated across the membrane into the cytosol where it acts as a specific endopeptidase against either of the SNARE proteins. BoNTs cleave their substrates before the formation of SNARE complex. Copyright © 2009. Caister Academic Press. Reproduced from Kukreja R, Singh BR. Botulinum neurotoxins-structure and mechanism of action. In: Proft T, editor. Microbial Toxins: Current Research and Future Trends. Norfolk: Caister Academic Press; 2009:15–40.75
Abbreviations: SNARE, sensitive factor attachment protein receptor; BoNTs, botulinum neurotoxins.

The remarkable therapeutic utility of botulinum toxin lies in its ability to specifically and potently inhibit involuntary muscle activity for an extended duration. Intoxication of the nerve terminal by BoNTs is fully reversible and does not lead to neurodegeneration.34 Upon synaptic blockade of cholinergic nerve terminals by therapeutic BoNT, the neuron forms new synapses that replace its original ones in a process known as sprouting. As the nerve terminals eventually recover, original synapses are regenerated, the sprouts retreat, and the synaptic contact is reestablished leading to restoration of exocytosis.35

Depending on the target tissue, BoNT can block the cholinergic autonomic innervation of the tear, salivary, and sweat glands or the cholinergic neuromuscular innervation of striated and smooth muscles.36 After intramuscular injection, the dose-dependent paralytic effect of BoNT can be detected within 2–3 days. It reaches its maximal effect in <2 weeks and gradually begins to decline in a few months due to the ongoing turnover of the synapses at the neuromuscular junction.35 The duration of effect lasts somewhere between 3 months and 6 months, and the benefits have been observed to increase with time.37 There has been no evidence of any long-term or permanent degeneration or atrophy of muscles in patients with repeated injections of BoNTs over an extended period.35

Current therapeutic BoNT formulations

Despite a plethora of research on the molecular action and the medical uses of BoNTs, currently only two serotypes of BoNTs are commercially being used as therapeutics, type A (BoNT/A) and type B (BoNT/B). There are three preparations of BoNT/A that are approved by the FDA, namely, Botox® (onabotulinumtoxinA) manufactured by Allergan Inc., USA; Dysport® (abobotulinumtoxinA) by Ipsen Ltd, UK; and Xeomin® (incobotulinumtoxinA) manufactured by Merz Pharmaceuticals, Germany. BoNT serotype B (MYOBLOC®, rimabotulinumtoxinB; Solstice Neurosciences, USA) was approved by the FDA in year 2000.13 The remarkable therapeutic utility of BoNT lies in its ability to specifically and potently inhibit involuntary muscle activity for an extended duration. The major differences between the botulinum toxin drug preparations include the bacterial strains from which they are produced, their manufacturing processes, composition, and presence of NAPs, and the type and quantity of excipients used in each formulation.

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A Tribute to Johannes Everse

Author: Larry H Bernstein, MD, FCAP

 

Johannes Everse was a retired Tenured Professor at Texas Tech University Health Sciences Center in Lubbock, Texas, who dies on June 10, 2013.  He survived the Nazi invasion of Netherlands during World War II, and worked in the pharmaceutical industry after finishing a unique technical education the surpassed any that existed in United States that included an extensive knowledge of analytical instruments and expertise in organic chemical syntheses.  Given a unique opportunity, he applied for and obtained a position as a technician in the Laboratory of Nathan O Kaplan’s Laboratory at the time of Kaplan’s move from John’s Hopkins University to Brandeis University, where Kaplan with Sidney Colowick established the prestigious Methods in Enzymology series, and in a few short years built a worldclass Graduate Department of Biochemistry.  Kaplan was very sharp in selecting graduate students, postdoctoral students, and at administration, but his ability to recognioze potential talent was seen in his recruitment of Francis Stolzenbach and Johannes Everse.  He also gave considerable support to those who he had confidence in.  Consequently, Everse was able to take exams completing a BS degree, and eventually, the PhD degree at the University of California, San Diego, in the 1970s. When Prof. Kaplan was recruited to the UCSD campus by Martin Kamens, he was also installed in the National Academy of Sciences.

I worked with Jo Everse for several years as postdoctoral biochemist and resident-USPHS Fellow in  Pathology on the mechanism of the malate dehydrogenase (MDH) reaction and the regulatory function of the mitochondrial and cytoplasmic MDHs.   These were important formative years in my scientific training, and it was by no accident that I was sent to work in that laboratory by my previous mentor, a pathologist and biochemist who had worked on adenylate kinases, as I had been attracted to that problem as a medical student working on the ontogeny of the lactic dehydrogenases in the embryonic lens.  Jo Everse was responsible for synthesizing the pyridine nucleotide adducts that proved to be critical to understanding the pyridine nucleotide related dehydrogenase reactions.  Jo was undoubtedly a driving force in that laboratory.

It was at that time that my first daughter was born, and she had the opportunity to play with the Everse children, who as adults are both PhD biochemists.  I have been fortunate to live through a dynamic period in the history of scientific discovery, and most amazingly, at a time of decline in funding for science that has not been deterred since the Vietnam War.  You may consider it the cost of hegemony after the treaty that ended WWII and brought us the cold war.

Jo went on to a tenured faculty position at TTUHSS, and his retirement came shortly before his death at 80. While he stayed longer than his superiors wanted, his welcome was not so warm after he criticized the administration of the graduate program.  Unfortunately, he did not have the kind of backing that a colleague at Berkeley, Howard Schachman, Professor of the Graduate School Division of Biochemistry, Biophysics and Structural Biology, enjoyed.  It should not be a surprise how good health, power and money makes a difference in how it plays out.

Schachman was asked to retire in 2002 having a busy, well-funded study, that involved allostery and precisely – in the structure, function, assembly and interactions of biological macromolecules, with particular emphasis on the regulatory enzyme, aspartate transcarbamylase (ATCase).  The studies challenged earlier studies that designated the complex of ATCase with a bisubstrate ligand as the R state of the enzyme. but changes in the conformation were reinterpreted to be the result of the actual binding event rather than the allosteric transition whereby the enzyme is converted from an inactive, taut (T) state to the activated R conformation and they developed methods for understanding the formation of domains and the effect of deletions of helical regions on stability and the folding and assembly pathways.

Jo Everse came out of the depression in Europe (1931 birth), lived through WWII, and he managed to get a unique technical education that took him to Boston.  He became an excellent teacher.  He had a good marriage and father of two children.  He collected Packard automobiles and rebuilt them.  He also played the organ, and he made and maintained an organ for his home.  He lived a good life.

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