Topoisomerase Inhibitors

Inhibiting enzymes involved in DNA synthesis should stop cancer cells, because cancer is uncontrolled cell division. Topoisomerases are enzymes that help to relieve the twists on double helical DNA as it unwinds preparatory to replication. It appears logical that topoisomerase inhibitors should be cancer inhibitors. Unfortunately targeting DNA-binding proteins also targets most of the signal receptors that are the targets for the evolution of plant alkaloids.

Drugs are designed to be specific in their interactions with a particular target protein, but they are too small to be specific and end up binding to many other related proteins. Hence, drugs have side reactions that are to some extent unpredictable, because the interacting proteins are not known.

Aspirin, for example, is supposed to bind to and inhibit COX-2, the enzyme that converts omega-3 and omega-6, long-chain fatty acids into corresponding anti-inflammatory and inflammatory prostaglandins, resp. Aspirin also binds to proteins that inhibit NFkB, the transcription factor that controls expression of inflammatory genes. Aspirin binds to dozens of other proteins. Aspirin does lots of other things than just blunt inflammation, but those side reactions are usually not significant enough to get our attention.

Heparin is one of the most commonly used drugs. It binds to and activates an inhibitor of thrombin, an enzyme that activates fibrin and mediates clotting. Heparin also binds to other components of the clotting system, as well as a dozen components of the complement system, and most of the cytokines that control communications throughout the body. When patients are given heparin injections, heparin binds continually to all of these components and must be constantly supplemented and monitored. Inflammation depletes the heparin components throughout the body, so it is not known prior to injection, how much heparin will be needed to saturate other serum proteins before the desired level of clotting inhibition is achieved. This illustrates rather dramatically that most drugs have only limited specificity.

One of my students provided another example of the minimal specificity of small molecules, especially the alkaloids and phenolics produced by plants. He brought to me a research article espousing the use of phenolics from yerba mate, which serves as a coffee-like stimulant in Argentina, as a topoisomerase inhibitor and potential anti-tumor treatment. Sure enough, phenolics extracted from this plant inhibit topoisomerase, and they may well be able to inhibit the growth of tumors, but it is doubtful that the binding of the phenolics to topoisomerase in the tumor nuclei has anything to do with inhibition of tumor growth.

Topoisomerase binds to nuclear DNA as the DNA unwinds during replication to produce two new double helical DNA molecules. Topoisomerase is a DNA-binding protein, i.e. a protein that binds to a negatively charged polymer of small deoxyribose sugars and flat purine and pyrimidine bases. Proteins bind to DNA in two ways. Amino acids of the protein either bind along the edges of the hydrophobic stack of base pairs, e.g. sequence-specific transcription factors, or they provide hydrophobic, flat surfaces that bind to the hydrophobic faces of the separated bases. Topoisomerase does both, because it deals with single-stranded regions of DNA and therefore binds to both the phosphates, as well as the bases. The important point here is that both aromatic amino acids, with flat hydrophobic rings, and the hydrophobic tails of basic amino acids, i.e. lysine and arginine, bind to the hydrophobic faces of nucleic acid bases.

I have illustrated the binding of a “topoisomerase inhibitor” to show the arginine (blue) in the active site cleft of the topoisomerase that binds across the hydrophobic face of the inhibitor (grey and red). Many plant phenolics and alkaloids would be expected to similarly bind and act as inhibitors of topoisomerase. This observation and the ease by which alkaloids enter cells (attached to circulating heparan sulfate?) suggests that a major function of the nuclear envelope may be to minimize access of alkaloid and related molecules to the nucleic acid binding proteins of the nucleus.

The binding promiscuity of secondary plant products is further exemplified by berberine. Berberine is an alkaloid found in goldenseal and is an herbal remedy used to treat a variety of inflammatory diseases. It also binds to heparin (and nucleic acids) to produce a fluorescent complex. Thus, mast cells that store and secrete histamine and heparin to produce the symptoms of allergy, can be vividly stained with berberine.

I could not resist the temptation to check to see if berberine also binds to topoisomerase. A quick search of the research literature showed that berberine is in fact a topoisomerase inhibitor.

The numerous cross reactions of drugs are further illustrated by metformin, the common drug used in the treatment of type II diabetes. Metformin is approximately planar and provides a surface that cannot hydrogen bond, i.e. it is hydrophobic. I expected that metformin would bind to tryptophans that I observed as common substrate-binding amino acids in the active sites of proteins that bound to polysaccharides, e.g. lectins, glycosidases and glycanases. To test this, I had students in one of my courses examine the inhibitory activity of metformin on E. coli beta-galactosidase. They found measurable inhibition and support for competitive binding to the active site that contains a pair of the predicted tryptophans.

My protein modeling and structural studies show the basis for numerous interactions between plant secondary compounds, drugs, nucleic acids, polysaccharides (glycosaminoglycans, e.g. heparin) and proteins. Unpredicted cross reactions abound and every drug can be expected to interact with multiple proteins. This provides a note of caution to the use of any drug and encourages minimal exposure, since many unobserved and unanticipated side effects are occurring. These observations also question routine ingestion of herbal remedies, after all, plants use their secondary products as potent defenses against being eaten. Alkaloids disrupt nervous systems and cellular signaling. Plants are not naturally safe.

Asthma Nitric Oxide

Arginine is the source of nitric oxide using the enzyme NOS. Inducible NOS is expressed in response to triggers of inflammation mediated by NFkB. NO in turn activates guanylate cyclase to increase cyclic GMP and cGMP relaxes muscle actin and myosin.

Studies are being made to assess the status of asthma by measuring nitric oxide (NO) in respired air. Since NO is produced as a product of the action of induced nitric oxide synthase (iNOS), and iNOS is synthesized when the inflammatory transcription factor NFkB is activated, then the inflammation that is a characteristic of asthma should be signaled by the production of NO.

If NO is involved in asthma, then one would expect relaxation of muscle to occur. That is the opposite of what is observed; contraction of the muscle cells surrounding airways is responsible for airway constriction is asthmatic episodes. Thus, during asthmatic episodes NO must be reduced.

Since NO is made from the amino acid arginine, then the ability to produce NO can be decreased by reducing available arginine. Arginine can be reduced by the enzyme arginase. The product is ornitine that can in turn be converted into purtrescine, the simplest of the polyamines that are used as counterions in the secretion of heparan and chondroitin sulfates.

Arginase is produced in high levels in asthmatics, but not in normal lungs. Also elevated putrescine is found in asthmatic lungs, indicating that arginine is being converted by arginase into ornithine and on to purtrescine. Ornithine is the precursor for proline that is a key amino acid in collagen production and the increased extracellular matrix that thickens the tissue of asthmatic lungs is rich in collagen.

A major point of this discussion is that NO can only be effectively lowered if there is a limited supply of arginine. Thus arginine supplementation should be helpful to asthmatics. Nitroglycerine patches may also be a means of enhancing sources of NO. Another possible approach would be the use of NO donors conjugated to NSAIDS. These conjugates delivered in an atomized form could provide the lungs with both NO and anti-inflammatory agents. Asthmatics also respond well to rigorous application of the anti-inflamatory diet and lifestyle.

Heparin Binding

This little video clip that I put together shows how a growth factor binds to its receptor. Heparin mediates the process. The growth factors bind to receptors in pairs with the heparin threaded through all four proteins and holding the complex together. The receptors would be embedded in the membrane at the bottom. This clip starts with a single receptor to which the heparin binds first, followed by the first growth factor.  The basic amino acids, arginine and lysine are shown in blue.  In the first image, the amino acid backbone is shown as a ribbon with blue bars.  In the second, the atoms of the amino acid side chains are filled in and the blue bars become blue atomic balls that reveal the surface of the protein.  The blue areas are positively charged and groups on the surface are heparin-binding domains.  The heparin is shown as a polysaccharide with atoms colored by element, O-red, S-orange, N-blue.  The proteins bind to the heparin like beads on a string.  Pairing of the receptors in a particular orientation determined by the heparin and growth factors, is responsible for triggering the cytoplasmic signal that the growth factor is present.