Ion pumps as targets for therapeutic intervention

Public Health Research Institute, New York, NY 10016
E-mail: perlin@phri.nyu.edu
Tel. 212 578-0820

The development of an effective target for therapeutic intervention remains a critical part of the drug discovery process. Ion pumps, which regulate ion flow across cells, serve as selective targets for common drugs used to treat heart disease and gastrointestinal ulcers. The clinical success of these drugs continues to make ion pumps desirable as targets for developing new therapeutics including antiulcer and antifungal drugs.

Introduction

The modern drug discovery route starts with a disease and the identification of a therapeutic target, which can be assayed quantitatively for assessing lead compound development. Ultimately, the success of a given therapeutic will be a direct result of its effectiveness against its target, which in turn leads to an amelioration or elimination of a given disease state. Target specificity is critical to limit toxicity, although absolute specificity (one drug-one target) is difficult to achieve and even more difficult to prove. Clearly, the more that is known about the fundamental biochemical and physiological properties of a specific target, the greater the chance that the drug discovery processes will lead to a successful end-product. One such target class is the P-type ion pumps, which are enzymes involved in ionic balance in all cells. Ion pumps use the cellular energy currency ATP to function.

Representative examples include the sodium pump of cardiac cells, which is blocked by the action of cardiac glycosides like digoxin (lanoxin) used to treat heart disease, and the proton pump of stomach, which is selectively blocked by the action of omeprazole (prilosec), an acid blocker used to treat gastric ulcers and other gastrointestinal disorders. Lanoxin and prilosec are two of the top ten leading prescribed therapeutics worldwide, and collectively account for more than $3 billion in sales. Their market success reflects a positive clinical outcome for therapeutic usage. However, given the fact that the targets of these drugs are very closely related it is the specificity of the drug-target interaction that ensures clinical outcome. This brief review will focus on essential properties of P-type ion pumps that make them successful targets for existing therapeutics, and will discuss how these properties are being exploited for the development of new classes of therapeutics directed against pathogenic fungi.

Overview of P-type Ion Pumps

The P-type ion pumps comprise a large family of proteins in animal, plant, bacterial and fungal cells that are responsible for the active movement of ions in cells. P-type ion pumps frequently play prominent roles in cell and tissue physiology, and are plentiful in most cells (Pedersen and Carafoli, 1987). At the structural level, these enzymes share a common organisational motif (Lutsenko and Kaplan, 1995). They form a mushroom-like structure with the stalk portion of the enzyme traversing the cell membrane. Structural conservation is strongly maintained for P-type ion pumps, even when their building blocks, the primary amino acid sequence varies. All P-type enzymes maintain certain critical structural features that are essential to function. However, significant diversity occurs in the region outside the cell, which aids the diversity of drug interactions. Structural conservation helps define P-type ion pumps and enables them to behave mechanistically in a similar manner. Yet, it is the subtle diversity between members of the P-type class that accounts for differences in enzyme function and drug interaction. It is this embedded diversity within a standardised organised structure that facilitates pharmacological approaches to these enzymes.

Cardiac Glycosides and the Sodium Pump.

Congestive heart failure remains one of the most common causes of death and disability in industrialised countries, accounting for several hundred thousand deaths each year. Pharmacological intervention at an early stage of clinical disease is essential to prolong life, although mortality rates for 5 year survival still approach 50 % (Kelly and Smith, 1996). Heart failure may result from ventricular dysfunction, and is most commonly observed in individuals with advanced atherosclerosis. Therapeutic regimens must be tailored to suit individual conditions.

For centuries, dating back to the description of the foxglove plant Digitalis purpurea in 1785, digitalis and other related cardiac glycosides have been used to treat congestive heart failure. Cardiac glycosides have a positive affect on heart output by increasing the speed of cardiac muscle function. The increased cardiac output ameliorates the disturbances characteristic of heart failure (Brunton, 1996). The molecular basis for the increased force of contraction is largely due to an increase in cellular calcium levels that increases the velocity and extent of muscle shortening. The rise in calcium concentration is an indirect result of inhibition of the sodium pump by cardiac glycosides. Careful patient dosing leading to controlled inhibition of sodium pump activity is critical to avoid toxicity as small increases in sodium concentration can have pronounced effects on subsequent calcium availability. The toxicity associated with cardiac glycosides may actually result from excessive cellular calcium.

Inhibition of the sodium pump by cardiac glycosides occurs by binding to a specific site on the outside of the cell. The sodium pump is more than 10,000 times more sensitive to cardiac glycosides than related ion pumps like the calcium pump of muscle and the proton-pump of the stomach. Finally, it is clear that the pronounced affinity of cardiac glycosides for the sodium pump over related animal cell ion pumps resides in subtle differences in the structural properties comprising the outer portions of the enzymes. This diversity facilitates specific interactions that confer drug selectivity.

Ulcers and acid blockers

Excessive gastric acid production is closely linked to a number of disease states including duodenal and gastric ulcers, Zollinger-Ellison Syndrome, Gastroesophageal Reflux Disease, and an assortment of other conditions. About 20 million Americans develop at least one ulcer during their lifetime, and each year ulcers affect about 4 million people. More than 40,000 people have surgery because of persistent symptoms or problems from ulcers, and about 6,000 people die of ulcer-related complications. Peptic ulcers generally arise due to an imbalance of acid secretory mechanisms and mucosal protective factors. Type I ulcers, which typically occur higher in the stomach frequently result from impaired mucosal protective factors and are not linked to hyperacid secretion. In contrast, Type II ulcers gastric and duodenal ulcers are associated with elevated acid secretion. The aetiology of gastric and duodenal ulcers is complex, but it is closely correlated with infection by the bacterium Helicobacter pylori, malignancy, and use of nonsteroid anti-inflammatory drugs like aspirin. Mucosal colonisation by Helicobacter pylori contributes to defects in mucosal defences, and the eradication of the bacterium is an essential part of antiulcer therapy. It is estimated that global Helicobacter pylori infection rates vary between 20-90% and are linked to age and socio-economic status.

The major pathways regulating acid secretion by the proton pump in the stomach include neural stimulation via the vagus nerve, endocrine stimulation via gastrin release, and paracrine stimulation by local release of histamine. Therapeutic regimens intended to reduce acid secretion have exploited numerous aspects of this pathway. For example, the H₂ receptor antagonists in clinical use such as cimetidine and ranitidine competitively inhibit the interaction of histamine with H₂ receptors. In recent years, agents that block the proton-pump have become the preferred therapy due to their high degree of target specificity and clinical efficacy.

The proton pump inhibitors omeprazole (prilosec), lansoprazole (prevacid), and pantoprazole exist as pro-drugs that need activation for interaction with the proton pump (Lorentzon et al., 1997). The stable pro-drugs reach acid secreting cells from the blood and are transported into the strongly acidic environment of the stomach where they are converted to an active form that blocks the proton pump.

Unlike classical drugs, the highly selective nature of proton pump antagonists is derived from their accumulation in the acid environment, which activates pro-drug in close proximity to its target. In principle, the highly reactive drug species generated can react with any protein. But its accessibility to a reactive moiety on the proton pump, and its relative instability when uncomplexed, confers exquisite specificity to this drug (Sachs et al., 1995).

As new anti-ulcer compounds progress through clinical trials, their value as therapeutics will ultimately depend on being potent and selective inhibitors of the stomach proton pump.

Developing new antifungals to the proton pump

Opportunistic fungal infections are frequent complications of HIV-infected and other immunocompromised patients. Candida albicans is the principal pathogen responsible for such infections, although other pathogens such as Cryptococcus neoformans, Aspergillus fumigatus, Histoplasma capsulatum, Pneumocystis carinii and other Candida species are important as well (Dixon et al., 1996). These organisms have become major hospital acquired causes of morbidity and mortality in the immunocompromised (Sternberg, 1994). Candida albicans is normally found on mucosal surfaces of the mouth, gastrointestinal tract and female reproductive system. It is usually contained through competition with other organisms, and the action of host defence systems such as an intact epithelium, salivary secretions, and antibody and cell-mediated immunity. Suppression of these systems by antibiotic therapy and or by disease provides a competitive advantage that allows Candida albicans to dominate the topical flora. Mucosal Candida infections are frequently observed in AIDS and other immuno-compromised patients as oral and esophageal thrush, and as vaginitis in women, which accounts for more than 10 million cases per year. Mucosal infections can be effectively treated with existing antifungal agents such as nystatin, miconazole, fluconazole, etc. In contrast, systemic or invasive fungal disease is a more serious condition that is commonly observed in severely neutropenic patients resulting from cancer chemotherapy and organ or bone marrow transplant therapy. The percentage of cancer patients with evidence of an invasive fungal infection ranges from 5-30 percent (Meunier, 1996), and fungemia now accounts for 15% of all hospital acquired bloodstream infections. Candida albicans is the fourth most common cause of all bloodstream infections accounting for 8% of all infections (Pfaller et al., 1998). Other organisms such as Aspergillus fumigatus frequently cause disease in cancer patients and have associated mortality rate exceeding 85%.

Therapeutic agents that are effective for superficial or mucosal infections are most often ineffective for use with invasive disease. The treatment options for invasive infections are extremely limited and almost always involve the use amphotericin B (Armstrong, 1993). However, recovery rates are poor and amphotericin B suffers from extreme toxicity resulting in severe side effects, including short- and long-term kidney dysfunction. Newer therapeutics, like fluconazole (Diflucan) and itraconazole (Sporonox) are highly successful in treating mucosal disease. But, their long-term efficacy in treating disseminated disease remains uncertain due to the presence of naturally resistant Candida species (Candida cruseii, etc.) and the development of resistance in previously susceptible species.

As the mortality from fungal infections grows worldwide, there is an urgent need to develop more effective therapeutics to deal with fungal disease. The present worldwide market for medical antifungals exceeds $3 billion, and it is expected that this market will experience significant increases in the next 5-10 years, as recognition of fungal infections as an important clinical problem continues to emerge. It is imperative that new targets be identified to expand the repertoire of antifungal options.

A New Mechanistic Class

Several years ago, we proposed that the fungal proton pump could serve as a target for a new class of antifungal drugs (Monk and Perlin, 1994). The fungal proton pump helps regulate pH within the cell and maintains the energy gradient necessary for nutrient uptake. The proton pump is one of the few antifungal targets that have been demonstrated to be essential by gene disruption techniques, and it has been linked to the ability of fungi to cause disease.

Finally, many of the important antifungal drugs in clinical use today are limited by their inability to kill fungal cells. These drugs prevent additional growth of cells but have little affect on existing cell populations. Thus, a competent (or partially competent) immune system is required to clear infections. In the case of severely immuno-compromised individuals, this clearing is not possible and large cell populations often remain as potential sources of new infection. It is preferable that antifungal agents be able to kill existing cells. The proton pump is an essential enzyme that is needed for growth and it is likely that specific inhibitors will kill the cells. In fact, the importance of the H⁺-ATPase as an antifungal target was recently demonstrated by showing that agents capable of blocking the proton pump resulted in cell death. In the past few years, high through-put screening of natural product and combinatorial chemical libraries have resulted in the identification of several classes of compounds that show proton pump-directed antifungal properties. These compounds are highly active on a broad range of pathogenic fungi including Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus.
Target specificity is a central issue for the effectiveness of proton pump antagonists. However, it is becoming clear that there is sufficient diversity between the various ion pumps to provide a selective window of interaction leading to safer and more effective therapeutics.

Conclusion

The search for new therapeutic targets remains a primary goal of the pharmaceutical and biotechnology industries, and a great deal of emphasis has been placed on innovative approaches for target development such as those emerging from large-scale sequencing of genomes, DNA chip-based gene expression systems, and high resolution structure analyses. However, certain existing targets offer promise for future therapeutic development because drug selectivity can be achieved between like family members. The cardiac sodium pump and stomach proton pump are P-type ion pumps that serve as targets for two of the most clinically successful therapeutics, digoxin and omeprazole, respectively. These therapeutics, which are used to treat heart disease and gastrointestinal disorders, exhibit a high degree of enzyme selectivity that is essential to their clinical success. The stomach proton pump remains a target for a new generation of non-covalent proton pump antagonists, and the fungal proton pump has emerged as an important new developmental target for a new class of antifungal therapeutics. Finally, the large number of P-type enzymes in plants, other fungi, parasites, and bacteria that remain unexploited offers promise for expanded therapeutic development.

Acknowledgements

The Perlin Lab is generously funded by National Institutes of Health Grant GM 38225 and by grants from Astra Hassle and Phytera.

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