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Effect of 4-ethyl-4-aza-5[alpha]-cholestane(ND-497) on carbohydrate metabolism of Staphylococcus aureus.

ABSTRACT

The steadily increasing number of antibiotic resistant strains of infectious microorganisms is frightening. There is special concern over Staphlococcus aureus, the principal cause of hospital infections, boils, cellulitis, toxic shock syndrome and death from infection. Novel potent antimicrobial azasteroids, active against bacteria, molds and yeasts, have been discovered in our steroid research. We wish to describe recent studies on ND-497 (4-ethyl-4-aza-5[alpha]-cholestane) against a penicillin-resistant strain of Staph. It was found to be a more powerful inhibitor of this resistant strain than erythromycin, streptomycin, polymyxin B, neomycin or tetracycline.

These azasteroids inhibit growth of gram-positive bacteria and fungi at 1.0 [micro]g/ml or less. It appeared at first that activity resulted from damage to cellular membranes and a loss of essential constituents. We have now discovered that the azasteroids are specific in their action and toxic at concentrations that do not cause leakage. They disrupt energy production at low concentrations. Future research will seek to identify affected enzymes and proteins.

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INTRODUCTION

Increased personal hygiene, safe water supplies, vaccines, sulfa drugs and antibiotics had increased life expectancy from 47 to almost 80 years in a century. Now, emerging new infectious disease problems, the development of pathogens resistant to anti-infective drugs and the spectre of bio-terrorism are frightening. During the past 25 years few new antimicrobial drugs were introduced and most of these were "cousins" of drugs in use and prime candidates for microbial resistance. Adverse side effects are also being observed with increasing frequency prohibiting the use of certain drugs on some patients. Enserink (2003) describes a new strain of resistant staph, producing painful boils on many parts of the body, pain and death, which is epidemic in Aids communities and prisons. He expresses the fear that this strain will become established in our hospitals. Shnoyerson and Plotkin (2002) shared a frightening scenario in their book, "the Deadly Rise of Drug Resistant Bacteria." The possibility of being exposed to dangerous and difficult to treat infectious diseases has become a reality.

Since the mid-1950's, Doorenbos's laboratory has been developing procedures for synthesizing target heterocyclic steroids as potential medicinal substances. Novel androgen, progesterone, and aldosterone inhibitors, as well as anti-inflammatory, antibacterial, antifungal, neuromuscular blocking, and birth control products have been discovered in these studies.

Azasteroids, steroids in which one or more of the skeletal carbon atoms is replaced by nitrogen, proved to be the most interesting of the heterocyclic steroids. Azasteroids do not occur in nature. They must be synthesized. Beginning with position 4, methods for introducing a nitrogen atom at almost every position were developed. The first designed 4-aza-steroids were prepared in our laboratory. It was in the late 1950's. 4-Aza-5-androsten-17[beta]-ol-3-one and several derivatives were prepared It was anticipated that they would be competitive antagonists of testosterone rendering them useful in delaying the development of enlarged prostate glands and prostate cancer as well as treating these conditions. These studies were supported by the National Cancer Institute. Finasteride and Proscar, first marketed around 1980, are two analogues of these 4-azasteroids in use today.

An antimicrobial screening program, inaugurated specifically for our heterocyclic steroids in 1960, identified several very active compounds (Doorenbos 1962 1964, Shay 1962, Smith 1963 1964, Varrichio 1966). Most were azasteroids. The first announcement of antimicrobial azasteroids reported the inhibitory activity of five steroids on resting and growing cells of Sarcina lutea and Saccharomyces cerevisiae at concentrations of 1 to 100 [micro]g/ml. Additional studies with sixteen nitrogen-containing steroids (Smith 1964) revealed that four 4-azacholesanes inhibited a large number of gram-positive bacteria, yeasts and molds at concentrations often less than 1 [micro]g/ml. Growth of Gaffkya tetragena, the most sensitive microorganism, was inhibited at 0.02 [micro]g/ml. Brucella abortus was the only observed sensitive gram-negative bacterium in these initial studies.

An investigation of effects of five of these steroids on Streptococcus pyogenes (Smith 1963) demonstrated a similarity with cationic quaternary ammonium salts. Each are complexed by serum proteins, are hemolytic, inhibited by lecithin and fatty acids, and lower surface tension. However, the many observed differences made it clear that there was much more to mechanisms of action.

Many variations in structural features of azasteroids markedly affected activity suggesting interactions with enzymes, proteins or other receptors are significant in their mechanisms of action. For example, azaandrostanes and azapregnanes are inactive, unlike the azacholestanes (Smith 1963 1964)). Azacholestane derivatives with a 5[beta]-configuration are inactive (Scott 1966). A 3[beta]-alkyl group enhances activity and a 6[beta]-alkyl group destroys activity (Doorenbos 1965).

Two mechanisms affecting cell permeability were revealed in studies with the yeast, Saccharomyces cerevisiae. Sublethal concentrations of azasteroid blocked uptake of glucose and alanine by viable resting cells as did uranyl nitrate (Smith 1964). Uranyl nitrate is known to be a specific inhibitor of glucose transport. Yeast cells, pretreated with uranyl nitrate, were protected from the lethal action of these steroids. Thus it appears that these substances competitively bind at the same site which is involved in nutrient uptake.

Nutrient uptake inhibition would be growth limiting but would not explain the primary lethal action of the azasteroid. It was postulated that azasteroid inhibition of cell growth was due to effects on essential enzymes or other proteins inhibiting essential functions such as energy production. Examination of the effects of ND-497 on S. lutea revealed that it inhibited dehlydrogenase of whole cells or lysates regardless of whether the systems were using endogenous, glucose or succinate metabolism (Smith 1965).

METHODS

The azasteroid selected for these studies was 4-ethyl-4-aza-5[alpha]-cholestane (ND-497), Figure 1, (Doorenbos 1962), prepared synthetically from cholesterol.

[FIGURE 1 OMITTED]

A penicillin resistant strain of S. aureus K257 culture was selected for the respiration studies. Stock cultures were maintained on nutrient agar slopes in the cold and subcultured bimonthly.

Media: Media were obtained from Baltimore Biological Laboratories and sterilized by autoclaving, Bacteria were maintained on slants of brain heart infusion agar and yeasts and molds on Sabouraud dextrose agar.

Medium used for S. aureus contained: 20 g. pancreatic digest of casein, 110 [micro]g biotin, 50 [micro]g nicotinic acid, 50 [micro]g thiamine hydrochloride in 1.0 l. distilled water adjusted to pH 7.6 with sodium hydroxide and autoclaved. Fresh egg white was added to achieve 1.0% v/v. Cultures were incubated 12 hours at 37[degrees]C. Cells were washed with a phosphate buffer (pH 7) prior to respiration experiments.

Substrates and Test Compounds: 0.05 M solutions of the following were sterilized by filtration: glucose, sucrose, D-glucose-6-phosphate disodium salt, D-glucose-1-phosphate dipotassium salt, sodium citrate, cis-aconitic acid, and [alpha]-ketoglutaric acid. The following test compounds wee prepared at 120 [micro]g/ml and 60 [micro]g/ml: ND-197, uranyl nitrate, polymyxin B sulfate, streptomycin sulfate, neomycin sulfate, eythromycin and tetracycline hydrochloride.

Antimicrobial Activities of ND-497: Minimum inhibitory concentrations against the various microorganisms were determined, repeating each four times, by the two-fold serial dilution method in Eugon broth.

Respiration Studies: Duplicate measurements utilizing standard Warburg manometric techniques were used for measuring oxygen uptake.. Cells were harvested by centrifugation and washed twice with phosphate buffer prior to use. Oxygen uptake was measured in microliters at 15 min. intervals at 37[degrees]C.

RESULTS

This penicillin resistant strain of S. aureus exhibited four times the sensitivity to ND-497 of the other seven microorganism species assayed. It was sensitive at 10 [micro]g/ml, the lowest concentration used in these studies. The rate of respiration of S. aureus was highest for cells metabolizing glucose, intermediate for fructose and lowest for sucrose. ND-497 substantially inhibited the metabolism of each of these sugars at concentrations well below that producing lysis. The lowest concentration used in these studies was 10 [micro] ug/ml. Similarly, ND-497 inhibited the metabolism of pyruvate, glucose-1-phosphate, glucose-6-phosphate, Krebs cycle and pentose cycle intermediates. This is illustrated by averages of duplicate respiration measurements of microliters of oxygen uptake per flask in 105 minutes (Table 1).

Comparisons of inhibtion of glucose metabolism were made between ND497 and several antimicrobial agents known to inhibit carbohydrate metabolism.. It was discovered that not only was ND-497 the most potent of these substances, but also had the greatest effect in reducing glucose metabolism at each of several concentrations over the 105 minutes of each experiment. No evidence of the presence of resistant cells was observed in a series of gradient plate studies. Combinations of each of these antimicrobial agents and ND-497 were also evaluated. Tetracycline hydrochloride, erythromycin and uranyl nitrate each inhibited the actions of ND-497. Synergistic responses were observed when ND-497 was used in combination with neomycin sulfate, streptomycin sulfate or polymyxin B sulfate (Table 2).

DISCUSSION

ND-497 is one of over thirty novel potent antimicrobial substances discovered in Doorenbos's research. They are heterocyclic derivatives of natural steroids active against gram-positive bacteria, molds and yeasts. The goal of this study was to learn more about possible mechanisms of action.

Earlier studies had demonstrated that these substances damage the microbial cell membranes causing leakage of cellular constituents.. There was evidence of antimicrobial activity at concentrations which did not induce cell leakage. This observation is confirmed in this study which demonstrated inhibition of metabolism of glucose, fructose, sucrose, pyruvate, glucose-1-phosphate and glucose-6-phosphae without inducing cell leakage. These results demonstrate effects upon enzymes involved in oxidation/reduction

The discovery of antagonism of the effects of ND-497 by tetracycline, erythromycin and uranyl nitrate suggests that similar enzymes or proteins are involved. The synergistic effects of neomycin, streptomycin and polymyxin B indicates that they have different mechanisms of action and affect different enzymes and proteins. It is noteworthy that ND-497 was a more potent antimicrobial agent and inhibitor of carbohydrate metabolism of this strain of penicillin resistant Staphlococcus aureus than any of the antibiotics included in this study.

Research to identify affected proteins, enzymes, and other receptor sites, structural requirements, active sites and mechanisms of action are in progress.
TABLE 1 Warburg Respiration Measurements (Average of two measurements)

Substrates Oxygen Uptake
 ([micro]L 105 min.)

Glucose 190
Fructose 175
Sucrose 155
Glucose + 20 [micro]g ND-497 135
Fructose + 20 [micro]g ND-497 130
Sucrose + 20 [micro]g ND-497 105
Glucose-6-phosphate 47
Glucose-6-phosphate + 20 [micro]g ND-497 18
Glucose-1-phosphate 39
Glucose-1-phosphate + 20 [micro]g ND-497 35
Citrate 75
Citrate + 20 [micro]g ND-497 67
Pyruvate 73
Pyruvate + 20 [micro] ND-497 64
cis-Aconitate 48
cis-Aconitate + 20 [micro]g ND-497 18
[alpha]-Ketoglutarate 33
[alpha]-Ketoglutarate + 20 [micro]g ND-497 28

TABLE 2 Warburg Respiration Measurements (Average of Two Measurements)

Antimicrobial Substrate Oxygen Uptake ([micro]L 105 min.)

20 [micro]g. tetracycline
 hydrochloride 89
10 [micro]g tetracycline
 hydrochloride + 10 [micro]g ND-497 71
10 [micro]g ND-497 66
20 [micro]g ND-497 64
20 [micro]g erythromycin 91
10 [micro]g erythromycin + 10
 [micro]g ND-497 80
20 [micro]g streptomycin sulfate 70
10 [micro]g streptomycin sulfate + 10
 [micro]g ND-497 62
20 [micro]g polymyxin B sulfate 72
10 [micro]g polymyxin B sulfate + 10
 [micro]g ND-497 62
20 [micro]g neomycin sulfate 87
10 [micro]g neomycin sulfate + 10
 [micro]g ND-497 62


ACKNOWLEDGEMENTS

We wish to acknowledge the support of research grant A-106798 from the National Institute of Allergy and Infectious Diseases and facilities of the College of Pharmacy and Biology Dept., University of Mississippi and the Harrison School of Pharmacy, Auburn University for this research.

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Tinney, F. 1966 Synthesis and Antimicrobial Study of Steroidal Amines and Amides. Ph.D. Dissertation, University of Maryland.

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Norman J. Doorenbos

Department of Pharmacal Sciences

Harrison School of Pharmacy

Auburn University

Auburn University, AL 36849

doorenj@auburn.edu

Samia A. El Dardiry

Tanta University

Tanta, Egypt

Lyman A. Magee

Department of Biology

University of Mississippi

University, MS 38655
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Author:Doorenbos, Norman J.; El Dardiry, Samia A.; Magee, Lyman A.
Publication:Journal of the Alabama Academy of Science
Date:Jan 1, 2003
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