Effect of Azasteroids on Gram-Positive Bacteria

Abstract

A group of nitrogen-containing steroids closely related in structure was screened for antibacterial activity, by use of Bacillus subtilis and Sarcina lutea as the test organisms. The most active compounds were cholesterol derivatives containing a tertiary or quaternary nitrogen in, or attached to, the A ring. Similar methyltestosterone or progesterone derivatives were inactive. All of the cholesterol derivatives that inhibited growth were surfactant, and, structurally, they would be classified as cationic detergents. Some of the inactive compounds were surfactant, but, structurally, they would be classified as nonionic detergents. Certain features of the antibacterial activity of one of the active steroids-ND 212 (4-dimethylaminoethyl-4-aza-5-cholesten-3-one methiodide)-were studied. Growth of a culture of B. subtilis containing 5 X 107 cells per milliliter was inhibited by 1 Ag/ml (1.7 x 10-6 M) of ND 212. The amount of growth inhibition was directly related to both cell and steroid concentration. Loss of viability was rapid and irreversible. With B. subtilis, cell lysis was observed. With S. lutea grown in C'4-glucose, ND 212 caused release into the media of up to 25 % of the cellular radioactivity. Extensive leakage occurred before loss of viability was observed. At bacteriostatic azasteroid concentrations, there was little leakage. ND 212 was readily bound in large amounts to B. subtilis cells. Inactive azasteroids were bound poorly. C'4-cholestanone was also bound, whereas C'4-methyltestosterone and C'4-progesterone were not bound in significant amounts. At least 50% of the bound C'4-cholestanone was associated with the membrane fraction. Smith, Shay, and Doorenbos (22) were the first to report antimicrobial properties of steroids containing nitrogen in the nucleus. The compounds, 4-aza-cholestanes, inhibited the growth of various gram-positive bacteria, yeasts, and molds. The growth of gram-negative bacteria was not inhibited. Further studies by the same authors have delineated certain features of the mechanism of action of the compounds. The azacholestanes were found to be lethal to microorganisms (22) and to be surfactant (23). The authors concluded that there was no positive relationship between surface activity and antimicrobial activity (23). More recently, the compounds have been found to cause lysis of protoplasts of Sarcina lutea (21) and loss of cellular consitutents from yeast (R. F. Smith, Ph.D. Thesis, Univ. Maryland, College Park, 1965), suggesting that their primary site of action is the cytoplasmic membrane.

Effect of Progesterone on Neisseria gonorrhoeae

“…Previous reports have shown that only the growth of gram-positive organisms was inhibited by gonadal steroids (1,26,27) or by related azasteroids (18,21). No inhibitory effects were detected with a variety of gram-negative organisms.…”

Binding of cholesterol by Neisseria gonorrhoeae

“…Cholesterol and progesterone bind to different sites on the gonococcal membrane. In eucaryotic systems, progesterone binds to either protein (1) or phospholipids (18,19). Miller and Morse (8) showed that progesterone bound readily to isolated gonococcal membrane proteins, although a protein receptor on the intact membrane could not be conclusively demonstrated.…”

Synthesis and Evaluation of Antimicrobial Properties of Amidinoazaandrostanes and Guanidinoazaandrostanes

“…Many substances that are inactive in dc polarography give characteristic ac oscillopolarograms. The technique supplements classical polarography but has its own established theory and instrumentation (3)(4)(5)(6).…”