Dihydropteridine

Ptaridyt group

Glutamyl motaty

Figure 2-3. Folic acid.

-SO-C6H4NH2-p, -S-S-C5H4-NH2-p, -CONH2, or even -P03H2 groups yielded compounds retaining some antimicrobial properties. Activity approached or exceeded the original sulfonamides in a few instances. Other pioneering studies to relate antibacterial activity to the degree of ionization (pKa), and thus to structural parameters, were carried out. Increased activity was observed as the pKa value reached 6.7 and lessened again with further increases. With time, very active compounds outside this optimal range were discovered.5

Unraveling the full mechanism of action of the "sulfas" took over two decades. An early finding was that the action of sulfanilamide against Streptococcus haemoliticus can be antagonized by an extract of the bacteria, as well as pus and yeast extracts. It was proposed that the drug was inhibiting an enzyme whose normal substrate was also a low-molecular-weight compound. Subsequent investigations involving extractions and testing of a variety of tissues yielded a weak acid with a diazotable (aromatic) amine, which was identified as p-aminobenzoic acid (PABA). It was suggested that PABA was the naturally occurring substrate and that it was an essential metabolite for (sulfonamide-sensitive) bacteria. It was also demonstrated that PABA-containing extracts were able to reverse the sulfanilamide-induced inhibition of bacterial growth in a competitive manner.

A comparison of the structures of PABA (as the carboxylate) and sulfanilamide (as the anion) indicated that this competitive inhibition was due to the amazing congruence of structural and electronic features between the two molecules.

Findings of the wide natural distribution of PABA and the establishment of the structure of folic acid (Fig. 2-3) seemed to confirm Wood's earlier prediction.

A complete understanding of sulfonamide action evolved over a 20-year period. The biosynthesis of the various folates in living cells had to be elucidated; their functions in the "scheme of things" had to be worked out. The following discourse will consider the effects of sulfonamides, as well as that of another group of important enzyme inhibitors—the dihydrofolic acid reductase inhibitors. Figure 2-4 outlines the stratagem as it is presently understood.

Guanine (A) is converted in several steps to the pteridyl alcohol 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine (B). A two-step phosphorylation results in the pyrophosphate (D). The amine function of PABA is now in position to displace nucle-

5 For a comprehensive review of sulfonamides, see Seydel (1968).

Dihydropteridine Pyrophosphate
Figure 2-4. Biosynthesis and its interference of tetrahydrofolic acid.

ophilicaily the pyrophosphate, yielding 7,8-dihydropteroic acid (E). This reaction is catalyzed by the enzyme dihydropteroate synthetase (DHPS). It is at this point that sulfonamides (SA) are believed to compete with PABA by binding to what is probably two sites on the DHPS enzyme that are "reserved" for -NH2 function of PABA and its carboxylate function, now occupied by the drug's -NH2 group and sulfonamide anionic moiety, which are also about 6.7 A apart. The next step involves the incorporation of glutamic acid (F) resulting in 7,8-dihydrofolic acid, FH2 (G). Finally, the enzyme dihydrofolic acid reductase (DHFR or FH2R), catalyzes the reduction of FH2 to 5,6,7,8-tetrahydrofolic acid, FH4. There exist dihydrofolic acid reductase inhibitors such as trimethoprin (TM) that can prevent this step from occurring, thus preventing FH4 synthesis as well. As will be seen (Chapter 4), FH4 is a crucial coenzyme in the biosynthesis of purines and thymidilic acid, and therefore of RNA and DNA. The interference by sulfonamides in this biosynthesis therefore deprives the bacterial cell of the building blocks for new nucleic acids and, ultimately, inhibits bacterial reproduction (bacteriostasis). The selectivity of this cellular toxicity is due to the fact that the mammalian host cells cannot carry out this synthesis but must obtain folic acid from exogenous sources (diet) and reduce it to FHt by folate reductase.

The scheme of sulfonamide mechanism as outlined so far fails to answer an important question. If we are really dealing with a classic competitive inhibition by an inhibitor (the sulfa drug) of a substrate (PABA), why is the inhibition not overcome as the level of normal substrate rises under in vivo conditions? Actually, if a sulfonamide and PABA are added to a growing bacterial culture simultaneously, then no growth inhibition is observed. However, delaying the addition of PABA to the sulfa-treated bacterial colony decreased its effectiveness as the drug's antagonist to the point that reversal of growth inhibition ceases after 1 to 2 hours. This means that we are not dealing simply with competitive inhibition; something else must be happening as well.

Several possibilities come to mind. One is that the sulfonamide actually forms a pteridinyl derivative such as I, which in turn may tend to reduce the availability of the pteridyl pyrophosphate ester D, and decrease the production of G. A condensation product of sulfamethoxazole and the pteridyl alcohol B was actually obtained, raising the possibility of a sulfonamide-containing "imposter" of dihydropteroic acid arising. A second viable possibility is to consider a tautometric equilibrium between the pteridine alcohol B and the aldehyde form C, which will very likely form the Schiff base derivative J, with the p-amino function of a sulfonamide. This would also lead to a pteridyl alcohol depletion from the path of the FH2 synthesis (Seydel, 1968).

The fact that the FH4 synthesis can be blocked at two different enzymic steps (i.e., by sulfonamides at DHPS and by FH2tf inhibitors at that reductive step) resulted in a pharmaceutical product (Co-Trimoxazole) consisting of sulfamethoxazole and trimethoprim. The assumption is that a sequential double blockade is at work allowing these two drugs to act synergistically and minimize the emergence of resistance dramatically. Clinical effectiveness appears to bear this out, yet the matter is not readily settled. It was demonstrated that not all FH2/? inhibitors act synergistically with sulfonamides. There is now evidence that sulfonamides act on FH2/? as well as on DHPS, suggesting that as the reason for the actual synergism observed. Thus a simultaneous multiple inhibition, rather than a true sequential blockade, may be operative here.

Was this article helpful?

0 0

Post a comment