ANALOGS OF PTEROYLGLUTAMATE (fOLATE) 581 



The most thoroughly studied folate analogs are aminopterin (4-hydroxy 

 group replaced by amino group) and amethopterin (10-methylaminopterin) 

 because of their importance in the chemotherapy of cancer. These substances 

 produce states of folate deficiency in all types of organism and, in most 

 instances, the symptoms of this deficiency can be prevented by providing 

 tetrahydrofolate or 5-formyltetrahydrofolate, but not with folate, indicating 

 that the site of block lies somewhere in the pathway of the transformation 

 of folate to its coenzymically active forms. Although this block produces a 

 general disturbance in tetrahydrofolate function and, it appears, nucleic 

 acid and protein synthesis, there are two reasons for some degree of speci- 

 ficity. In the first place, the syntheses of the various substances requiring 

 Ci units from folate coenzymes are not necessarily all depressed equally, 

 since it is a general rule that a lowering of the concentration of some sub- 

 stance from which several pathways lead will produce varying effects on 

 these pathways, depending mainly on the nature of the enzymes involved 

 and the supply of reactants for each pathway (in this case C^ unit accep- 

 tors). In the second place, those cells or tissues with the highest rates of 

 synthesis and dependent on these synthetic reactions for growth or multi- 

 plication wiU be most adversely affected by the folate analogs. Here it is 

 not a matter of degree of functional activity but of proliferation; the heart 

 is not readily affected by these analogs whereas the hematopoietic system is. 

 We shall not discuss the more biological aspects of the actions of these 

 analogs, since this is covered adequately in a number of books and reviews 

 (e.g. Holland, 1961; Delmonte and Jukes, 1962), but confine attention to 

 the basic enzyme and metabolic effects. 



Inhibition of the Reduction of Folate to Tetrahydrofolate 



This reduction occurs in two steps and in most instances it appears that 

 each step is catalyzed by a specific enzyme, but possibly in other cases a 

 single enzyme is responsible. Most assays of folate reduction for analog 

 inhibition have involved determination of the formation of either tetrahy- 

 drofolate or folinate, or the disappearance of folate, and it is difficult to 

 differentiate between the two steps with regard to inhibition. In some re- 

 ports the term "folate reductase" is applied to the over-all reaction. One 

 thing is certain: Dihydrofolate reductase, which has been better purified 

 and more thorougly studied than folate reductase, is very potently inhibited 

 by aminopterin and amethopterin. The enzyme from chicken liver is inhib- 

 ited 74% by 0.000053 mM aminopterin (Futterman, 1957) and from hu- 

 man leukemic leucocytes 64% by 0.00001 mM (Bertino et al, 1960), in 

 both cases the substrate being 10,000-fold or more in excess of the analog. 

 Osborn et al. (1958) calculated the K's for aminopterin and amethopterin 

 to be 0.000001 mM and 0.0000023 mM, respectively, using the chicken liver 

 enzyme, Blakley and McDougall (1961) reported a value of 0.0000024 mM 



