Table 1.

Inhibitory concentrations (IC₅₀, µM) against isolated TS and DHFR^a

	TS			DHFR
compd	Humanb	E. colib	T.gondiic	Humand	E.colie	T. gondiic
4	0.54	>180	1.8	0.21	0.016	0.17
5	5.4	230	27	4.0	0.020	2.2
Raltitrexedf	0.38	5.7	0.9	21	23	2.3
Pemetrexedg	9.5	76	2.8	6.6	230	0.46
MTX	29	90	18	0.022	0.0066	0.011

^aThe percent inhibition was determined at a minimum of four inhibitor concentrations within 20% of the 50% point. The standard deviations for determination of 50% points are within ±10% of value given.

^bKindly provided by Dr. Frank Maley, New York State Department of Health, Albany, NY.

^cKindly provided by Dr. K. Anderson, Yale University.

^dKindly provided by Dr. J. H. Freisheim, Medical College of Ohio, Toledo, OH.

^eKindly provided by Dr. R. L. Blakley, Jude Children’s hospital, Memphis TN.

^fKindly provided by Dr. Ann Jackman, Institute of Cancer Research, Sutton, Surrey, UK.

^gKindly provided by Dr. Chuan Shih, Eli Lilly and Co.

Dihydrofolate Reductase (DHFR) Assay.42 All enzymes were assayed spetrophotometrically in a solution containing 50 µM dihydrofolate, 80 µM NADPH, 0.05 M Tris-HCl, 0.001 M 2-mercaptoethanol, and 0.001 M EDTA at pH 7.4 and 30 °C. The reaction was initiated with an amount of enzyme yielding a change in OD at 340 nm of 0.015/min.

Thymidylate Synthase (TS) Assay. TS was assayed spectrophotometrically at 30 °C and pH 7.4 in a mixture containing 0.1 M 2-mercaptoethanol, 0.0003 M (6R,S)-tetrahydrofolate, 0.012 M formaldehyde, 0.02 M MgCl₂, 0.001 M dUMP, 0.04 M Tris-HCl, and 0.00075 M NaEDTA. This was the assay described by Wahba and Friedkin,43 except that the dUMP concentration was increased 25-fold according to the method of Davisson et al.44 The reaction was initiated by the addition of an amount of enzyme yielding a change in absorbance at 340 nm of 0.016/min in the absence of inhibitor.