Pneumocystis carinii IMPDH was originally reported to be approximately 70 amino acids shorter at the amino terminus than IMPDH from other species1, but recent work in our laboratory has demonstrated that four splicing variants of theprotein are possible2. Three of these variants, including one identical to the originally described mRNA, contain a stop codon in the 5' region of the pre-mRNA that results in loss of regions of the amino terminus thought to be important for enzyme activity. The fourth variant codes for a form of IMPDH that contains 529 amino acids and retains key amino terminal sequences (GeneBank Accession No. 196975)2. In P. carinii isolated from infected rats, the splicing variant coding for the long form predominated and only the long form of the protein was detected; in organisms from culture, splicing variants coding for the short form of IMPDH predominated but only the long form of the protein was detected. The catalytic activity of this longer form of IMPDH from P. carinii was confirmed by a complementation study in bacteria lacking native IMPDH. P. carinii IMPDH long form was expressed with a his-tag on the amino terminal end and purified by metal-affinity chromatography. The protein retains catalytic activity and, like the native protein in P. carinii, exists as a tetramer in solution. The his-tagged protein showed a Km for NAD of 24 ± 1 micromolar and a Km for IMP of 51 ± 8 micromolar, similar to forms of the enzyme from other species. Substrate inhibition was observed at millimolar concentrations of both NAD and IMP. The enzyme activity was strongly inhibited by mycophenolic acid (IC50 0.066 micromolar) and showed a different pattern of inhibition with other inhibitors than the mammalian enzyme, suggesting selective drug design is possible.
Trimetrexate and BW301U (piritrexim isethionate), lipid-soluble inhibitors of dihydrofolate reductase, are potent inhibitors of the growth of Pneumocystis carinii in culture with WI-38 cells. Inhibition was observed with 0.1 microgram of trimetrexate or BW301U per ml. Trimethoprim is ineffective at 100 micrograms/ml in this culture system. Both trimetrexate and BW301U were effective as prophylactic agents against P. carinii pneumonia in rats; trimetrexate at 7.5 mg/kg protected 9 of 10 rats, and BW301U at 5 mg/kg protected 4 of 10.
The novel furopyrimidine N-(4-{N-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)methyl]methylamino}benzoyl)-L- glutamate (MTXO), a classical antifolate with antitumor activity comparable to that of methotrexate (MTX), has been studied as inhibitor-cofactor ternary crystal complexes with wild-type Pneumocystis carinii (pc) and recombinant human wild-type dihydrofolate reductase (hDHFR). These structural data provide the first direct comparison of the binding interactions of the same antifolate inhibitor in the active site for pc and human DHFR. The human ternary DHFR complex crystallizes in the rhombohedral space group R3 and is isomorphous to the ternary complex reported for a gamma-tetrazole methotrexate analogue, MTXT. The pcDHFR complex crystallizes in the monoclinic space group P2(1) and is isomorphous to that reported for a trimethoprim (TMP) complex. Interpretation of difference Fourier electron-density maps for these ternary complexes revealed that MTXO binds with its 2,4-diaminofuropyrimidine ring interacting with Glu32 in pc and Glu30 in human DHFR, as observed for MTXT. The presence of the 6-5 furopyrimidine ring instead of the 6-6 pteridine ring results in a different bridge conformation compared with that of MTXT. The bridge torsion angles for MTXO, i.e. C(4a)-C(5)-C(8)-N(9) and C(5)-C(8)-N(9)-C(1'), are -156.5/51.9 degrees and -162.6/51.8 degrees, respectively for h and pc, compared with -146.8/57.4 degrees for MTXT. In each case, the p-aminobenzoylglutamate conformation is similar to that observed for MTXT. In the pcDHFR complex, the active-site region is conserved and the additional 20 residues in the sequence compared with the human enzyme are located in external loop regions. There is a significant change in the nicotinamide ribose conformation of the cofactor which places the nicotinamide O atom close to the 4NH(2) group of MTXO (2.7 A), a shift not observed in hDHFR structures. As a consequence of this, there is a loss of a hydrogen bond between the nicotinamide carbonyl group and the backbone of Ala12 in pcDHFR. In the human ternary complexes, the cofactor NADPH is bound with a more extended conformation, and the nicotinamide O atom makes a 3.5 A contact with the 4NH(2) group of MTXO. Although the novel classical antifolate MTXO is not highly active against pcDHFR, there are correlations between its binding interactions consistent with its lower potency as an inhibitor of h and pcDHFR compared with MTX.
