Kinetically controlled drug resistance: how Penicillium brevicompactum survives mycophenolic acid

Abstract

The filamentous fungus Penicillium brevicompactum produces the immunosuppressive drug mycophenolic acid (MPA), which is a potent inhibitor of eukaryotic IMP dehydrogenases (IMPDHs). IMPDH catalyzes the conversion of IMP to XMP via a covalent enzyme intermediate, E-XMP*; MPA inhibits by trapping E-XMP*. P. brevicompactum (Pb) contains two MPA-resistant IMPDHs, PbIMPDH-A and PbIMPDH-B, which are 17- and 10(3)-fold more resistant to MPA than typically observed. Surprisingly, the active sites of these resistant enzymes are essentially identical to those of MPA-sensitive enzymes, so the mechanistic basis of resistance is not apparent. Here, we show that, unlike MPA-sensitive IMPDHs, formation of E-XMP* is rate-limiting for both PbIMPDH-A and PbIMPDH-B. Therefore, MPA resistance derives from the failure to accumulate the drug-sensitive intermediate.

FIGURE 1.

IMPDH reaction. A, mechanism of MPA inhibition. R5P, 1-β-d-ribofuranosyl 5-phosphate. B, the alignment of IMPDH-A and IMPDH-B from P. brevicompactum 23078 and Penicillium chrysogenum, IMPDH-A from A. nidulans, and IMPDH2 from Chinese hamster was mapped onto the x-ray crystal structure of the MPA complex of Chinese hamster IMPDH (Protein Data Bank code 1JR1 (8)). Dark magenta, 100% conserved; tan, 58% conserved; dark cyan, 17% conserved. MPA is shown in blue ball and stick, the IMP intermediate in gray ball and stick, K⁺ in purple, and residues within 4 Å of MPA and IMP in stick. Ala-249 is also shown because mutations at this residue confer MPA resistance.

FIGURE 2.

Mechanism of MPA inhibition of fungal IMPDHs. A and B, AnImdA, [MPA] = 0 (●), 10 (○), 25 (▾), and 50 (▿) nm; [E] = 39 nm. A, versus NAD⁺, [IMP] = 500 μm, and K_m = 10 μm. Data were fit to Equation 5. B, AnImdA versus IMP, [NAD⁺] = 500 μm, and K_m = 170 μm. Data were fit to Equation 5. C and D, PbIMPDH-A, [MPA] = 0 (●), 200 (○), and 400 (▾) nm; [E] = 76 nm. C, versus NAD⁺, [IMP] = 2 mm, and K_m = 130 μm. Data were fit to Equation 3. D, versus IMP, [NAD⁺] = 1 mm, and K_m = 340 μm. Data were fit to Equation 4. E and F, PbIMPDH-B, [MPA] = 0 (●), 10 (○), and 25 (▾) μm; [E] = 71 nm. E, versus NAD⁺, [IMP] = 5 mm, and K_m = 1.4 mm. Data were fit to Equation 3. F, versus IMP, [NAD⁺] = 5 mm, and K_m = 0.79 mm. Data were fit to Equation 5. All fits were performed to nonlinear equations using SigmaPlot. Lineweaver-Burk plots are shown for inspection only.