QM/MM Study of Cytochrome P450 BM3 Catalysis Mechanism and Application in Drug Design

P450: Super family of monooxygenases Function: metabolism of carbon-source organic molecules: hormones, vitamin D etc. detoxification of xenobiotic compounds synthesis of biologically active compounds

Why interesting in drug design The eve of ADME in High-Throughput Screening Absorption by the intestine Diffusion to the organism Metabolism by the liver Excretion by the kidney & Toxicology

One-half of potential drugs fail because of ADME/Tox issues Of the estimated $600 million cost of bringing a new drug to market, more than $400 million of that is wasted pursuing leads that turn out to be dogs. P450 (CYP) enzymes--a class of enzymes responsible for the metabolism of more than 50% of all known drugs

Enzyme P450 BM3 The function of P450 enzyme in drug metabolism is responsible for the failure of a good inhibitor to become a useful drug P450 BM3: the closet bacterial analog to the mammalian enzyme, soluble, three domains (BMP heme domain, a FAD- and FMN- containing NADPH-cytochrome P450 reductase) Archives of Biochemistry and Biophysics 1999, 369(1), RH + O 2 +2e - +2H + R-OH+H 2 O

Structure: heme Fe, S-Cys and H2O as ligands Figure shows the porphyrin ring in the active site.

Putative conformational change Electron transfer: NADPH->FAD->FMN ->BMP (heme) Conformational change facilitated by the “ hinge ” moving between a “ closed ” conformation and an “ open ” conformation FMN-heme distance:18 Å ->8 Å

Dutton’s line: electron transfer Productive electron transfer: within 14 – 15 Å k ET =k 0 ET exp[-  R(t)] Munro, Andrew et al Trends in Biochemical Sciences 2002

Catalytic cycle of cytochrome P450 6a, 6b, and 7: putative, no experimental structure RDS: 2 nd e - injection Intermediate compound I : oxyferryl/porphyrin π-cation radical

X-ray structure and product 3200 mol min -1 (mol of enzyme) -1 (NADPH rate, 37°C), 50% conversion D. C. Haines et al., Biochemistry, JPZ, X-ray 1.65 Å, BMP with N-Palmitoylgolycine

Computational modeling Computational modeling proposed the following binding structure, B: Jovanovic et al, JACS (2005)

Solid State NMR Experiment 2D solid-state NMR 15 N 13 CO SPECIFIC CP spectrum of 13 COLeu; 15 N-Gly, 15 N-Phe labeled cytochrome P450 BM-3 bound with NPG at 0 (red) and -30 °C (blue). The L86-F87 pair exhibits a pronounced shift as a function of temperature. The L86-F87 pair is in the binding pocket, and in fact F87 plays a “ gatekeeper ” role in that its bulky side chain must be rotated in order to allow for substrate binding.

QM/MM Combined Quantum mechanics/ Molecular mechanics (QM/MM) is a hybrid technique to model enzyme- catalyzed reactions. A small reactive part of the system is treated quantum mechanically (i.e., by an electronic structure method); this allows the electronic rearrangements involved in a chemical reaction, namely bond breaking and making, to be modeled. The large non-reactive part is described by molecular mechanics, and the two regions are able to interact. The combination of the efficiency and speed of the MM force field with the versatility and range of applicability of the QM method allows reactions in large systems to be studied.

Docking structure and QM/MM modeling QM region of P450 BM3: Total charge= -3 Spin multiplicity= 4 MM region: protein, explicit water shell Docking structure: Schrodinger, Inc. (2004)

Activation Barrier in QM/MM Doublet, QM/MM, U-DFT, without zero-point energy correction

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