Ligand-based Drug Design: Homework (Winter 2009)
Influenza is a serious viral disease that is often resistant to drug treatments. It is very likely that were the worldwide avian influenza (H5N1) pandemic take place, the virus will acquire resistance to the current first-line influenza drug oseltamivir (Tamiflu). Thus, it is necessary to develop analogs of oseltamivir that can fight resistant strains of the virus.
The three-dimensional structure of oseltamivir (in active carboxylate form) bound to its target (influenza neuraminidase) is known. This structure of the bioactive conformer bound to neuraminidase is available from PDB (ID code 2HT8). Visualization of the bound inhibitor shows that the cyclohexene ring adopts a conformation in which the alkyl ether side-chain is in the equatorial position.
![CC(C)O[C@@H]1C=CCCC1](images/3iPrO_cHexene.png)
To pursue the fragment-based drug discovery effort, a small analog containing the cyclohexene ring and the isopropyl side-chain was created. This molecule, (S)3-isopropoxy-cyclohexene, is shown on the right. However, this fragment did not show a good binding to neuraminidase. Conformational analysis of this molecule was performed with the MM3 force field. It was found that isopropoxy side-chain in this fragment can exist in either equatorial or axial ring position. Thus, an hypothesis was proposed that the weak binding of the fragment could be due to the fragment adopting a different conformation than the bioactive conformation of known good binder oseltamivir.
With the help of literature searches and/or computer modeling, answer the following questions:
- Provide a rational justification on why molecules like oseltamivir inhibit viral neuraminidase.
- Discuss in general terms what is known about the equatorial/axial stereo preference of substituent in cyclohexane and cyclohexene. What are some of the general strategies to force a particular substituent into equatorial position? Provide literature references when appropriate.
- Generate 3D structure of the 3-isopropylether analog of cyclohexene (see the image above) in which the ether arm is in the equatorial position using a method of your choice.
- Generate 3D structure of the 3-isopropylether analog of cyclohexene (see the image above) in which the ether arm is in the axial position using a method of your choice.
- Minimize each of these two structures with MOLDEN's Force Field optimizer tool and MM3 Tinker force field. Make sure to give a unique names, such as 3iPrO_cHxe_eq and 3iPrO_cHxe_axto the two jobs. The minimized structures will be saved in files 3iPrO_cHxe_eq_2 and 3iPrO_cHxe_ax.xyz_2. If you choose so, you are free to use programs other than MOLDEN for this task.
- Perform conformational analysis of equatorial and axial conformers of the fragment using TINKER's scan program. You may restrict the number of conformers generated by using 8 kcal/mol Energy Threshold.
- Use the TINKER's program analyze to calculate Total Potential Energy of each conformer (this is also listed during the scan in the last column). Note that you can give the answers to questions TINKER asks on the command line. For example, to calculate the Total Potential Energy of equatorial conformer 3, type analyze 3iPrO_cHxe_eq.003 E.
- Create a table showing two lowest-energy equatorial conformers and two lowest-energy axial conformers. For each conformer, give the name, the value of the C=C-C-O dihedral, the value of the =C-C-O-C dihedral, the Total Potential Energy, and the relative energy with respect to the lowest energy conformer.
- Based on the relative energies of each of the four conformers in the previous table, calculate the probability of each of these conformers. The probabilities can be calculated from Boltzmann's distribution. When evaluating the partition function, you may ignore the presence of other conformers as they are significantly higher in energy (this means that i = 1, 2, 3, 4 in the partition function formula). Assume room temperature (kT = 0.593 kcal/mol) and no degeneracy (gi=1 as this is asymmetric molecule).
- Modify structure of the cyclohexene ring in the fragment in a rational attempt to make the equatorial position for the ether arm more favorable. Keep in mind that the MM3 force field cannot handle substituents that are too unusual (but Amber/GAFF in Molden can). Also, long flexible substituents will quickly increase the number of conformations to unmanageable numbers. Make the same change in equatorial and axial structures; make sure that the absolute stereochemistry of the two disubstituted molecules is the same.
- Minimize the equatorial and axial conformers of your modified structures using MM3 force field and MOLDEN. Do not worry if the axial conformer converts into equatorial during minimization.
- Perform conformational analysis of your modified molecules using TINKER's scan program. You may observe that the search from axial conformer also finds equatorial minima. This is the expected behavior for conformational analysis programs.
- Use the TINKER's program analyze to calculate Total Potential Energy of each conformer (this is also listed during the scan in the last column). Note that you can give the answers to questions TINKER asks on the command line. For example, to calculate the Total Potential Energy of equatorial conformer 3, type analyze 3iPrO_X_cHxe_eq.003 E.
- Create a table showing two lowest-energy equatorial conformers and two lowest-energy axial conformers of your modified compound. For each conformer, give the name, the value of the C=C-C-O dihedral, the value of the =C-C-O-C dihedral, the Total Potential Energy, and the relative energy with respect to the lowest energy conformer.
- Based on the relative energies of each of the four conformers in the previous table, calculate the probability of each of these conformers. The probabilities can be calculated from Boltzmann's distribution. When evaluating the partition function, you may ignore the presence of other high-energy conformers (this means that i = 1, 2, 3, 4). Assume room temperature (kT = 0.593 kcal/mol) and no degeneracy (gi=1 as this is asymmetric molecule).
- Overlay one of your best modified fragments with the structure of the bioactive conformer of oseltamivir from PDB file 2HT8 using a program of your choice (Chimera, PyMOL, and Sybyl are available in the computer lab). You can convert the Tinker's XYZ file to PDB file using either MOLDEN or TINKER's xyzpdb tool.
- Discuss if your attempt to increase the fraction of bioactive equatorial conformers by modifying the cyclohexene ring was successful.