Computational Investigation of a Series of Small Molecules as Potential Compounds for Lysyl Hydroxylase-2 (LH2) Inhibition

Abstract

The catalytic function of lysyl hydroxylase-2 (LH2), a member of the Fe(II)/αKG-dependent oxygenase superfamily, is to catalyze the hydroxylation of lysine to hydroxylysine in collagen, resulting in stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs). Reports show that high amounts of LH2 lead to the accumulation of HLCCs, causing fibrosis and specific types of cancer metastasis. Some members of the Fe(II)/αKG-dependent family have also been reported to have intramolecular O₂ tunnels, which aid in transporting one of the required cosubstrates into the active site. While LH2 can be a promising target to combat these diseases, efficacious inhibitors are still lacking. We have used computational simulations to investigate a series of 44 small molecules as lead compounds for LH2 inhibition. Tunneling analyses indicate the existence of several intramolecular tunnels. The lengths of the calculated O₂-transporting tunnels in holoenzymes are relatively longer than those in the apoenzyme, suggesting that the ligands may affect the enzyme's structure and possibly block (at least partially) the tunnels. The sequence alignment analysis between LH enzymes from different organisms shows that all of the amino acid residues with the highest occurrence rate in the oxygen tunnels are conserved. Our results suggest that the enolate form of diketone compounds establishes stronger interactions with the Fe(II) in the active site. Branching the enolate compounds with functional groups such as phenyl and pyridinyl enhances the interaction with various residues around the active site. Our results provide information about possible leads for further LH2 inhibition design and development.

Figure 1.

(A) Crystal structure of full-length human lysyl hydroxylase LH3 and its active site complexed with αKG (PDB ID: 6FXK). (B) The general catalytic mechanism of the αKG-dependent hydroxylase superfamily. (C) Hydroxylation of lysine to hydroxylysine by the LH2 in the presence of molecular oxygen.

Figure 2.

(A) Calculated QM/MM interaction energies (kcal mol⁻¹) for the first set of test compounds (ID: 1–16). (B) Average QM/MM interaction energies ($\mathrm{\Delta }\mathrm{\Delta }{E}_{\text{Avg.}}^{\text{Intert.}}$) and the standard deviation for the compounds with the most negative interaction energies. All of the calculations are at the ωB97X-D/6-31G(d,p) level of theory and AMOEBAbio18 force field. (C) Averaged noncovalent index (aNCI) and (D) thermal fluctuation index (TFI) analyses for cpd12 (ID: 14). The isosurface cutoff for NCI and TFI is 0.4 au, and the data is plotted in the color range of $-0.05<\mathrm{s}\mathrm{i}\mathrm{g}\mathrm{n}\left({\lambda }_2\right)\rho <0.05\mathrm{a}\mathrm{u}$.

Figure 3.

(A) Calculated QM/MM interaction energies (kcal mol⁻¹) for the second set of the studied ligands (ID: 17–36) at the ωB97X-D/6-31G(d,p) level of theory and AMOEBAbio 18 force field. The plot of the noncovalent interactions (NCIs) between the ligand and surrounding amino acid residues for (B) cpd27p, (C) cpd26p, and (D) cpd2ph. The isosurface cutoff for the NCI is 0.35 au, and the data is plotted in the color range of $-0.05<\mathrm{s}\mathrm{i}\mathrm{g}\mathrm{n}\left({\lambda }_2\right)\rho <0.05\mathrm{a}\mathrm{u}$. Residues H666, D668, and H718 are shown in thin sticks, ligand atoms are in ball and sticks (with different color codes), ${\mathrm{F}\mathrm{e}}^{2+}$ is in pink vdW sphere, and the surrounding residues with the noncovalent interactions are in thick sticks. Hydrogen atoms are not shown for more clarity.

Figure 4.

(A) O₂-transporting tunnels with the largest calculated percentages along the trajectory observed in the apoenzyme. Calculated tunnels are colored in blue, green, and red based on the tunnel’s length (blue: shortest, red: longest). The intersection of the tunnels inside the protein is the active site, which is not shown for clarity. The molecular surface of the protein is shown in magnification to give a better view of the enzyme’s cavities. (B) Close-up of the approximate positions of the tunnel-lining residues with more than ~80% presence in all of the calculated tunnels/representatives. (C) Condensed sequence alignment between human LH2 (UniProtKB: O00469) and selected members of the LH family. Key: “*” indicates residues conserved in all of the sequences, while “:” and “.” indicate highly and weakly conserved residues.

Figure 5.

O₂-transporting tunnels with the largest calculated percentages along the trajectory observed in the third set of lead compounds. The twodimensional (2D) structure of each ligand is also given next to its tunnel graph. Calculated tunnels are colored in blue, green, and red based on the increase in the tunnel’s length. The intersection of the tunnels inside the protein is the active site of the inhibition reaction, but the active site’s residues are not shown for more clarity.

Figure 6.

(A) Calculated QM/MM interaction energies (kcal mol⁻¹) for the most stable representative of the studied ligands of the third set (ID: 37–44) at the ωB97X-D/6-31G(d,p) level of theory and AMOEBAbio 18 force field. (B) Averaged noncovalent interactions (aNCIs) between the ligand and its surrounding residues for c28a, c28b, c2d, and c24. The isosurface cutoff for the NCI is 0.35 au, and the data is plotted in the color range of $-0.05<\mathrm{s}\mathrm{i}\mathrm{g}\mathrm{n}\left({\lambda }_2\right)\rho <0.05\mathrm{a}\mathrm{u}$ au. Residues H666, D668, and H718 are shown in thin sticks, ligand atoms are in ball and sticks (with different color codes), ${\mathrm{F}\mathrm{e}}^{2+}$ is in pink vdW sphere, and the surrounding residues with the noncovalent interactions are in thick sticks. Hydrogen atoms are not shown for more clarity.

Scheme 1.. (A) First Set of Test Compounds^a and (B) Possible Coordination Geometries of the Ligands to Fe(II) in the Active Site

^aThe numbers in parentheses show the ligand’s charge and number of coordinated waters to the Fe cation in the active site in MD1 and MD2, respectively.

Scheme 2.

(A) Second Set of the Studied Ligands^a and (B) Different Geometries of the Active Site in Coordination with the Lead Compounds ^a Numbers in parentheses are ligand’s charge, ligand’s coordination mode (monodentate or bidentate), and coordination number of the complex, respectively.