Merging Allosteric and Active Site Binding Motifs: De novo Generation of Target Selectivity and Potency via Natural-Product-Derived Fragments

The de novo design of molecules from scratch with tailored biological activity is still the major intellectual challenge in chemical biology and drug discovery. Herein we validate natural-product-derived fragments (NPDFs) as excellent molecular seeds for the targeted de novo discovery of lead structures for the modulation of therapeutically relevant proteins. The application of this de novo approach delivered, in synergy with the combination of allosteric and active site binding motifs, highly selective and ligand-efficient non-zinc-binding (3: 4-{[5-(2-{[(3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl]methyl}benzoic acid) as well as zinc-binding (4: 4-({5-[2-({[3-(3-carboxypropoxy)phenyl]methyl}carbamoyl)eth-1-yn-1-yl]-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl}methyl)benzoic acid) uracil-based MMP-13 inhibitors presenting IC50 values of 11 nm (3: LE=0.35) and 6 nm (4: LE=0.31).

Uracil as NPDF interacts via its cis amide bonds with the backbone NH and CO of Met232, as well as the side chain amino group of Lys228 in the top ranked poses.
After a thorough investigation of the binding site for possible binding partners and the vectors provided by the uracil fragment for synthetic modifications, we introduced a benzylic group by N1-alkylation of the uracil in order to interact with the aromatic side chains of Tyr225 and Phe231 via CH-pi interactions. The top-ranked pose for this elongation shows the unique H-bonding pattern to Met232 as well as the intended CH-pi interactions in a sandwich-type binding orientation.
Consequently, we aimed to bind deeper into the S1'-binding site of the target protein to further improve the affinity of the emerging inhibitor. C5 of the uracil fragment offered an attractive vector to take advantage of the linear S1'-binding site by the introduction of a linear propiolic acid fragment addressing the backbone NH of Thr224 via the carboxylic acid of the emerging inhibitor.
Adding a benzyl amine to the carboxylic acid terminus in order to interact with His201 via pipi-interaction, furnished the final scaffold of our design approach. Again, the top-ranked poses confirmed all the intended interactions between the de novo designed inhibitor scaffold and the target protein.

Structure Activity Relationship (SAR)
A directed compound library was designed, synthesized, and tested in vitro against  in order to verify the docked binding motif of the scaffold 2 (not all data shown). The top ranked poses in the docking experiments of all library compounds showed the same binding motif as the scaffold 2. Though, the inhibitory potency of all compounds containing a substituent in the ortho-position on the benzyl group on the right hand side of the scaffold was eliminated completely at c(inhibitor) = 6.5 µM. These findings correlate consistently regarding the narrowness of the S1'-binding site close to the active site and the clashes that occurred between the docked poses of the library compounds and MMP-13, shown using the example of the three representative library compounds 9a-c. (Table S1, Figure S3).

General Experimental Conditions
Reagents and solvents were purchased from commercial suppliers and used without further  (Table S2); Column temperature ϑ = 40 °C.

Biological Assays Enzo Life Sciences drug discovery kits
The following assay kits were purchased from Enzo Life Sciences