Application of Spark to the discovery of novel LpxC inhibitors

Novartis Institutes for Biomedical Research recently published the paper Application of Virtual Screening to the Identification of New LpxC Inhibitor Chemotypes, Oxazolidinone and Isoxazoline. They report using Spark™, Cresset’s scaffold hopping application, to find core replacements for the indazole moiety of compound 6 in Figure 1. Visual selection of the most promising top-ranking Spark results and further optimization studies led to the identification of novel LpxC inhibitors with subnanomolar binding to LpxC and in vivo antibacterial activity against P. aeruginosa and other Gram-negative bacteria.

Scaffold hopping

The bioactive conformation of compound 26, a simplified version of compound 6 originally reported by Actelion, was used as the starter molecule for the Spark scaffold hopping experiment. Aim of the search was to identify appropriate replacements of the indazole core (Figure 1).


Figure 1. Top: LpxC inhibitor 6. Bottom: The bioactive conformation of compound 26 was used as the starter molecule for the scaffold hopping experiment with Spark.

The allowed atom types for the ‘linker 1 atom’ and ‘atom 2’ (see Figure 2) were set respectively to ‘any carbon atom’ and ‘any atom’. It was further specified that all Spark designs must contain at least 1 ring and should not include any reactive functionalities. The Spark experiment was performed on fragment databases derived from ZINC,1 ChEMBL,2 and the VEHICLe3 collection of theoretical ring systems.

The similarity score of the Spark results towards compound 26 was calculated using 50% field and 50% shape similarity. The 100 top ranking clusters were manually reviewed with respect to synthetic feasibility, introduction of hydrophilic groups in the area of the indazole moiety and calculated physicochemical properties.


Figure 2. Allowed atom types for ‘linker 1 atom’ and ‘atom 2’  in the Spark experiment.

The oxazolidinone 13 and isoxazoline 25 scaffolds were shortlisted among several proposals which link ‘linker 1 atom’ and ‘atom 2’ with an hydrophilic linker (Figure 3).

Analogues of 13 and 25, where the methoxy group in para position was replaced by a bromine atom (Figure 3) showed cellular activity with Minimal Inhibitory Concentrations (MIC) values below 4 μg/mL against P. aeruginosa. Accordingly, these series were selected for further investigation.


Figure 3. Oxazolidinone 13a and isoxazoline 25a showed  MIC <4 μg/mL against P. aeruginosa.

SAR optimization

Further investigation and expansion of both oxazolidinone and isoxazoline series led to the identification of compounds with potent in vitro activity against P. aeruginosa and other Gram-negative bacteria. Representative compound 13f (Figure 4) demonstrated excellent efficacy against P. aeruginosa in an in vivo mouse neutropenic thigh infection model.

The crystal structure of 13f complexed with the P. aeruginosa LpxC enzyme (PDB: 6MAE) shows that the hydroxamic acid moiety is bound to the zinc atom in the active site, and involved in interactions with H78, H237, T190, E77, and D241 (Figure 4). The hydrophobic tail (phenyl group) interacts with several hydrophobic side chains, and the cyclopropyl group further extends to the solvent exposed region. The sulfone oxygen atoms interacts with well-defined crystallographic water molecules (not shown in Figure 4), and the methyl group attached to the sulfone functionality is engaged in hydrophobic interactions with F191. The carbonyl oxygen of the oxazolidinone forms a favorable polar interaction with the C2 CH group of H19.

The crystal structure of 13f thus confirms that the oxazolidinone group can favourably orient the sulfone/hydroxamic acid portion of the inhibitor with respect to the hydrophobic phenyl group while maintaining a low-energy conformation, as predicted by Spark.

Figure 4. X-ray crystal structure of 13f complexed with P. aeruginosa LpxC enzyme (PDB: 6MAE)



This paper from NIBR shows that Spark can guide the rational design and discovery of new drug candidates. In this case, a traditional scaffold hopping experiment led to the identification of novel oxazolidinone and isoxazoline LpxC inhibitors with potent antibacterial activity against Gram-negative bacteria.

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  1. Irwin, J. J.; Sterling, T.; Mysinger, M. M.; Bolstad, E. S.; Coleman, R. G. ZINC: A Free Tool to Discover Chemistry for Biology. J. Chem. Inf. Model. 2012, 52 (7), 1757–1768.
  2. Gaulton, A.; Bellis, L. J.; Bento, A. P.; Chambers, J.; Davies, M.; Hersey, A.; Light, Y.; McGlinchey, S.; Michalovich, D.; Al-Lazikani, B.; Overington, J. P. ChEMBL: A Large- Scale Bioactivity Database for Drug Discovery. Nucleic Acids Res. 2012, 40 (D1), D1100–D1107.
  3. Pitt, W. R.; Parry, D. M.; Perry, B. G.; Groom, C. R. Heteroaromatic Rings of the Future. J. Med. Chem. 2009, 52 (9), 2952–2963

Application of Spark in the discovery of potent SOS1 inhibitors that block RAS activation via disruption of the RAS–SOS1 interaction

Bayer AG recently published an interesting paper on the discovery of potent and selective SOS1 inhibitors that block RAS activation via disruption of the RAS–SOS1 interaction.

They report using Spark™, Cresset’s bioisostere replacement tool, to rationally design linkers between active hit compounds from fragment screen and HTS. Herein, we review this interesting application of Spark to a ligand joining experiment that led to the discovery of BAY-293, a potent and selective inhibitor of KRAS-SOS1 interaction.

Fragment screen

Bayer decided on a two-pronged approach, running a HTS campaign and a fragment screen in parallel. The fragment screen, with a library of 3,000 fragments, led to the identification of fragments which bind to and can induce a conformational change at the KRAS-SOS1 protein-protein interaction site. By triggering a rotation of the Phe890 side chain, they open a new subpocket adjacent to the main binding pocket.

F1 was chosen as the starting point for further optimization. The crystal structure of F1 in complex with SOS1 (PDB: 6EPM, Figure 1) shows that the phenyl moiety makes a π–π interaction with Phe890 (Phe-out). The aminomethyl moiety forms hydrogen bonds to Asp887 and the backbone carbonyl of Tyr884 and makes an additional cation–π interaction with the Tyr884 side chain.

Figure 1. X-ray crystal structure of F1 complexed with KRASG12C–SOS1cat (PDB: 6EPM).

HTS and initial optimization

HTS of a Bayer library of 3 million compounds led to the identification of compound 1 (IC50 320 nM, Figure 2).

Figure 2. Compound 1 (HTS hit: IC50 320 nM).

Replacement of the naphthyl moiety in compound 1 with a pyrazolylphenyl group resulted in compound 17 (Figure 3), showing a good potency on SOS1 (IC50 140 nM) and improved aqueous solubility.

In terms of interactions with SOS1 (PDB: 5OVF, Figure 3), the quinazoline scaffold of compound 17 is sandwiched between His905 and Tyr884 (π–π stacking). The pyrazolylphenyl moiety occupies a hydrophobic pocket composed of Leu901 and Phe890 (Phe-in) and makes a T-stacking interaction with the side chain of Tyr884. The pyrazole moiety makes a water-bridged H-bond to Glu902. The central aniline NH makes a H-bond with the side chain of Asn879.

Figure 3: X-ray crystal structure of compound 17 in the SOS1SB active site (PDB: 5OVF).

Linking of the fragment and HTS hits with Spark

As the fragment screen identified a new subpocket that was not yet addressed by the HTS hits, scientists at Bayer attempted a ligand joining approach using Spark to try and further improve potency by combining both ligand series. A superimposition of the crystal structures of compound 17 (PDB: 5OVF) and F1 (PDB: 6EPM), shown in Figure 4 – A, suggests that this approach is feasible.

After cutting out the overlapping aryl groups of compounds 17 and F1, the authors performed ligand joining experiments with Spark (Figure 4 – B, C) to identify appropriate linkers that could correctly orientate both the tetrahydrocyclopenta[c]pyrazole moiety of F1 ­­­­and the aminoquinazoline group of 17 within the active site of SOS1. Among the top-scoring linkers suggested by Spark, the thiophene linker was chosen, synthesized, and found to be active.

Figure 4: Ligand-joining approach of fragment and HTS hits. A) Superimposition of the crystal structures of F1 bound to KRASG12C_SB–SOS1cat (SOS1 in gray, F1 and Phe890 with carbon atoms in magenta) and 17 bound to SOS1SB (17 and Phe890 shown; carbon atoms in green). B) Schematic depiction of the merging approach. C):  Representation of F1 and 17 with respective fields from Cresset.

Further optimization and discovery of BAY-293

Optimization of the hybrid hits containing the thiophene linker identified by Spark by introducing polar moieties (such as OH, NH2) that mimic the hydrogen bond interactions of the amino side chain of F1, and trigger the Phe-out conformation, led to the discovery of the optimized compound 23 (BAY-293, IC50 21nM). As shown in Figure 5, the side chain amino group of compound 23 interacts with SOS1 by making H-bond interactions with Asp887 and Tyr884 backbone carbonyl, as well as a cation-π interaction with the Tyr884 side chain.

Figure 5. X-ray crystal structure of compound 23 in the SOS1 active site (PDB: 5OVI).

Further screening and antiproliferative data proved that compound 23 (BAY-293) is a potent and selective inhibitor of KRAS-SOS1 interaction and indicates that inhibition of GEFs may represent a viable approach for targeting RAS-driven tumors.


This paper by Bayer shows the utility of the Spark approach. As well as being a superb bioisostere finder, Spark’s advanced capabilities include easy-to-use methods for water replacement, macrocyclization, fragment growing and fragment linking. In this case Spark was vital in suggesting how to join two disparate molecules occupying different parts of a protein binding site, transferring SAR from one series to another.

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