Targeting RAS oncogenesis with SOS1 inhibitors.

RAS proteins play major roles in many human cancers, but programs to develop direct RAS inhibitors so far have only been successful for the oncogenic KRAS mutant G12C. As an alternative approach, inhibitors for the RAS guanine nucleotide exchange factor SOS1 have been investigated by several academic groups and companies, and major progress has been achieved in recent years in the optimization of small molecule activators and inhibitors of SOS1. Here, we review the discovery and development of small molecule modulators of SOS1 and their molecular binding modes and modes of action. As targeting the RAS pathway is expected to result in the development of resistance mechanisms, SOS1 inhibitors will most likely be best applied in vertical combination approaches where two nodes of the RAS signaling pathway are hit simultaneously. We summarize the current understanding of which combination partners may be most beneficial for patients with RAS driven tumors.

Discovery of BAY-985, a Highly Selective TBK1/IKKε Inhibitor.

The serine/threonine kinase TBK1 (TANK-binding kinase 1) and its homologue IKKε are noncanonical members of the inhibitor of the nuclear factor κB (IκB) kinase family. These kinases play important roles in multiple cellular pathways and, in particular, in inflammation. Herein, we describe our investigations on a family of benzimidazoles and the identification of the potent and highly selective TBK1/IKKε inhibitor BAY-985. BAY-985 inhibits the cellular phosphorylation of interferon regulatory factor 3 and displays antiproliferative efficacy in the melanoma cell line SK-MEL-2 but showed only weak antitumor activity in the SK-MEL-2 human melanoma xenograft model.

Structure of wild-type Plk-1 kinase domain in complex with a selective DARPin.

As a key regulator of mitosis, the Ser/Thr protein polo-like kinase-1 (Plk-1) is a well validated drug target in cancer therapy. In order to enable structure-guided drug design, determination of the crystal structure of the kinase domain of Plk-1 was attempted. Using a multi-parallel cloning and expression approach, a set of length variants were identified which could be expressed in large amounts from insect cells and which could be purified to high purity. However, all attempts to crystallize these constructs failed. Crystals were ultimately obtained by generating designed ankyrin-repeat proteins (DARPins) selective for Plk-1 and using them for cocrystallization. Here, the first crystal structure of the kinase domain of wild-type apo Plk-1, in complex with DARPin 3H10, is presented, underlining the power of selective DARPins as crystallization tools. The structure was refined to 2.3 A resolution and shows the active conformation of Plk-1. It broadens the basis for modelling and cocrystallization studies for drug design. The binding epitope of 3H10 is rich in arginine, glutamine and lysine residues, suggesting that the DARPin enabled crystallization by masking a surface patch which is unfavourable for crystal contact formation. Based on the packing observed in the crystal, a truncated DARPin variant was designed which showed improved binding characteristics.

Detecting and overcoming hemihedral twinning during the MIR structure determination of Rna1p.

The structure of Rna1p was originally solved to 2.7 A resolution by MIRAS from crystals with partial hemihedral twinning in space group I4(1) [Hillig et al. (1999), Mol. Cell, 3, 781-791] by finding a low-twinned native crystal (twin fraction alpha=0.06) and after twin correction of all data sets. Rna1p crystals have now been used to examine how far twinning and twin correction affect MIR phasing with a higher resolution but highly twinned native data set. Even high hemihedral twinning [alphanative=0.39, alphaderivative=0.24] would not have hindered heavy-atom site identification of strong derivatives using difference Patterson maps. However, a weaker derivative could have been missed and refinement would have stalled at high R values had twinning not been identified and accounted for. Twin correction improved both site identification, experimental phasing statistics and MIR map quality. Different strategies were tested for refinement against twinned data. Using uncorrected twinned data and TWIN-CNS, Rna1p has now been refined to 2.2 A resolution (final twinned R and Rfree were 0.165 and 0.218, respectively). The increased resolution enabled release of the NCS restraints and allowed new conclusions to be drawn on the flexibility of the two molecules in the asymmetric unit. In the case of Rna1p, twinned crystal growth was possible owing to the presence of a twofold NCS axis almost parallel to the twin operator.