Non-Covalent Pan-KRAS Inhibitors

Mutations in KRas, a member of the Ras superfamily of small GTPases, are associated with approximately 30% of human tumors, making KRas mutants long-time targets for cancer treatment. Most drugs bind non-covalently to their targets. Articles published in the 1980s and 1990s, however reported affinities for GTP and KRas in the range of 10 pM and led scientists to conclude that the KRas GTP binding site could not be targeted by non-covalent inhibitors – the chemical nature of most drug/target interactions. It was deemed undruggable. 

Accordingly, KRas drug development efforts by pharmaceutical and biotechnology companies focused on alternative mechanisms for inhibiting mutant KRas signaling. Over time, opinions have shifted as to whether these compounds should specifically target just the different KRas mutants or not. Several covalently bound compounds targeting KRas G12C mutations (representing a meaningful but still relatively small number of patients) had impressive initial clinical effects, were approved by the FDA for sale, but, disappointingly, have proven in post-approval clinical use to have limited durations of effect. 

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Reasoning analogously based on his experience developing small molecule inhibitors of ATP binding to receptor tyrosine kinases, Dr. Hadari founded SHY with the expectations that:

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• previously reported affinity measurements for GTP/KRas binding were erroneous; 

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• small molecules could therefore be synthesized that compete non-covalently for GTP binding to KRas; 

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• such small molecules would be more therapeutically effective if they were not specific to just certain KRas mutants, but rather preferentially to KRas and also to other members of the protein superfamily of which KRas is one member; and, finally, 

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• notwithstanding the importance of KRas signaling to sustaining cellular functions, such non-covalently bound pan KRas inhibitors would not necessarily be toxic at therapeutically relevant doses.

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SHY has validated all these hypotheses and succeeded where others have not tried by utilizing state-of-the-art methods and combining proprietary high throughput screens with computationally tailored libraries and some inspired SAR.  

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• SHY conducted a series of measurements of GTP/KRas binding using more contemporary methods than the articles published in the 1980s. In 2020 we posted affinity measurements (Kd’s) for GTP/KRas and KRas mutant binding in the range of 250 nM on bioRxiv, Cold Spring Harbor Laboratory’s preprint server for biology. This Kd is approximately 10,000 times lower than the estimates published in the 1980s and 1990s. 

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• SHY has synthesized over 1,000 small molecules that compete non-covalently and differentiably for GTP for binding to KRas and other members of the KRas superfamily, and differentiably modulate KRas signaling. SHY employs a proprietary fluorescence-based cell-free assay to confirm competitive GTP binding. The inhibition of KRas signaling is further assessed in tumor-derived cell lines expressing both wild-type and mutant KRas.
   

• Select molecules from this portfolio are undergoing further in vitro, pharmacokinetic, and in vivo xenograft studies to evaluate their potential as clinical candidates. To date, as anticipated, in animal studies they have shown anti-tumor efficacy but not systemic toxicity.

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A newly published study in Molecular Cancer Therapeutics, with research led by SHY scientists, demonstrates SHY’s differentiated approach to RAS inhibition, providing the first evidence that the GTP-binding site of RAS proteins can be competitively targeted by small molecules in a non-covalent manner—a mechanism not employed by current clinical agents.

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Additional supplementary data: Identification of Small-Molecule Inhibitors that Block the GTP-Binding Pocket of K-Ras and Other Members of the Ras Superfamily of Small GTPases