Covalent Guanosine Mimetic Inhibitors of G12C KRAS

Xiong, Yuning; Lu, Jia; Hunter, John C.; Li, Lianbo; Scott, David A.; Choi, Hwan Geun; Lim, Sang Min; Manandhar, Anuj; Gondi, Sudershan; Sim, Taebo; Westover, Kenneth D.; Gray, Nathanael S.

doi:10.1021/acsmedchemlett.6b00373

Cited by 59 publications

(68 citation statements)

References 40 publications

(61 reference statements)

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“…The association and dissociation rate constants of SML-8-73-1 (k on = 4.1 × 10 6 M −1 s −1 , k off = 0.9 s −1 ) were similar to those seen for KRas WT , and the fitted rate constant for the covalent reaction was 0.1 s −1 . After initial submission of the current contribution, a publication appeared reporting a more thorough characterization of SML-8-73-1 than in the earlier publications 12 . In this publication, a rate constant of 0.014 s −1 was reported for the covalent reaction, with a K d value of 0.009 µM for the reversible binding step.…”

Section: Resultsmentioning

confidence: 99%

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Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity

Müller

Jeganathan

Heidrich

et al. 2017

Sci Rep

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Simple reversible competitive inhibition of nucleotide binding of GTP to Ras family GTPases has long been recognized as an unlikely approach to manipulating the activity of such proteins for experimental or therapeutic purposes. This is due to the high affinity of GTP to GTPases coupled with high cellular GTP concentrations, but also to problems of specificity for the highly conserved binding sites in GTPases. A recent approach suggested that these problems might be overcome by using GDP derivatives that can undergo a covalent reaction with disease specific mutants, in particular addressing inhibition of KRasG12C using GDP equipped with an electrophilic group at the β-phosphate. We show here that a major drawback to this approach is a loss of reversible affinity of such β-modified derivatives for Ras of at least 104 compared to GTP and GDP. With the help of a thorough kinetic characterization, we show that this leads to covalent reaction times that are too slow to make the compounds attractive for intracellular use, but that generation of a hypothetical reactive GDP derivative that retains the high reversible affinity of GDP/GTP to Ras might be a viable alternative.

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Section: Resultsmentioning

confidence: 99%

“…At the same time as the report of the Shokat group, a publication applying this approach utilized GDP molecules modified on the ß-phosphate with a reactive group 11, 12 . These derivatives were shown to be able to bind to and interact covalently with the cysteine group of the G12C mutant of KRas.…”

Section: Introductionmentioning

confidence: 99%

Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity

Müller

Jeganathan

Heidrich

et al. 2017

Sci Rep

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“…Drugs that target mutant alleles such as G12C, such as that described above, and another by Xiong et al (2016) obviously offer the best opportunity for safety and efficacy. However, strategies for targeting mutant proteins such as G12D and G12V have not yet been forthcoming.…”

Section: Therapeutic Approachesmentioning

confidence: 99%

RAS Proteins and Their Regulators in Human Disease

Simanshu

Nissley

McCormick

2017

Cell

1,396

1,350

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RAS proteins are binary switches, cycling between ON and OFF states during signal transduction. These switches are normally tightly controlled, but in RAS-related diseases, such as cancer, RASopathies, and many psychiatric disorders, mutations in the RAS genes or their regulators render RAS proteins persistently active. The structural basis of the switch and many of the pathways that RAS controls are well known, but the precise mechanisms by which RAS proteins function are less clear. All RAS biology occurs in membranes: a precise understanding of RAS’ interaction with membranes is essential to understand RAS action and to intervene in RAS-driven diseases.

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“…While this paints an optimistic picture of the prospects of allosteric KRAS inhibition, to the best of our knowledge none of these compounds has made it to clinical trial. Recent efforts toward developing covalent GDP analogues (30) or other small-molecule ligands (31) targeting G12C mutant KRAS may have a better chance of eventually treating specific tumor types (18). However, their application is likely limited to a few cancer cases such as small cell lung cancer (10).…”

Section: Introductionmentioning

confidence: 99%

Discovery of allosteric non-covalent KRAS inhibitors that bind with sub-micromolar affinity and disrupt effector binding

McCarthy

Pagba

Prakash

et al. 2018

Preprint

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Approximately 15% of all human tumors harbor mutant KRAS, a membrane-associated small GTPase and a notorious oncogene. Somatic mutations that render KRAS constitutively active lead to uncontrolled cell growth, survival, proliferation, and eventually cancer. KRAS is thus a critical anticancer drug target. However, despite aggressive efforts in recent years, there is no drug on the market that directly targets KRAS. In the current work, we combined molecular simulation and high-throughput virtual screening with a battery of cell-based and biophysical assays to discover a novel, pyrazolopyrimidine-based allosteric KRAS inhibitor that exhibits promising biochemical properties. The compound selectively binds to active KRAS with submicromolar affinity, slightly modulates exchange factor activity, disrupts effector Raf binding, significantly reduces signal transduction through mutant KRAS and inhibits cancer cell growth.Moreover, by studying two of its analogues, we identified key chemical features of the compound that are critical for affinity, effect on effector binding and mode of action. We propose a set of specific interactions with key residues at the switch regions of KRAS as critical for abrogating effector binding and reducing the rate of nucleotide exchange. Together, these findings not only demonstrate the viability of direct KRAS inhibition and offer guidance for future optimization efforts, but also show that pyrazolopyrimidine-based compounds may represent a first-in-class lead toward a clinically relevant targeting of KRAS by allosteric non-covalent inhibitors. Cell signalingThe inhibitory potential of compounds was tested in monoclonal Baby Hamster Kidney (BHK) cell lines stably expressing monomeric green fluorescence protein (mGFP)tagged KRAS G12D , KRAS G12V and HRAS G12V . Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Hyclone) supplemented with 10% v/v bovine calf serum and incubated with compound or vehicle (DMSO) for 3 h without serum. Cells were then harvested in lysis buffer (50 mM Tris (pH 7.5), 75 mM NaCl, 25 mM NaF, 5 mM MgCl 2 , 5 mM EGTA, 1 mM dithiothreitol, 100 µM NaVO 4 , 1% Nonidet P40 plus protease inhibitors) and subjected to Western analysis controlling protein loading by BCA (bicinchoninic acid) assay. Lysates were resolved with Bio-Rad polyacrylamide TGX 10% gel, transferred to polyvinylidene fluoride (PVDF) membrane and immunoblotted using pan-AKT (2920S), GFP (2956S), p-AKT S473 (4060L), p-cRaf S338 (9427S), p-ERK T202/Y204 (4370L), ERK1/2 (4695S) or β-actin antibodies (Cell Signaling Technology). IC 50 values were calculated with Prism 4-parameter fit. Pull-downWe pulled down GFP-RAS with GST-tagged RAS binding domain (RBD) of cRaf A85K (hereafter GST-Raf RBD ) to monitor RAS-Raf interaction. To prepare GST-Raf RBD bound to agarose beads, bacteria (BL21) transfected with a previously cloned GEX plasmid were grown in selection media to OD levels of 0.5-1.0 before protein expression was initiated with IPTG (1:1000). After 4 h, the sample was centrifuged at 6000 rpm ...

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Covalent Guanosine Mimetic Inhibitors of G12C KRAS

Cited by 59 publications

References 40 publications

Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity

Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity

RAS Proteins and Their Regulators in Human Disease

Discovery of allosteric non-covalent KRAS inhibitors that bind with sub-micromolar affinity and disrupt effector binding

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