RAS is the most commonly mutated oncogene in human cancers and RAS-driven tumors are amongst the most difficult to treat. Historically, discovery of therapeutics targeting this protein has proven challenging due to a lack of deep hydrophobic pockets to which a small molecule might bind. The single such pocket available is normally occupied by GDP or GTP which have millimolar cellular concentrations and picomolar affinities for KRAS and hence is challenging to target. The recent FDA approval of sotorasib, a covalent modifier of the KRAS^G12C mutant protein, has clinically validated KRAS as an oncology target. However, traditional challenges remain for targeting the more common KRAS mutations such as G12D and G12V. As an alternative approach, we investigated the superior binding capacity of macrocyclic peptides to identify KRAS inhibitory molecules. We focused on the recently reported disulfide-cyclized arginine-rich peptide KRpep-2d, discovered through phage display and previously independently confirmed by us as a bona fide KRAS binder. To mitigate intracellular disulfide reduction and loss of binding, we identified an alternate cyclization motif by inverting the configuration of Cys5 and linking it to Cys15 through a thioacetal bridge. The corresponding peptide bound KRAS through cis isomerization of the peptide bond between D-Cys5 and Pro6 as observed through x-ray crystallography. Prototypic molecules displayed measurable cellular inhibition of RAS signaling without membrane lysis and counter-screen off-target activity. An analogue containing azido-lysine confirmed the cell penetrant nature of this molecule using our recently reported NanoClick assay. To improve cellular activity, we sought to improve proteolytic stability. Structure-activity relationship studies identified key scaffold residues critical for binding and revealed that replacement of N- and C-terminal arginine residues with D-arginines and introduction of an α-methyl moiety at Ser10 resulted in a molecule with improved proteolytic stability as indicated by its persistence in whole cell homogenate. The resulting peptide MP-3995 had improved and sustained cell-based efficacy. On-target activity was validated through confirmation of target engagement, lack of signaling in irrelevant pathways, and use of non-binding control peptides. Mechanism of action studies suggested that peptide binding to both GDP- and GTP-states of KRAS may contribute to cellular activity. Although validated as bona fide and on-target inhibitors of cell based KRAS signaling, this series is unlikely to advance to the clinic in its current form due to its arginine-dependent cell entry mechanism. Indeed, we showed a strong correlation between net positive charge and histamine release in an ex vivo assay making this series challenging to study in vivo. Nonetheless, these molecules provide valuable templates for further medicinal chemistry efforts aimed at leveraging this unique inhibitory binding site on KRAS.

中文翻译：

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发现具有靶向抑制 KRAS 信号的细胞活性大环肽

RAS 是人类癌症中最常见的突变癌基因，RAS 驱动的肿瘤是最难治疗的肿瘤之一。从历史上看，由于缺乏小分子可能结合的深疏水口袋，因此发现靶向这种蛋白质的疗法具有挑战性。可用的单个此类口袋通常被 GDP 或 GTP 占据，它们对 KRAS 具有毫摩尔细胞浓度和皮摩尔亲和力，因此难以定位。最近 FDA 批准了索托拉西，一种 KRAS ^G12C的共价修饰剂突变蛋白，临床验证 KRAS 作为肿瘤学靶点。然而，针对更常见的 KRAS 突变，如 G12D 和 G12V，传统的挑战仍然存在。作为替代方法，我们研究了大环肽的优异结合能力，以识别 KRAS 抑制分子。我们专注于最近报道的二硫键环化的富含精氨酸的肽KRpep-2d，它是通过噬菌体展示发现的，并且之前被我们独立确认为真正的KRAS 粘合剂。为了减轻细胞内二硫键的还原和结合的丧失，我们通过反转 Cys5 的构型并通过硫缩醛桥将其连接到 Cys15 来确定替代的环化基序。如通过 X 射线晶体学观察到的，相应的肽通过 D-Cys5 和 Pro6 之间肽键的顺式异构化结合 KRAS。原型分子显示出可测量的 RAS 信号的细胞抑制作用，而没有膜裂解和反筛选脱靶活性。使用我们最近报道的 NanoClick 测定，含有叠氮基赖氨酸的类似物证实了该分子的细胞渗透性。为了提高细胞活性，我们寻求提高蛋白水解稳定性。构效关系研究确定了对结合至关重要的关键支架残基，并揭示用 D-精氨酸替换 N 端和 C 端精氨酸残基并在 Ser10 处引入 α-甲基部分导致分子具有改善的蛋白水解稳定性，如下所示它在全细胞匀浆中的持久性。所得肽MP-3995具有改进和持续的基于细胞的功效。通过确认目标参与、不相关途径中缺乏信号传导以及使用非结合控制肽来验证在靶活性。作用机制研究表明，与 KRAS 的 GDP 和 GTP 状态结合的肽可能有助于细胞活性。尽管被验证为基于细胞的 KRAS 信号传导的真正和靶向抑制剂，但由于其精氨酸依赖性细胞进入机制，该系列不太可能以其目前的形式进入临床。事实上，我们在离体试验中显示净正电荷与组胺释放之间存在很强的相关性，这使得该系列在体内研究具有挑战性. 尽管如此，这些分子为进一步的药物化学工作提供了有价值的模板，旨在利用 KRAS 上这种独特的抑制结合位点。