Tumour-associated KRAS mutations are the most prevalent in the three RAS-family isoforms and involve many different amino-acids. Therefore, molecules able to interfere with mutant KRAS protein are ...potentially important for wide-ranging tumour therapy. We describe the engineering of two RAS degraders based on protein macromolecules (macrodrugs) fused to specific E3 ligases. A KRAS-specific DARPin fused to the VHL E3 ligase is compared to a pan-RAS intracellular single domain antibody (iDAb) fused to the UBOX domain of the CHIP E3 ligase. We demonstrate that while the KRAS-specific DARPin degrader induces specific proteolysis of both mutant and wild type KRAS, it only inhibits proliferation of cancer cells expressing mutant KRAS in vitro and in vivo. Pan-RAS protein degradation, however, affects proliferation irrespective of the RAS mutation. These data show that specific KRAS degradation is an important therapeutic strategy to affect tumours expressing any of the range of KRAS mutations.
In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single ...domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.
RNase H2 is a specialized enzyme that degrades RNA in RNA/DNA hybrids and deficiency of this enzyme causes a severe neuroinflammatory disease, Aicardi Goutières syndrome (AGS). However, the molecular ...mechanism underlying AGS is still unclear. Here, we show that RNase H2 is associated with a subset of genes, in a transcription-dependent manner where it interacts with RNA Polymerase II. RNase H2 depletion impairs transcription leading to accumulation of R-loops, structures that comprise RNA/DNA hybrids and a displaced DNA strand, mainly associated with short and intronless genes. Importantly, accumulated R-loops are processed by XPG and XPF endonucleases which leads to DNA damage and activation of the immune response, features associated with AGS. Consequently, we uncover a key role for RNase H2 in the transcription of human genes by maintaining R-loop homeostasis. Our results provide insight into the mechanistic contribution of R-loops to AGS pathogenesis.
The RAS gene family is frequently mutated in human cancers, and the quest for compounds that bind to mutant RAS remains a major goal, as it also does for inhibitors of protein–protein interactions. ...We have refined crystallization conditions for KRAS169
Q61H-yielding crystals suitable for soaking with compounds and exploited this to assess new RAS-binding compounds selected by screening a protein–protein interaction-focused compound library using surface plasmon resonance. Two compounds, referred to as PPIN-1 and PPIN-2, with related structures from 30 initial RAS binders showed binding to a pocket where compounds had been previously developed, including RAS effector protein–protein interaction inhibitors selected using an intracellular antibody fragment (called Abd compounds). Unlike the Abd series of RAS binders, PPIN-1 and PPIN-2 compounds were not competed by the inhibitory anti-RAS intracellular antibody fragment and did not show any RAS-effector inhibition properties. By fusing the common, anchoring part from the two new compounds with the inhibitory substituents of the Abd series, we have created a set of compounds that inhibit RAS-effector interactions with increased potency. These fused compounds add to the growing catalog of RAS protein–protein inhibitors and show that building a chemical series by crossing over two chemical series is a strategy to create RAS-binding small molecules.
Inhibiting the RAS oncogenic protein has largely been through targeting the switch regions that interact with signalling effector proteins. Here, we report designed ankyrin repeat proteins (DARPins) ...macromolecules that specifically inhibit the KRAS isoform by binding to an allosteric site encompassing the region around KRAS-specific residue histidine 95 at the helix α3/loop 7/helix α4 interface. We show that these DARPins specifically inhibit KRAS/effector interactions and the dependent downstream signalling pathways in cancer cells. Binding by the DARPins at that region influences KRAS/effector interactions in different ways, including KRAS nucleotide exchange and inhibiting KRAS dimerization at the plasma membrane. These results highlight the importance of targeting the α3/loop 7/α4 interface, a previously untargeted site in RAS, for specifically inhibiting KRAS function.
Ras mutations are the oncogenic drivers of many human cancers and yet there are still no approved Ras-targeted cancer therapies. Inhibition of Ras nucleotide exchange is a promising new approach but ...better understanding of this mechanism of action is needed. Here we describe an antibody mimetic, DARPin K27, which inhibits nucleotide exchange of Ras. K27 binds preferentially to the inactive Ras GDP form with a K
of 4 nM and structural studies support its selectivity for inactive Ras. Intracellular expression of K27 significantly reduces the amount of active Ras, inhibits downstream signalling, in particular the levels of phosphorylated ERK, and slows the growth in soft agar of HCT116 cells. K27 is a potent, non-covalent inhibitor of nucleotide exchange, showing consistent effects across different isoforms of Ras, including wild-type and oncogenic mutant forms.
Targeting specific protein-protein interactions (PPIs) is an attractive concept for drug development, but hard to implement since intracellular antibodies do not penetrate cells and most ...small-molecule drugs are considered unsuitable for PPI inhibition. A potential solution to these problems is to select intracellular antibody fragments to block PPIs, use these antibody fragments for target validation in disease models and finally derive small molecules overlapping the antibody-binding site. Here, we explore this strategy using an anti-mutant RAS antibody fragment as a competitor in a small-molecule library screen for identifying RAS-binding compounds. The initial hits are optimized by structure-based design, resulting in potent RAS-binding compounds that interact with RAS inside the cells, prevent RAS-effector interactions and inhibit endogenous RAS-dependent signalling. Our results may aid RAS-dependent cancer drug development and demonstrate a general concept for developing small compounds to replace intracellular antibody fragments, enabling rational drug development to target validated PPIs.
The RAS family of proteins is amongst the most highly mutated in human cancers and has so far eluded drug therapy. Currently, much effort is being made to discover mutant RAS inhibitors and in vitro ...screening for RAS-binding drugs must be followed by cell-based assays. Here, we have developed a robust set of bioluminescence resonance energy transfer (BRET)-based RAS biosensors that enable monitoring of RAS-effector interaction inhibition in living cells. These include KRAS, HRAS and NRAS and a variety of different mutations that mirror those found in human cancers with the major RAS effectors such as CRAF, PI3K and RALGDS. We highlighted the utility of these RAS biosensors by showing a RAS-binding compound is a potent pan-RAS-effector interactions inhibitor in cells. The RAS biosensors represent a useful tool to investigate and characterize the potency of anti-RAS inhibitors in cells and more generally any RAS protein-protein interaction (PPI) in cells.
Pancreatic cancer is an aggressive cancer with a dismal prognosis. This is due to the difficulty to detect the disease at an early and curable stage. In addition, only limited treatment options are ...available, and they are confronted by mechanisms of resistance. Monoclonal antibody (mAb) molecules are highly specific biologics that can be directly used as a blocking agent or modified to deliver a drug payload depending on the desired outcome. They are widely used to target extracellular proteins, but they can also be employed to inhibit intracellular proteins, such as oncoproteins. While mAbs are a class of therapeutics that have been successfully employed to treat many cancers, they have shown only limited efficacy in pancreatic cancer as a monotherapy so far. In this review, we will discuss the challenges, opportunities and hopes to use mAbs for pancreatic cancer treatment, diagnostics and imagery.
Abstract Drug-tolerance has emerged as one of the major non-genetic adaptive processes driving resistance to targeted therapy (TT) in non-small cell lung cancer (NSCLC). However, the kinetics and ...sequence of molecular events governing this adaptive response remain poorly understood. Here, we combine real-time monitoring of the cell-cycle dynamics and single-cell RNA sequencing in a broad panel of oncogenic addiction such as EGFR-, ALK-, BRAF- and KRAS-mutant NSCLC, treated with their corresponding TT. We identify a common path of drug adaptation, which invariably involves alveolar type 1 (AT1) differentiation and Rho-associated protein kinase (ROCK)-mediated cytoskeletal remodeling. We also isolate and characterize a rare population of early escapers, which represent the earliest resistance-initiating cells that emerge in the first hours of treatment from the AT1-like population. A phenotypic drug screen identify farnesyltransferase inhibitors (FTI) such as tipifarnib as the most effective drugs in preventing relapse to TT in vitro and in vivo in several models of oncogenic addiction, which is confirmed by genetic depletion of the farnesyltransferase. These findings pave the way for the development of treatments combining TT and FTI to effectively prevent tumor relapse in oncogene-addicted NSCLC patients.
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