Mechanism of activation and the rewired network: New drug design concepts.

Precision oncology benefits from effective early phase drug discovery decisions. Recently, drugging inactive protein conformations has shown impressive successes, raising the cardinal questions of which targets can profit and what are the principles of the active/inactive protein pharmacology. Cancer driver mutations have been established to mimic the protein activation mechanism. We suggest that the decision whether to target an inactive (or active) conformation should largely rest on the protein mechanism of activation. We next discuss the recent identification of double (multiple) same-allele driver mutations and their impact on cell proliferation and suggest that like single driver mutations, double drivers also mimic the mechanism of activation. We further suggest that the structural perturbations of double (multiple) in cis mutations may reveal new surfaces/pockets for drug design. Finally, we underscore the preeminent role of the cellular network which is deregulated in cancer. Our structure-based review and outlook updates the traditional Mechanism of Action, informs decisions, and calls attention to the intrinsic activation mechanism of the target protein and the rewired tumor-specific network, ushering innovative considerations in precision medicine.

131I-C19 Iodide Radioisotope and Synthetic I-C19 Compounds as K-Ras4B-PDE6δ Inhibitors: A Novel Approach against Colorectal Cancer-Biological Characterization, Biokinetics and Dosimetry.

In 40-50% of colorectal cancer (CRC) cases, K-Ras gene mutations occur, which induce the expression of the K-Ras4B oncogenic isoform. K-Ras4B is transported by phosphodiesterase-6δ (PDE6δ) to the plasma membrane, where the K-Ras4B-PDE6δ complex dissociates and K-Ras4B, coupled to the plasma membrane, activates signaling pathways that favor cancer aggressiveness. Thus, the inhibition of the K-Ras4B-PDE6δ dissociation using specific small molecules could be a new strategy for the treatment of patients with CRC. This research aimed to perform a preclinical proof-of-concept and a therapeutic potential evaluation of the synthetic I-C19 and 131I-C19 compounds as inhibitors of the K-Ras4B-PDE6δ dissociation. Molecular docking and molecular dynamics simulations were performed to estimate the binding affinity and the anchorage sites of I-C19 in K-Ras4B-PDE6δ. K-Ras4B signaling pathways were assessed in HCT116, LoVo and SW620 colorectal cancer cells after I-C19 treatment. Two murine colorectal cancer models were used to evaluate the I-C19 therapeutic effect. The in vivo biokinetic profiles of I-C19 and 131I-C19 and the tumor radiation dose were also estimated. The K-Ras4B-PDE6δ stabilizer, 131I-C19, was highly selective and demonstrated a cytotoxic effect ten times greater than unlabeled I-C19. I-C19 prevented K-Ras4B activation and decreased its dependent signaling pathways. The in vivo administration of I-C19 (30 mg/kg) greatly reduced tumor growth in colorectal cancer. The biokinetic profile showed renal and hepatobiliary elimination, and the highest radiation absorbed dose was delivered to the tumor (52 Gy/74 MBq). The data support the idea that 131I-C19 is a novel K-Ras4B/PDE6δ stabilizer with two functionalities: as a K-Ras4B signaling inhibitor and as a compound with radiotherapeutic activity against colorectal tumors.

Oncogenic KRas mobility in the membrane and signaling response.

Ras signaling initiates at the plasma membrane. Thus, Ras behavior at the membrane and how it relates to its interactions with Raf and PI3Kα, are of immense interest. Here we review factors influencing Ras lateral diffusion. We then ask whether oncogenic Ras diffusion speed in the membrane is important for signaling response times and whether it affects ubiquitously all pathways. We suggest that if Ras expression is sufficiently high to dimerize (or form nanoclusters), signaling response of those pathways where dimers (or nanoclusters) are involved corresponds to the speed with which Ras molecules travel in the membrane. On average, the faster the rate at which Ras travels to dimerize, the shorter the time to MAPK signaling; but not PI3Kα. However, we argue that KRas speed may not play an important functional role because changes in mobility at this scale are unlikely to be significant. In line with this, despite the anchors' variability, lateral diffusion speeds of KRas and HRas are similar, as is that of Lck kinase; however, even though with similar anchor, Cdc42 mobility presents a different pattern, commensurate with its role in the positioning of the apical domain, suggesting that mobility evolved for function.

Is Nanoclustering essential for all oncogenic KRas pathways? Can it explain why wild-type KRas can inhibit its oncogenic variant?

Membrane-anchored oncogenic KRas can dimerize, form nanoclusters, and signal through the MAPK (Raf/MEK/ERK) and PI3Kα/Akt/mTOR. Both pathways are needed in KRAS-driven proliferation. Here we ask: Is oncogenic KRas nanoclustering (or dimerization) essential for all KRas signaling pathways? Raf kinase domain dimerization, thus MAPK activation, requires KRas nanoclusters. By contrast, the PI3Kα heterodimer acts as a monomeric unit; thus, does PI3Kα activation and PI3Kα/Akt/mTOR signaling require nanoclustering? Further, calmodulin binds only to oncogenic KRas4B. Here we ask: Does calmodulin downregulate KRas4B cancer development as suggested early on, or promote it? We also ask: Why is oncogenic KRas4B the most abundant isoform? Does wild-type Ras indeed inhibit its oncogenic variants as data appeared to suggest? And related to the last question, why is wild-type KRas a more potent inhibitor of its oncogenic form than wild-type NRas of its oncogenic form? Resolving these cardinal questions, and others, such as how exactly does RASSF5 (NORE1A) act as tumor suppressor, and why Ras isoforms tend to occur in distinct cancer types are crucial for effective pharmacology. In this review, we take a nanoclustering/dimerization-centric outlook and show that many questions can be explained by simply considering Ras nanoclustering.

Dissociation of the Signaling Protein K-Ras4B from Lipid Membranes Induced by a Molecular Tweezer.

Oncogenic Ras mutations occur in more than 30 % of human cancers. K-Ras4B is the most frequently mutated isoform of Ras proteins. Development of effective K-Ras4B inhibitors has been challenging, hence new approaches to inhibit this oncogenic protein are urgently required. The polybasic domain of K-Ras4B with its stretch of lysine residues is essential for its plasma membrane targeting and localization. Employing CD and fluorescence spectroscopy, confocal fluorescence, and atomic force microscopy we show that the molecular tweezer CLR01 is able to efficiently bind to the lysine stretch in the polybasic domain of K-Ras4B, resulting in dissociation of the K-Ras4B protein from the lipid membrane and disintegration of K-Ras4B nanoclusters in the lipid bilayer. These results suggest that targeting of the polybasic domain of K-Ras4B by properly designed tweezers might represent an effective strategy for inactivation of K-Ras4B signaling.

The Hypervariable Region of K-Ras4B Governs Molecular Recognition and Function.

The flexible C-terminal hypervariable region distinguishes K-Ras4B, an important proto-oncogenic GTPase, from other Ras GTPases. This unique lysine-rich portion of the protein harbors sites for post-translational modification, including cysteine prenylation, carboxymethylation, phosphorylation, and likely many others. The functions of the hypervariable region are diverse, ranging from anchoring K-Ras4B at the plasma membrane to sampling potentially auto-inhibitory binding sites in its GTPase domain and participating in isoform-specific protein-protein interactions and signaling. Despite much research, there are still many questions about the hypervariable region of K-Ras4B. For example, mechanistic details of its interaction with plasma membrane lipids and with the GTPase domain require further clarification. The roles of the hypervariable region in K-Ras4B-specific protein-protein interactions and signaling are incompletely defined. It is also unclear why post-translational modifications frequently found in protein polylysine domains, such as acetylation, glycation, and carbamoylation, have not been observed in K-Ras4B. Expanding knowledge of the hypervariable region will likely drive the development of novel highly-efficient and selective inhibitors of K-Ras4B that are urgently needed by cancer patients.

Allosteric Activation of GDP-Bound Ras Isoforms by Bisphenol Derivative Plasticisers.

The protein family of small GTPases controls cellular processes by acting as a binary switch between an active and an inactive state. The most prominent family members are H-Ras, N-Ras, and K-Ras isoforms, which are highly related and frequently mutated in cancer. Bisphenols are widespread in modern life because of their industrial application as plasticisers. Bisphenol A (BPA) is the best-known member and has gained significant scientific as well as public attention as an endocrine disrupting chemical, a fact that eventually led to its replacement. However, compounds used to replace BPA still contain the molecular scaffold of bisphenols. BPA, BPAF, BPB, BPE, BPF, and an amine-substituted BPAF-derivate all interact with all GDP-bound Ras-Isoforms through binding to a common site on these proteins. NMR-, SOScat-, and GDI- assay-based data revealed a new bisphenol-induced, allosterically activated GDP-bound Ras conformation that define these plasticisers as Ras allosteric agonists.

Computational Insights into the Interactions between Calmodulin and the c/nSH2 Domains of p85α Regulatory Subunit of PI3Kα: Implication for PI3Kα Activation by Calmodulin.

Calmodulin (CaM) and phosphatidylinositide-3 kinase (PI3Kα) are well known for their multiple roles in a series of intracellular signaling pathways and in the progression of several human cancers. Crosstalk between CaM and PI3Kα has been an area of intensive research. Recent experiments have shown that in adenocarcinoma, K-Ras4B is involved in the CaM-PI3Kα crosstalk. Based on experimental results, we have recently put forward a hypothesis that the coordination of CaM and PI3Kα with K-Ras4B forms a CaM-PI3Kα-K-Ras4B ternary complex, which leads to the formation of pancreatic ductal adenocarcinoma. However, the mechanism for the CaM-PI3Kα crosstalk is unresolved. Based on molecular modeling and molecular dynamics simulations, here we explored the potential interactions between CaM and the c/nSH2 domains of p85α subunit of PI3Kα. We demonstrated that CaM can interact with the c/nSH2 domains and the interaction details were unraveled. Moreover, the possible modes for the CaM-cSH2 and CaM-nSH2 interactions were uncovered and we used them to construct a complete CaM-PI3Kα complex model. The structural model of CaM-PI3Kα interaction not only offers a support for our previous ternary complex hypothesis, but also is useful for drug design targeted at CaM-PI3Kα protein-protein interactions.

Membrane-associated Ras dimers are isoform-specific: K-Ras dimers differ from H-Ras dimers.

Are the dimer structures of active Ras isoforms similar? This question is significant since Ras can activate its effectors as a monomer; however, as a dimer, it promotes Raf's activation and MAPK (mitogen-activated protein kinase) cell signalling. In the present study, we model possible catalytic domain dimer interfaces of membrane-anchored GTP-bound K-Ras4B and H-Ras, and compare their conformations. The active helical dimers formed by the allosteric lobe are isoform-specific: K-Ras4B-GTP favours the α3 and α4 interface; H-Ras-GTP favours α4 and α5. Both isoforms also populate a stable ß-sheet dimer interface formed by the effector lobe; a less stable ß-sandwich interface is sustained by salt bridges of the ß-sheet side chains. Raf's high-affinity ß-sheet interaction is promoted by the active helical interface. Collectively, Ras isoforms' dimer conformations are not uniform; instead, the isoform-specific dimers reflect the favoured interactions of the HVRs (hypervariable regions) with cell membrane microdomains, biasing the effector-binding site orientations, thus isoform binding selectivity.

Principles of K-Ras effector organization and the role of oncogenic K-Ras in cancer initiation through G1 cell cycle deregulation.

Illustrated here is the critical role of oncogenic KRAS in the initiation of cancer through deregulation of the G1 cell cycle, and elements and scenarios taking place under physiological conditions and in KRAS-driven cancer. Raf, PI3K and RalGDS are major K-Ras effectors. They bind at the same Ras site. What decides the cell selection among them? This temporal and spatial decision is critical since in some cellular context the outcome of their signaling pathways may oppose each other. Key among them is the concentration of calcium/calmodulin, negative feedback loops, where a downstream member of the pathway inhibits its upstream activator and cross-inhibition, where inhibition entails blocking another pathway. These three elements, in addition to spatial restrictions by K-Ras-membrane interactions, are not independent; they integrate to provide blueprints for cell decisions. Importantly, elucidation of signaling requires not only K-Ras binary interactions; but the structures and dynamics of its multiprotein complexes.

Antineoplastic effect of compounds C14 and P8 on TNBC and radioresistant TNBC cells by stabilizing the K-Ras4BG13D/PDE6δ complex.

Introduction: Breast cancer (BC) is the leading cause of cancer-related deaths among women, with triple-negative breast cancer (TNBC) representing one of the most aggressive and treatment-resistant subtypes. In this study, we aimed to evaluate the antitumor potential of C14 and P8 molecules in both TNBC and radioresistant TNBC cells. These compounds were chosen for their ability to stabilize the complex formed by the overactivated form of K-Ras4BG13D and its membrane transporter (PDE6δ). Methods: The antitumor potential of C14 and P8 was assessed using TNBC cell lines, MDA-MB-231, and the radioresistant derivative MDA-MB-231RR, both carrying the K-Ras4B> G13D mutation. We investigated the compounds' effects on K-Ras signaling pathways, cell viability, and tumor growth in vivo. Results: Western blotting analysis determined the negative impact of C14 and P8 on the activation of mutant K-Ras signaling pathways in MDA-MB-231 and MDA-MB-231RR cells. Proliferation assays demonstrated their efficacy as cytotoxic agents against K-RasG13D mutant cancer cells and in inducing apoptosis. Clonogenic assays proven their ability to inhibit TNBC and radioresistant TNBC cell clonogenicity. In In vivo studies, C14 and P8 inhibited tumor growth and reduced proliferation, angiogenesis, and cell cycle progression markers. Discussion: These findings suggest that C14 and P8 could serve as promising adjuvant treatments for TNBC, particularly for non-responders to standard therapies. By targeting overactivated K-Ras and its membrane transporter, these compounds offer potential therapeutic benefits against TNBC, including its radioresistant form. Further research and clinical trials are warranted to validate their efficacy and safety as novel TNBC treatments.

Autopromotion of K-Ras4B Feedback Activation Through an SOS-Mediated Long-Range Allosteric Effect.

The Ras-specific guanine nucleotide exchange factors Son of Sevenless (SOS) regulates Ras activation by converting inactive GDP-bound to active GTP-bound states. The catalytic activity of Ras is further allosterically regulated by GTP-Ras bound to a distal site through a positive feedback loop. To address the mechanism underlying the long-range allosteric activation of the catalytic K-Ras4B by an additional allosteric GTP-Ras through SOS, we employed molecular dynamics simulation of the K-Ras4BG13D•SOScat complex with and without an allosteric GTP-bound K-Ras4BG13D. We found that the binding of an allosteric GTP-K-Ras4BG13D enhanced the affinity between the catalytic K-Ras4BG13D and SOScat, forming a more stable conformational state. The peeling away of the switch I from the nucleotide binding site facilitated the dissociation of GDP, thereby contributing to the increased nucleotide exchange rate. The community networks further showed stronger edge connection upon allosteric GTP-K-Ras4BG13D binding, which represented an increased interaction between catalytic K-Ras4BG13D and SOScat. Moreover, GTP-K-Ras4BG13D binding transmitted allosteric signaling pathways though the Cdc25 domain of SOS that enhanced the allosteric regulatory from the K-Ras4BG13D allosteric site to the catalytic site. This study may provide an in-depth mechanism for abnormal activation and allosteric regulation of K-Ras4BG13D.

Synthesis, Biological Evaluation, and Binding Mode of a New Class of Oncogenic K-Ras4b Inhibitors.

Ras proteins are implicated in some of the most common life-threatening cancers. Despite intense research during the past three decades, progress towards small-molecule inhibitors of mutant Ras proteins still has been limited. Only recently has significant progress been made, in particular with ligands for binding sites located in the switch II and between the switch I and switch II region of K-Ras4B. However, the structural diversity of inhibitors identified for those sites to date is narrow. Herein, we show that hydrazones and oxime ethers of specific bis(het)aryl ketones represent structurally variable chemotypes for new GDP/GTP-exchange inhibitors with significant cellular activity.

K-RAS4A: Lead or Supporting Role in Cancer Biology?

The RAS oncogene is one of the most frequently mutated genes in human cancer, with K-RAS having a leading role in tumorigenesis. K-RAS undergoes alternative splicing, and as a result its transcript generates two gene products K-RAS4A and K-RAS4B, which are affected by the same oncogenic mutations, are highly homologous, and are expressed in a variety of human tissues at different levels. In addition, both isoforms localise to the plasma membrane by distinct targeting motifs. While some evidence suggests nonredundant functions for both splice variants, most work to date has focused on K-RAS4B, or even just K-RAS (i.e., without differentiating between the splice variants). This review aims to address the most relevant evidence published regarding K-RAS4A and to discuss if this "minor" isoform could also play a leading role in cancer, concluding that a significant body of evidence supports a leading role rather than a supporting (or secondary) role for K-RAS4A in cancer biology.

Sequence-Selective Covalent CaaX-Box Receptors Prevent Farnesylation of Oncogenic Ras Proteins and Impact MAPK/PI3†K Signaling.

Oncogenic Ras proteins are implicated in the most common life-threatening cancers. Despite intense research over the past two decades, the progress towards small-molecule inhibitors has been limited. One reason for this failure is that Ras proteins interact with their effectors only via protein-protein interactions, which are notoriously difficult to address with small organic molecules. Herein we describe an alternative strategy, which prevents farnesylation and subsequent membrane insertion, a prerequisite for the activation of Ras proteins. Our approach is based on sequence-selective supramolecular receptors which bind to the C-terminal farnesyl transferase recognition unit of Ras and Rheb proteins and covalently modify the essential cysteine in the so-called CaaX-box.

Ras isoform-specific expression, chromatin accessibility, and signaling.

The anchorage of Ras isoforms in the membrane and their nanocluster formations have been studied extensively, including their detailed interactions, sizes, preferred membrane environments, chemistry, and geometry. However, the staggering challenge of their epigenetics and chromatin accessibility in distinct cell states and types, which we propose is a major factor determining their specific expression, still awaits unraveling. Ras isoforms are distinguished by their C-terminal hypervariable region (HVR) which acts in intracellular transport, regulation, and membrane anchorage. Here, we review some isoform-specific activities at the plasma membrane from a structural dynamic standpoint. Inspired by physics and chemistry, we recognize that understanding functional specificity requires insight into how biomolecules can organize themselves in different cellular environments. Within this framework, we suggest that isoform-specific expression may largely be controlled by the chromatin density and physical compaction, which allow (or curb) access to "chromatinized DNA." Genes are preferentially expressed in tissues: proteins expressed in pancreatic cells may not be equally expressed in lung cells. It is the rule-not an exception, and it can be at least partly understood in terms of chromatin organization and accessibility state. Genes are expressed when they can be sufficiently exposed to the transcription machinery, and they are less so when they are persistently buried in dense chromatin. Notably, chromatin accessibility can similarly determine expression of drug resistance genes.

Inhibition of Nonfunctional Ras.

Intuitively, functional states should be targeted; not nonfunctional ones. So why could drugging the inactive K-Ras4BG12Cwork-but drugging the inactive kinase will likely not? The reason is the distinct oncogenic mechanisms. Kinase driver mutations work by stabilizing the active state and/or destabilizing the inactive state. Either way, oncogenic kinases are mostly in the active state. Ras driver mutations work by quelling its deactivation mechanisms, GTP hydrolysis, and nucleotide exchange. Covalent inhibitors that bind to the inactive GDP-bound K-Ras4BG12C conformation can thus work. By contrast, in kinases, allosteric inhibitors work by altering the active-site conformation to favor orthosteric drugs. From the translational standpoint this distinction is vital: it expedites effective pharmaceutical development and extends the drug classification based on the mechanism of action. Collectively, here we postulate that drug action relates to blocking the mechanism of activation, not to whether the protein is in the active or inactive state.

Facile Synthesis of Boc-Protected Selenocystine and its Compatibility with Late-Stage Farnesylation at Cysteine Site.

BACKGROUND: The unique hypervariable C-terminal region (HVR) of K-Ras4B, one of the most frequently mutated proteins in many powerful cancers, contains a C-terminal farnesylated and methylated Cys and a poly-lysine motif, which decides the association of K-Ras4B to the inner leaflet of plasma membrane for activating the downstream signaling activity. In our previous work, we inserted an additional Cys in K-Ras4B HVR peptide synthesis for NCL in the semi-synthesis of K-Ras4b protein, but it is not suitable for application in protein dimerization research. The recently developed selenocysteine (Sec, U) mediated native chemical ligation reaction followed by selective deselenization, which can help to broaden the scope of protein synthesis, requires the generation of the peptide fragment with an N-terminal Sec. OBJECTIVE: To synthesize K-Ras4B HVR peptide containing both N-terminal Sec and C-terminal farnesylated and methylated Cys to achieve traceless protein semi-synthesis. METHODS AND RESULTS: We have developed a facile synthesis approach for producing Boc-Sec)2-OH using economic Se powder, which can facilitate scaling up preparation of peptides containing Sec at the N-terminus. Furthermore, we synthesized K-Ras4B HVR peptide containing selenocystine by utilization of Boc-Sec)2-OH. Finally, we took K-Ras4B HVR peptide as an example to test the compatibility of farnesylation reaction at Cys with the N-terminal Sec)2, and the farnesyl group was successfully added to the thiol group of Cys.

Oncogenic K-Ras4B Dimerization Enhances Downstream Mitogen-activated Protein Kinase Signaling.

Ras recruits and activates effectors that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, Raf's activation requires dimerization, which can be facilitated by Ras dimerization. Previously, we showed that active K-Ras4B dimerizes in silico and in vitro through two major interfaces: (i) ß-interface, mapped to Switch I and effector-binding regions, (ii) α-interface at the allosteric lobe. Here, we chose constitutively active K-Ras4B as our control and two double mutants (K101D and R102E; and R41E and K42D) in the α- and ß-interfaces. Two of the mutations are from The Cancer Genome Atlas (TCGA) and the Catalogue Of Somatic Mutations In Cancer (COSMIC) data sets. R41 and R102 are found in several adenocarcinomas in Ras isoforms. We performed site-directed mutagenesis, cellular localization experiments, and molecular dynamics (MD) simulations to assess the impact of the mutations on K-Ras4B dimerization and function. α-interface K101D/R102E double mutations reduced dimerization but only slightly reduced downstream phosphorylated extracellular signal-regulated kinase (ERK) (pERK) levels. While ß-interface R41E/K42D double mutations did not interfere with dimerization, they almost completely blocked K-Ras4B-mediated ERK phosphorylation. Both double mutations increased downstream phosphorylated Akt (pAkt) levels in cells. Changes in pERK and pAkt levels altered ERK- and Akt-regulated gene expressions, such as EGR1, JUN, and BCL2L11. These results underscore the role of the α-interface in K-Ras4B homodimerization and the ß-surface in effector binding. MD simulations highlight that the membrane and hypervariable region (HVR) interact with both α- and ß-interfaces of K-Ras4B mutants, respectively, inhibiting homodimerization and probably effector binding. Mutations at both interfaces interfered with mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase signaling but in different forms and extents. We conclude that dimerization is not necessary but enhances downstream MAPK signaling.