A Novel Pan-RAS Inhibitor with a Unique Mechanism of Action Blocks Tumor Growth in Mouse Models of GI Cancer.

Here, we describe a novel pan-RAS inhibitor, ADT-007, that potently inhibited the growth of RAS mutant cancer cells irrespective of the RAS mutation or isozyme. RAS WT cancer cells with activated RAS from upstream mutations were equally sensitive. Conversely, cells from normal tissues or RAS WT cancer cells harboring downstream BRAF mutations were insensitive. Insensitivity to ADT-007 was attributed to low activated RAS levels and metabolic deactivation by UDP-glucuronosyltransferases expressed in normal cells but repressed in RAS mutant cancer cells. Cellular, biochemical, and biophysical experiments show ADT-007 binds nucleotide-free RAS to block GTP activation of RAS and MAPK/AKT signaling. Local administration of ADT-007 strongly inhibited tumor growth in syngeneic immune-competent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancer while activating innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug inhibited tumor growth, supporting further development of this novel class of pan-RAS inhibitors for treating RAS-driven cancers. SIGNIFICANCE: ADT-007 is a 1 st -in-class pan-RAS inhibitor with ultra-high potency and unique selectivity for cancer cells with mutant or activated RAS capable of circumventing resistance and activating antitumor immunity. Further development of ADT-007 analogs or prodrugs with oral bioavailability as a generalizable monotherapy or combined with immunotherapy is warranted.

Development of tolerance to chemokine receptor antagonists: current paradigms and the need for further investigation.

Chemokine G-protein coupled receptors are validated drug targets for many diseases, including cancer, neurological, and inflammatory disorders. Despite much time and effort spent on therapeutic development, very few chemokine receptor antagonists are approved for clinical use. Among potential reasons for the slow progress in developing chemokine receptor inhibitors, antagonist tolerance, a progressive reduction in drug efficacy after repeated administration, is likely to play a key role. The mechanisms leading to antagonist tolerance remain poorly understood. In many cases, antagonist tolerance is accompanied by increased receptor concentration on the cell surface after prolonged exposure to chemokine receptor antagonists. This points to a possible role of altered receptor internalization and presentation on the cell surface, as has been shown for agonist (primarily opioid) tolerance. In addition, examples of antagonist tolerance in the context of other G-protein coupled receptors suggest the involvement of noncanonical signal transduction in opposing the effects of the antagonists. In this review, we summarize the available progress and challenges in therapeutic development of chemokine receptor antagonists, describe the available knowledge about antagonist tolerance, and propose new avenues for future investigation of this important phenomenon. Furthermore, we highlight the modern methodologies that have the potential to reveal novel mechanisms leading to antagonist tolerance and to propel the field forward by advancing the development of potent "tolerance-free" antagonists of chemokine receptors.

Conformational Dynamics Allows Sampling of an "Active-like" State by Oncogenic K-Ras-GDP.

Mutations in K-Ras GTPase replacing Gly12 with either Asp or Val are common in cancer. These mutations decelerate intrinsic and catalyzed GTP hydrolysis, leading to accumulation of K-Ras-GTP in cells. Signaling cascades initiated by K-Ras-GTP promote cell proliferation, survival, and invasion. Despite functional differences between the most frequent G12D mutation and the most aggressive and chemotherapy resistant G12V mutation, their long-suspected distinct structural features remain elusive. Using NMR, X-ray structures, and computational methods, we found that oncogenic mutants of K-Ras4B, the predominant splice variant of K-Ras, exhibit distinct conformational dynamics when GDP-bound, visiting the "active-like" conformational state similar to the one observed in GTP-bound K-Ras. This behavior distinguishes G12V from wild type and G12D K-Ras4B-GDP. The likely reason is interactions between the aliphatic sidechain of V12 and the Switch II region of K-Ras4BG12V-GDP, which are distinct in K-Ras4BG12D-GDP. In the X-ray structures, crystal contacts reduce the dynamics of the sidechain at position 12 by stabilizing the Switch I region of the protein. This explains why structural differences between G12V and G12D K-Ras have yet not been reported. Together, our results suggest a previously unknown mechanism of K-Ras activation. This mechanism relies on conformational dynamics caused by specific oncogenic mutations in the GDP-bound state. Our findings also imply that the therapeutic strategies decreasing the level of K-Ras-GTP by interfering with nucleotide exchange or by expediting GTP hydrolysis may work differently in different oncogenic mutants.

The dynamic nature of the K-Ras/calmodulin complex can be altered by oncogenic mutations.

Oncogenic mutant K-Ras promotes cancer cell proliferation, migration, invasion, and survival by assembling signaling complexes. To date, the functional and structural roles of K-Ras mutations within these complexes are incompletely understood despite their mechanistic and therapeutic significance. Here, we review recent advances in understanding specific binding between K-Ras and the calcium sensor calmodulin. This interaction positively and negatively regulates diverse functions of K-Ras in cancer, suggesting flexibility in K-Ras/calmodulin complex formation. Also, structural data suggest that oncogenic K-Ras likely samples several conformational states, influencing its distinct assemblies with calmodulin and with other proteins. Understanding how K-Ras interacts with calmodulin and with other partners is essential to discovering novel inhibitors of K-Ras in cancer.

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.

Natural Products Attenuating Biosynthesis, Processing, and Activity of Ras Oncoproteins: State of the Art and Future Perspectives.

RAS genes encode signaling proteins, which, in mammalian cells, act as molecular switches regulating critical cellular processes as proliferation, growth, differentiation, survival, motility, and metabolism in response to specific stimuli. Deregulation of Ras functions has a high impact on human health: gain-of-function point mutations in RAS genes are found in some developmental disorders and thirty percent of all human cancers, including the deadliest. For this reason, the pathogenic Ras variants represent important clinical targets against which to develop novel, effective, and possibly selective pharmacological inhibitors. Natural products represent a virtually unlimited resource of structurally different compounds from which one could draw on for this purpose, given the improvements in isolation and screening of active molecules from complex sources. After a summary of Ras proteins molecular and regulatory features and Ras-dependent pathways relevant for drug development, we point out the most promising inhibitory approaches, the known druggable sites of wild-type and oncogenic Ras mutants, and describe the known natural compounds capable of attenuating Ras signaling. Finally, we highlight critical issues and perspectives for the future selection of potential Ras inhibitors from natural sources.

SPHK2-Generated S1P in CD11b+ Macrophages Blocks STING to Suppress the Inflammatory Function of Alveolar Macrophages.

Acute lung injury (ALI) is a lethal inflammatory lung disorder whose incidence is on the rise. Alveolar macrophages normally act to resolve inflammation, but when dysregulated they can provoke ALI. We demonstrate that monocyte-derived macrophages (CD11b+ macrophages) recruited into the airspace upregulate the anti-inflammatory function of alveolar macrophages by suppressing their stimulator of type 1 interferon gene (STING) signaling. Depletion of CD11b+ macrophages in mice (macrophagedep mice) after endotoxin or after Pseudomonas aeruginosa causes expansion of the inflammatory alveolar macrophage population, leading to neutrophil accumulation, irreversible loss of lung vascular barrier function, and lethality. We show that CD11b+ macrophages suppress alveolar macrophage-STING signaling via sphingosine kinase-2 (SPHK2) generation of sphingosine-1-phosphate (S1P). Thus, adoptive transfer of wild-type (WT) or STING-/-, but not SPHK2-/-, CD11b monocytes from murine bone marrow into injured macrophagedep mice rescue anti-inflammatory alveolar macrophages and reverse lung vascular injury. SPHK2-induced S1P generation in CD11b+ macrophages has the potential to educate alveolar macrophages to resolve ALI.

Characterization of heteromeric complexes between chemokine (C-X-C motif) receptor 4 and α1-adrenergic receptors utilizing intermolecular bioluminescence resonance energy transfer assays.

Recently, we reported that chemokine (C-X-C motif) receptor 4 (CXCR4) heteromerizes with α1-adrenergic receptors (AR) on the cell surface of vascular smooth muscle cells, through which the receptors cross-talk. Direct biophysical evidence for CXCR4:α1-AR heteromers, however, is lacking. Here we utilized bimolecular luminescence/fluorescence complementation (BiLC/BiFC) combined with intermolecular bioluminescence resonance energy transfer (BRET) assays in HEK293T cells to evaluate CXCR4:α1a/b/d-AR heteromerization. Atypical chemokine receptor 3 (ACKR3) and metabotropic glutamate receptor 1 (mGlu1R) were utilized as controls. BRET between CXCR4-RLuc (Renilla reniformis) and enhanced yellow fluorescent protein (EYFP)-tagged ACKR3 or α1a/b/d-ARs fulfilled criteria for constitutive heteromerization. BRET between CXCR4-RLuc and EYFP or mGlu1R-EYFP were nonspecific. BRET50 for CXCR4:ACKR3 and CXCR4:α1a/b/d-AR heteromers were comparable. Stimulation of cells with phenylephrine increased BRETmax of CXCR4:α1a/b/d-AR heteromers without affecting BRET50; stimulation with CXCL12 reduced BRETmax of CXCR4:α1a-AR heteromers, but did not affect BRET50 or BRETmax/50 for CXCR4:α1b/d-AR. A peptide analogue of transmembrane domain (TM) 2 of CXCR4 reduced BRETmax of CXCR4:α1a/b/d-AR heteromers and increased BRET50 of CXCR4:α1a/b-AR interactions. A TM4 analogue of CXCR4 did not alter BRET. We observed CXCR4, α1a-AR and mGlu1R homodimerization by BiFC/BiLC, and heteromerization of homodimeric CXCR4 with proto- and homodimeric α1a-AR by BiFC/BiLC BRET. BiFC/BiLC BRET for interactions between homodimeric CXCR4 and homodimeric mGlu1R was nonspecific. Our findings suggest that the heteromerization affinity of CXCR4 for ACKR3 and α1-ARs is comparable, provide evidence for conformational changes of the receptor complexes upon agonist binding and support the concept that proto- and oligomeric CXCR4 and α1-ARs constitutively form higher-order hetero-oligomeric receptor clusters.

Correction: Mixed lineage kinase 3 promotes breast tumorigenesis via phosphorylation and activation of p21-activated kinase 1.

The original version of this Article did not acknowledge Pradeep Sathyanarayana as an author. His affiliation is Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine, USA.

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.

The Structural Basis of the Farnesylated and Methylated KRas4B Interaction with Calmodulin.

Ca2+-calmodulin (CaM) extracts KRas4B from the plasma membrane, suggesting that KRas4B/CaM interaction plays a role in regulating Ras signaling. To gain mechanistic insight, we provide a computational model, supported by experimental structural data, of farnesylated/methylated KRas4B1-185 interacting with CaM in solution and at anionic membranes including signaling lipids. Due to multiple interaction modes, we observe diverse conformational ensembles of the KRas4B-CaM complex. A highly populated conformation reveals the catalytic domain interacting with the N-lobe and the hypervariable region (HVR) wrapping around the linker with the farnesyl docking to the extended CaM's C-lobe pocket. Alternatively, KRas4B can interact with collapsed CaM with the farnesyl penetrating CaM's center. At anionic membranes, CaM interacts with the catalytic domain with large fluctuations, drawing the HVR. Signaling lipids establishing strong salt bridges with CaM prevent membrane departure. Membrane-interacting KRas4B-CaM complex can productively recruit phosphatidylinositol 3-kinase α (PI3Kα) to the plasma membrane, serving as a coagent in activating PI3Kα/Akt signaling.

Vemurafenib Inhibits Active PTK6 in PTEN-null Prostate Tumor Cells.

Protein tyrosine kinase 6 (PTK6, also called BRK) is overexpressed and activated in human prostate cancer. Loss of the tumor suppressor PTEN, a frequent event in prostate cancer, leads to PTK6 activation at the plasma membrane and its oncogenic signaling. The small molecule inhibitor vemurafenib, also known as PLX4032, and its tool analog PLX4720 were designed to inhibit constitutively active BRAF V600E, yet they also have potent effects against PTK6. Vemurafenib is used in the treatment of metastatic melanoma, but its efficacy in prostate cancer has not been assessed. When activated at the plasma membrane, PTK6 promotes signaling through FAK, EGFR, and ERK1/2, and we show this can be blocked by vemurafenib. In addition, PTK6-mediated cell growth, migration, and invasion are inhibited upon vemurafenib administration. Using a flank xenograft model, vemurafenib treatment reduced tumor burden. Using saturation transfer difference NMR and molecular docking, we demonstrate that vemurafenib binds in the active site of PTK6, inhibiting its activation. These structural studies provide insight into the PTK6-vemurafenib complex, which can be utilized for further refinement chemistry, whereas functional studies demonstrate that active PTK6 is a viable drug target in prostate cancer.

Mixed lineage kinase 3 promotes breast tumorigenesis via phosphorylation and activation of p21-activated kinase 1.

Mixed lineage kinase 3 (MLK3), a MAP3K member has been envisioned as a viable drug target in cancer, yet its detailed function and signaling is not fully elucidated. We identified that MLK3 tightly associates with an oncogene, PAK1. Mammalian PAK1 being a Ste20 (MAP4K) member, we tested whether it is an upstream regulator of MLK3. In contrast to our hypothesis, MLK3 activated PAK1 kinase activity directly, as well as in the cells. Although, MLK3 can phosphorylate PAK1 on Ser133 and Ser204 sites, PAK1S133A mutant is constitutively active, whereas, PAK1S204A is not activated by MLK3. Stable overexpression of PAK1S204A in breast cancer cells, impedes migration, invasion, and NFĸB activity. In vivo breast cancer cell tumorigenesis is significantly reduced in tumors expressing PAK1S204A mutant. These results suggest that mammalian PAK1 does not act as a MAP4K and MLK3-induced direct activation of PAK1 plays a key role in breast cancer tumorigenesis.

Novel peptide nanoparticle-biased antagonist of CCR3 blocks eosinophil recruitment and airway hyperresponsiveness.

BACKGROUND: Chemokine signaling through CCR3 is a key regulatory pathway for eosinophil recruitment into tissues associated with allergic inflammation and asthma. To date, none of the CCR3 antagonists have shown efficacy in clinical trials. One reason might be their unbiased mode of inhibition that prevents receptor internalization, leading to drug tolerance. OBJECTIVE: We sought to develop a novel peptide nanoparticle CCR3 inhibitor (R321) with a biased mode of inhibition that would block G protein signaling but enable or promote receptor internalization. METHODS: Self-assembly of R321 peptide into nanoparticles and peptide binding to CCR3 were analyzed by means of dynamic light scattering and nuclear magnetic resonance. Inhibitory activity on CCR3 signaling was assessed in vitro by using flow cytometry, confocal microscopy, and Western blot analysis in a CCR3+ eosinophil cell line and blood eosinophils. In vivo effects of R321 were assessed by using a triple-allergen mouse asthma model. RESULTS: R321 self-assembles into nanoparticles and binds directly to CCR3, altering receptor function. Half-maximal inhibitory concentration values for eotaxin-induced chemotaxis of blood eosinophils are in the low nanomolar range. R321 inhibits only the early phase of extracellular signal-regulated kinase 1/2 activation and not the late phase generally associated with ß-arrestin recruitment and receptor endocytosis, promoting CCR3 internalization and degradation. In vivo R321 effectively blocks eosinophil recruitment into the blood, lungs, and airways and prevents airway hyperresponsiveness in a mouse eosinophilic asthma model. CONCLUSIONS: R321 is a potent and selective antagonist of the CCR3 signaling cascade. Inhibition through a biased mode of antagonism might hold significant therapeutic promise by eluding the formation of drug tolerance.

The bacterial Ras/Rap1 site-specific endopeptidase RRSP cleaves Ras through an atypical mechanism to disrupt Ras-ERK signaling.

The Ras-extracellular signal-regulated kinase pathway is critical for controlling cell proliferation, and its aberrant activation drives the growth of various cancers. Because many pathogens produce toxins that inhibit Ras activity, efforts to develop effective Ras inhibitors to treat cancer could be informed by studies of Ras inhibition by pathogens. Vibrio vulnificus causes fatal infections in a manner that depends on multifunctional autoprocessing repeats-in-toxin, a toxin that releases bacterial effector domains into host cells. One such domain is the Ras/Rap1-specific endopeptidase (RRSP), which site-specifically cleaves the Switch I domain of the small GTPases Ras and Rap1. We solved the crystal structure of RRSP and found that its backbone shares a structural fold with the EreA/ChaN-like superfamily of enzymes. Unlike other proteases in this family, RRSP is not a metalloprotease. Through nuclear magnetic resonance analysis and nucleotide exchange assays, we determined that the processing of KRAS by RRSP did not release any fragments or cause KRAS to dissociate from its bound nucleotide but instead only locally affected its structure. However, this structural alteration of KRAS was sufficient to disable guanine nucleotide exchange factor-mediated nucleotide exchange and prevent KRAS from binding to RAF. Thus, RRSP is a bacterial effector that represents a previously unrecognized class of protease that disconnects Ras from its signaling network while inducing limited structural disturbance in its target.

Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3.

We observed in PRESTO-Tango ß-arrestin recruitment assays that the α1-adrenergic receptor (AR) antagonist prazosin activates chemokine (C-X-C motif) receptor (CXCR)4. This prompted us to further examine this unexpected pharmacological behavior. We screened a panel of 14 α1/2- and ß1/2/3-AR antagonists for CXCR4 and atypical chemokine receptor (ACKR)3 agonist activity in PRESTO-Tango assays against the cognate agonist CXCL12. We observed that multiple α1-AR antagonists activate CXCR4 (CXCL12 = prazosin = cyclazosin > doxazosin) and ACKR3 (CXCL12 = prazosin = cyclazosin > alfuzosin = doxazosin = phentolamine > terazosin = silodosin = tamsulosin). The two strongest CXCR4/ACKR3 activators, prazosin and cyclazosin, were selected for a more detailed evaluation. We found that the drugs dose-dependently activate both receptors in ß-arrestin recruitment assays, stimulate ERK1/2 phosphorylation in HEK293 cells overexpressing each receptor, and that their effects on CXCR4 could be inhibited with AMD3100. Both α1-AR antagonists induced significant chemical shift changes in the 1H-13C-heteronuclear single quantum correlation spectrum of CXCR4 and ACKR3 in membranes, suggesting receptor binding. Furthermore, prazosin and cyclazosin induced internalization of endogenous CXCR4/ACKR3 in human vascular smooth muscle cells (hVSMC). While these drugs did not in induce chemotaxis in hVSMC, they inhibited CXCL12-induced chemotaxis with high efficacy and potency (IC50: prazosin-4.5 nM, cyclazosin 11.6 pM). Our findings reveal unexpected pharmacological properties of prazosin, cyclazosin, and likely other α1-AR antagonists. The results of the present study imply that prazosin and cyclazosin are biased or partial CXCR4/ACKR3 agonists, which function as potent CXCL12 antagonists. Our findings could provide a mechanistic basis for previously observed anti-cancer properties of α1-AR antagonists and support the concept that prazosin could be re-purposed for the treatment of disease processes in which CXCR4 and ACKR3 are thought to play significant pathophysiological roles, such as cancer metastases or various autoimmune pathologies.

Calmodulin (CaM) Activates PI3Kα by Targeting the "Soft" CaM-Binding Motifs in Both the nSH2 and cSH2 Domains of p85α.

PI3Kα is a key lipid kinase in the PI3K/Akt pathway. Its frequent oncogenic mutations make it a primary drug target. Calmodulin (CaM) activates PI3Kα independently of extracellular signals, indicating a significant role in oncogenic PI3Kα activation. Here, we reveal the atomic-scale structures of CaM in complexes with the nSH2 and cSH2 domains of the regulatory p85α subunit of PI3Kα, and illustrate how CaM activates PI3Kα by targeting the "soft 1-5-10" CaM-binding motifs in both nSH2 and cSH2 domains. Experiment observed CaM binding cSH2 first, followed by nSH2 binding hours later. CaM typically prefers binding helical peptides. Here we observe that, unlike in cSH2, the CaM-binding motif in nSH2 populates a mixed ß-sheet/α-helix/random coil structure. The population shift from a ß-sheet toward CaM's favored α-helical conformation explains why the nSH2 domain needs a longer time for CaM binding in the experiments. The "soft" CaM-binding motifs in both nSH2 and cSH2 domains establish strong CaM-PI3Kα interactions, collectively facilitating PI3Kα activation. This work uncovers the structural basis for CaM-driven PI3Kα activation.

Interaction of Calmodulin with the cSH2 Domain of the p85 Regulatory Subunit.

Calmodulin (CaM) is a calcium sensor protein that directly interacts with the dual-specificity (lipid and protein) kinase PI3Kα through the SH2 domains of the p85 regulatory subunit. In adenocarcinomas, the CaM interaction removes the autoinhibition of the p110 catalytic subunit of PI3Kα, leading to activation of PI3Kα and promoting cell proliferation, survival, and migration. Here we demonstrate that the cSH2 domain of p85α engages its two CaM-binding motifs in the interaction with the N- and C-lobes of CaM as well as the flexible central linker, and our nuclear magnetic resonance experiments provide structural details. We show that in response to binding CaM, cSH2 exposes its tryptophan residue at the N-terminal region to the solvent. Because of the flexible nature of both CaM and cSH2, multiple binding modes of the interactions are possible. Binding of CaM to the cSH2 domain can help release the inhibition imposed on the p110 subunit, similar to the binding of the phosphorylated motif of RTK, or phosphorylated CaM (pCaM), to the SH2 domains. Amino acid sequence analysis shows that CaM-binding motifs are common in SH2 domains of non-RTKs. We speculate that CaM can also activate these kinases through similar mechanisms.

Honokiol is a FOXM1 antagonist.

Honokiol is a natural product and an emerging drug for a wide variety of malignancies, including hematopoietic malignancies, sarcomas, and common epithelial tumors. The broad range of activity of honokiol against numerous malignancies with diverse genetic backgrounds suggests that honokiol is inhibiting an activity that is common to multiple malignancies. Oncogenic transcription factor FOXM1 is one of the most overexpressed oncoproteins in human cancer. Here we found that honokiol inhibits FOXM1-mediated transcription and FOXM1 protein expression. More importantly, we found that honokiol's inhibitory effect on FOXM1 is a result of binding of honokiol to FOXM1. This binding is specific to honokiol, a dimerized allylphenol, and was not observed in compounds that either were monomeric allylphenols or un-substituted dihydroxy phenols. This indicates that both substitution and dimerization of allylphenols are required for physical interaction with FOXM1. We thus demonstrate a novel and specific mechanism for FOXM1 inhibition by honokiol, which partially may explain its anticancer activity in cancer cells.

Phosphorylated Calmodulin Promotes PI3K Activation by Binding to the SH2 Domains.

How calmodulin (CaM) acts in KRAS-driven cancers is a vastly important question. CaM binds to and stimulates PI3Kα/Akt signaling, promoting cell growth and proliferation. Phosphorylation of CaM at Tyr99 (pY99) enhances PI3Kα activation. PI3Kα is a lipid kinase. It phosphorylates PIP2 to produce PIP3, to which Akt binds. PI3Kα has two subunits: the regulatory p85 and the catalytic p110. Here, exploiting explicit-solvent MD simulations we unveil key interactions between phosphorylated CaM (pCaM) and the two SH2 domains in the p85 subunit, confirm experimental observations, and uncover PI3Kα's mechanism of activation. pCaMs form strong and stable interactions with both nSH2 and cSH2 domains, with pY99 being the dominant contributor. Despite the high structural similarity between the two SH2 domains, we observe that nSH2 prefers an extended CaM conformation, whereas cSH2 prefers a collapsed conformation. Notably, collapsed CaM is observed after binding of an extended CaM to K-Ras4B. Thus, the more populated extended pCaM conformation targets nSH2 to release its autoinhibition of p110 catalytic sites. This executes the key activation step of PI3Kα. Independently, K-Ras4B allosterically activates p110. These events are at the cell membrane, which contributes to tighten the PI3Kα Ras binding domain/K-Ras4B interaction, to accomplish K-Ras4B allosteric activation, with a minor contribution from cSH2.