Interaction with WDR5 promotes target gene recognition and tumorigenesis by MYC.

MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies. The oncogenic potential of MYC stems from its ability to bind regulatory sequences in thousands of target genes, which depends on interaction of MYC with its obligate partner, MAX. Here, we show that broad association of MYC with chromatin also depends on interaction with the WD40-repeat protein WDR5. MYC binds WDR5 via an evolutionarily conserved "MYC box IIIb" motif that engages a shallow, hydrophobic cleft on the surface of WDR5. Structure-guided mutations in MYC that disrupt interaction with WDR5 attenuate binding of MYC at ∼80% of its chromosomal locations and disable its ability to promote induced pluripotent stem cell formation and drive tumorigenesis. Our data reveal WDR5 as a key determinant for MYC recruitment to chromatin and uncover a tractable target for the discovery of anticancer therapies against MYC-driven tumors.

Drugging an undruggable pocket on KRAS.

The 3 human RAS genes, KRAS, NRAS, and HRAS, encode 4 different RAS proteins which belong to the protein family of small GTPases that function as binary molecular switches involved in cell signaling. Activating mutations in RAS are among the most common oncogenic drivers in human cancers, with KRAS being the most frequently mutated oncogene. Although KRAS is an excellent drug discovery target for many cancers, and despite decades of research, no therapeutic agent directly targeting RAS has been clinically approved. Using structure-based drug design, we have discovered BI-2852 (1), a KRAS inhibitor that binds with nanomolar affinity to a pocket, thus far perceived to be "undruggable," between switch I and II on RAS; 1 is mechanistically distinct from covalent KRASG12C inhibitors because it binds to a different pocket present in both the active and inactive forms of KRAS. In doing so, it blocks all GEF, GAP, and effector interactions with KRAS, leading to inhibition of downstream signaling and an antiproliferative effect in the low micromolar range in KRAS mutant cells. These findings clearly demonstrate that this so-called switch I/II pocket is indeed druggable and provide the scientific community with a chemical probe that simultaneously targets the active and inactive forms of KRAS.

High-throughput screening identifies small molecules that bind to the RAS:SOS:RAS complex and perturb RAS signaling.

K-RAS is mutated in approximately 30% of human cancers, resulting in increased RAS signaling and tumor growth. Thus, RAS is a highly validated therapeutic target, especially in tumors of the pancreas, lung and colon. Although directly targeting RAS has proven to be challenging, it may be possible to target other proteins involved in RAS signaling, such as the guanine nucleotide exchange factor Son of Sevenless (SOS). We have previously reported on the discovery of small molecules that bind to SOS1, activate SOS-mediated nucleotide exchange on RAS, and paradoxically inhibit ERK phosphorylation (Burns et al., PNAS, 2014). Here, we describe the discovery of additional, structurally diverse small molecules that also bind to SOS1 in the same pocket and elicit similar biological effects. We tested >160,000 compounds in a fluorescence-based assay to assess their effects on SOS-mediated nucleotide exchange. X-Ray structures revealed that these small molecules bind to the CDC25 domain of SOS1. Compounds that elicited high levels of nucleotide exchange activity in vitro increased RAS-GTP levels in cells, and inhibited phospho ERK levels at higher treatment concentrations. The identification of structurally diverse SOS1 binding ligands may assist in the discovery of new molecules designed to target RAS-driven tumors.

Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

A method for the second-site screening of K-Ras in the presence of a covalently attached first-site ligand.

K-Ras is a well-validated cancer target but is considered to be "undruggable" due to the lack of suitable binding pockets. We previously discovered small molecules that bind weakly to K-Ras but wanted to improve their binding affinities by identifying ligands that bind near our initial hits that we could link together. Here we describe an approach for identifying second site ligands that uses a cysteine residue to covalently attach a compound for tight binding to the first site pocket followed by a fragment screen for binding to a second site. This approach could be very useful for targeting Ras and other challenging drug targets.

Fragment Optimization of Reversible Binding to the Switch II Pocket on KRAS Leads to a Potent, In Vivo Active KRASG12C Inhibitor.

Activating mutations in KRAS are the most frequent oncogenic alterations in cancer. The oncogenic hotspot position 12, located at the lip of the switch II pocket, offers a covalent attachment point for KRASG12C inhibitors. To date, KRASG12C inhibitors have been discovered by first covalently binding to the cysteine at position 12 and then optimizing pocket binding. We report on the discovery of the in vivo active KRASG12C inhibitor BI-0474 using a different approach, in which small molecules that bind reversibly to the switch II pocket were identified and then optimized for non-covalent binding using structure-based design. Finally, the Michael acceptor containing warhead was attached. Our approach offers not only an alternative approach to discovering KRASG12C inhibitors but also provides a starting point for the discovery of inhibitors against other oncogenic KRAS mutants.

Structure-based design of high affinity peptides inhibiting the interaction of p53 with MDM2 and MDMX.

MDM2 and MDMX function as key regulators of p53 by binding to its N terminus, inhibiting its transcriptional activity, and promoting degradation. MDM2 and MDMX overexpression or hyperactivation directly contributes to the loss of p53 function during the development of nearly 50% of human cancers. Recent studies showed that disrupting p53-MDM2 and p53-MDMX interactions can lead to robust activation of p53 but also revealed a need to develop novel dual specific or MDMX-specific inhibitors. Using phage display we identified a 12-residue peptide (pDI) with inhibitory activity against MDM2 and MDMX. The co-crystal structures of the pDI and a single mutant derivative (pDI6W) liganded with the N-terminal domains of human MDMX and MDM2 served as the basis for the design of 11 distinct pDI-derivative peptides that were tested for inhibitory potential. The best derivative (termed pDIQ) contained four amino acid substitutions and exhibited a 5-fold increase in potency over the parent peptide against both MDM2 (IC(50) = 8 nm) and MDMX (IC(50) = 110 nm). Further structural studies revealed key molecular features enabling the high affinity binding of the pDIQ to these proteins. These include large conformational changes of the pDIQ to reach into a hydrophobic site unique to MDMX. The findings suggest new strategies toward the rational design of small molecule inhibitors efficiently targeting MDMX.

Physical Therapy for Gait, Balance, and Cognition in Individuals with Cognitive Impairment: A Retrospective Analysis.

OBJECTIVES: The purpose of this study was to determine if a pragmatic physical therapy (PT) program was associated with improved cognition, gait, and balance in individuals with cognitive impairment. This study investigated these associations for individuals with Alzheimer disease (AD), vascular dementia (VaD), dementia with Lewy bodies (DLB), and mild cognitive impairment (MCI) in order to better characterize outcomes to PT for each diagnostic group. METHODS: Data before and after one month of physical therapy were extracted from patient records (67 with AD, 34 with VaD, 35 with DLB, and 37 with MCI). The mean number of PT sessions over a month was 3.4 (±1.8). Outcomes covered the domains of gait, balance, and cognition with multiple outcomes used to measure different constructs within the balance and gait domains. RESULTS: All groups showed improvements in balance and at least one gait outcome measure. Those with MCI improved in every measure of gait and balance performance. Lastly, cognition as measured by Montreal Cognitive Assessment improved in individuals in the AD, VaD, and MCI groups. CONCLUSION: While this retrospective analysis is not appropriate for causal inference, results of one month of physical therapy were associated with decreases in gait, balance, and cognitive impairment in individuals with AD, VaD, DLB<, and MCI. Clinical Implications. While physical therapy is not typically a primary treatment strategy for individuals with cognitive impairment, the results of this study are consistent with the literature that demonstrates improvement from physical therapy for other neurodegenerative diseases. Further clinical and research exploration for physical therapy as a primary treatment strategy in these populations is warranted.

Discovery of Sulfonamide-Derived Agonists of SOS1-Mediated Nucleotide Exchange on RAS Using Fragment-Based Methods.

The nucleotide exchange factor Son of Sevenless (SOS) catalyzes the activation of RAS by converting it from its inactive GDP-bound state to its active GTP-bound state. Recently, we have reported the discovery of small-molecule allosteric activators of SOS1 that can increase the amount of RAS-GTP in cells. The compounds can inhibit ERK phosphorylation at higher concentrations by engaging a feedback mechanism. To further study this process, we sought different chemical matter from an NMR-based fragment screen using selective methyl labeling. To aid this process, several Ile methyl groups located in different binding sites of the protein were assigned and used to categorize the NMR hits into different classes. Hit to lead optimization using an iterative structure-based design paradigm resulted in compounds with improvements in binding affinity. These improved molecules of a different chemical class increase SOS1cat-mediated nucleotide exchange on RAS and display cellular action consistent with our prior results.

Discovery of WD Repeat-Containing Protein 5 (WDR5)-MYC Inhibitors Using Fragment-Based Methods and Structure-Based Design.

The frequent deregulation of MYC and its elevated expression via multiple mechanisms drives cells to a tumorigenic state. Indeed, MYC is overexpressed in up to ∼50% of human cancers and is considered a highly validated anticancer target. Recently, we discovered that WD repeat-containing protein 5 (WDR5) binds to MYC and is a critical cofactor required for the recruitment of MYC to its target genes and reported the first small molecule inhibitors of the WDR5-MYC interaction using structure-based design. These compounds display high binding affinity, but have poor physicochemical properties and are hence not suitable for in vivo studies. Herein, we conducted an NMR-based fragment screening to identify additional chemical matter and, using a structure-based approach, we merged a fragment hit with the previously reported sulfonamide series. Compounds in this series can disrupt the WDR5-MYC interaction in cells, and as a consequence, we observed a reduction of MYC localization to chromatin.

Small Molecule SOS1 Agonists Modulate MAPK and PI3K Signaling via Independent Cellular Responses.

Activating mutations in RAS can lead to oncogenesis by enhancing downstream signaling, such as through the MAPK and PI3K pathways. Therefore, therapeutically targeting RAS may perturb multiple signaling pathways simultaneously. One method for modulating RAS signaling is to target the activity of the guanine nucleotide exchange factor SOS1. Our laboratory has discovered compounds that bind to SOS1 and activate RAS. Interestingly, these SOS1 agonist compounds elicit biphasic modulation of ERK phosphorylation and simultaneous inhibition of AKT phosphorylation levels. Here, we utilized multiple chemically distinct compounds to elucidate whether these effects on MAPK and PI3K signaling by SOS1 agonists were mechanistically linked. In addition, we used CRISPR/Cas9 gene-editing to generate clonally derived SOS1 knockout cells and identified a potent SOS1 agonist that rapidly elicited on-target molecular effects at substantially lower concentrations than those causing off-target effects. Our findings will allow us to further define the on-target utility of SOS1 agonists.

Discovery and Optimization of Salicylic Acid-Derived Sulfonamide Inhibitors of the WD Repeat-Containing Protein 5-MYC Protein-Protein Interaction.

The treatment of tumors driven by overexpression or amplification of MYC oncogenes remains a significant challenge in drug discovery. Here, we present a new strategy toward the inhibition of MYC via the disruption of the protein-protein interaction between MYC and its chromatin cofactor WD Repeat-Containing Protein 5. Blocking the association of these proteins is hypothesized to disrupt the localization of MYC to chromatin, thus disrupting the ability of MYC to sustain tumorigenesis. Utilizing a high-throughput screening campaign and subsequent structure-guided design, we identify small-molecule inhibitors of this interaction with potent in vitro binding affinity and report structurally related negative controls that can be used to study the effect of this disruption. Our work suggests that disruption of this protein-protein interaction may provide a path toward an effective approach for the treatment of multiple tumors and anticipate that the molecules disclosed can be used as starting points for future efforts toward compounds with improved drug-like properties.

Displacement of WDR5 from Chromatin by a WIN Site Inhibitor with Picomolar Affinity.

The chromatin-associated protein WDR5 is a promising target for pharmacological inhibition in cancer. Drug discovery efforts center on the blockade of the "WIN site" of WDR5, a well-defined pocket that is amenable to small molecule inhibition. Various cancer contexts have been proposed to be targets for WIN site inhibitors, but a lack of understanding of WDR5 target genes and of the primary effects of WIN site inhibitors hampers their utility. Here, by the discovery of potent WIN site inhibitors, we demonstrate that the WIN site links WDR5 to chromatin at a small cohort of loci, including a specific subset of ribosome protein genes. WIN site inhibitors rapidly displace WDR5 from chromatin and decrease the expression of associated genes, causing translational inhibition, nucleolar stress, and p53 induction. Our studies define a mode by which WDR5 engages chromatin and forecast that WIN site blockade could have utility against multiple cancer types.

Discovery of Quinazolines That Activate SOS1-Mediated Nucleotide Exchange on RAS.

Proteins in the RAS family are important regulators of cellular signaling and, when mutated, can drive cancer pathogenesis. Despite considerable effort over the last 30 years, RAS proteins have proven to be recalcitrant therapeutic targets. One approach for modulating RAS signaling is to target proteins that interact with RAS, such as the guanine nucleotide exchange factor (GEF) son of sevenless homologue 1 (SOS1). Here, we report hit-to-lead studies on quinazoline-containing compounds that bind to SOS1 and activate nucleotide exchange on RAS. Using structure-based design, we refined the substituents attached to the quinazoline nucleus and built in additional interactions not present in the initial HTS hit. Optimized compounds activate nucleotide exchange at single-digit micromolar concentrations in vitro. In HeLa cells, these quinazolines increase the levels of RAS-GTP and cause signaling changes in the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway.

Discovery of Aminopiperidine Indoles That Activate the Guanine Nucleotide Exchange Factor SOS1 and Modulate RAS Signaling.

Deregulated RAS activity, often the result of mutation, is implicated in approximately 30% of all human cancers. Despite this statistic, no clinically successful treatment for RAS-driven tumors has yet been developed. One approach for modulating RAS activity is to target and affect the activity of proteins that interact with RAS, such as the guanine nucleotide exchange factor (GEF) son of sevenless homologue 1 (SOS1). Here, we report on structure-activity relationships (SAR) in an indole series of compounds. Using structure-based design, we systematically explored substitution patterns on the indole nucleus, the pendant amino acid moiety, and the linker unit that connects these two fragments. Best-in-class compounds activate the nucleotide exchange process at submicromolar concentrations in vitro, increase levels of active RAS-GTP in HeLa cells, and elicit signaling changes in the mitogen-activated protein kinase-extracellular regulated kinase (MAPK-ERK) pathway, resulting in a decrease in pERK1/2T202/Y204 protein levels at higher compound concentrations.

Discovery of Potent 2-Aryl-6,7-dihydro-5 H-pyrrolo[1,2- a]imidazoles as WDR5-WIN-Site Inhibitors Using Fragment-Based Methods and Structure-Based Design.

WDR5 is a chromatin-regulatory scaffold protein overexpressed in various cancers and a potential epigenetic drug target for the treatment of mixed-lineage leukemia. Here, we describe the discovery of potent and selective WDR5-WIN-site inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified several chemically distinct hit series that bind to the WIN site within WDR5. Members of a 6,7-dihydro-5 H-pyrrolo[1,2- a]imidazole fragment class were expanded using a structure-based design approach to arrive at lead compounds with dissociation constants <10 nM and micromolar cellular activity against an AML-leukemia cell line. These compounds represent starting points for the discovery of clinically useful WDR5 inhibitors for the treatment of cancer.

Discovery and Structure-Based Optimization of Benzimidazole-Derived Activators of SOS1-Mediated Nucleotide Exchange on RAS.

Son of sevenless homologue 1 (SOS1) is a guanine nucleotide exchange factor that catalyzes the exchange of GDP for GTP on RAS. In its active form, GTP-bound RAS is responsible for numerous critical cellular processes. Aberrant RAS activity is involved in ∼30% of all human cancers; hence, SOS1 is an attractive therapeutic target for its role in modulating RAS activation. Here, we describe a new series of benzimidazole-derived SOS1 agonists. Using structure-guided design, we discovered small molecules that increase nucleotide exchange on RAS in vitro at submicromolar concentrations, bind to SOS1 with low double-digit nanomolar affinity, rapidly enhance cellular RAS-GTP levels, and invoke biphasic signaling changes in phosphorylation of ERK 1/2. These compounds represent the most potent series of SOS1 agonists reported to date.

Heterologous desensitization of cardiac ß-adrenergic signal via hormone-induced ßAR/arrestin/PDE4 complexes.

AIMS: Cardiac ß-adrenergic receptor (ßAR) signalling is susceptible to heterologous desensitization by different neurohormonal stimuli in clinical conditions associated with heart failure. We aim to examine the underlying mechanism of cross talk between ßARs and a set of G-protein coupled receptors (GPCRs) activated by hormones/agonists. METHODS AND RESULTS: Rat ventricular cardiomyocytes were used to determine heterologous phosphorylation of ßARs under a series of GPCR agonists. Activation of Gs-coupled dopamine receptor, adenosine receptor, relaxin receptor and prostaglandin E2 receptor, and Gq-coupled α1 adrenergic receptor and angiotensin II type 1 receptor promotes phosphorylation of ß1AR and ß2AR at putative protein kinase A (PKA) phosphorylation sites; but activation of Gi-coupled α2 adrenergic receptor and activation of protease-activated receptor does not. The GPCR agonists that promote ß2AR phosphorylation effectively inhibit ßAR agonist isoproterenol-induced PKA phosphorylation of phospholamban and contractile function in ventricular cardiomyocytes. Heterologous GPCR stimuli have minimal to small effect on isoproterenol-induced ß2AR activation and G-protein coupling for cyclic adenosine monophosphate (cAMP) production. However, these GPCR stimuli significantly promote phosphorylation of phosphodiesterase 4D (PDE4D), and recruit PDE4D to the phosphorylated ß2AR in a ß-arrestin 2 dependent manner without promoting ß2AR endocytosis. The increased binding between ß2AR and PDE4D effectively hydrolyzes cAMP signal generated by subsequent stimulation with isoproterenol. Mutation of PKA phosphorylation sites in ß2AR, inhibition of PDE4, or genetic ablation of PDE4D or ß-arrestin 2 abolishes this heterologous inhibitory effect. Ablation of ß-arrestin 2 or PDE4D gene also rescues ß-adrenergic stimuli-induced myocyte contractile function. CONCLUSIONS: These data reveal essential roles of ß-arrestin 2 and PDE4D in a common mechanism for heterologous desensitization of cardiac ßARs under hormonal stimulation, which is associated with impaired cardiac function during the development of pathophysiological conditions.