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Fruit consumption may be beneficial for fighting infection. Although vitamin C is the celebrity component of fruit, its role in COVID-19 is unclear. Because spike S1 of SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) on host cells to enter the cell and initiate COVID-19, using an α-screen–based assay, we screened vitamin C and other components of fruit for inhibiting the interaction between spike S1 and ACE2. We found that prenol, but neither vitamin C nor other major components of fruit (e.g., cyanidin and rutin), reduced the interaction between spike S1 and ACE2. Thermal shift assays indicated that prenol associated with spike S1, but not ACE2, and that vitamin C remained unable to do so. Although prenol inhibited the entry of pseudotyped SARS-CoV-2, but not vesicular stomatitis virus, into human ACE2-expressing HEK293 cells, vitamin C blocked the entry of pseudotyped vesicular stomatitis virus, not SARS-CoV-2, indicating the specificity of the effect. Prenol, but not vitamin C, decreased SARS-CoV-2 spike S1–induced activation of NF-κB and the expression of proinflammatory cytokines in human A549 lung cells. Moreover, prenol also decreased the expression of proinflammatory cytokines induced by spike S1 of N501Y, E484K, Omicron, and Delta variants of SARS-CoV-2. Finally, oral treatment with prenol reduced fever, decreased lung inflammation, enhanced heart function, and improved locomotor activities in SARS-CoV-2 spike S1–intoxicated mice. These results suggest that prenol and prenol-containing fruits, but not vitamin C, may be more beneficial for fighting against COVID-19.

KEGG Pathway Enrichment Analysis of Differential Expressed Genes Between Thymus and T-ALL

KEGG Pathway Enrichment Analysis of Differential Expressed Genes Between CD4 T-Cells and T-ALL

Splicing alterations are common in diseases such as cancer, where mutations in splicing factor genes are frequently responsible for aberrant splicing. Here we present an alternative mechanism for splicing regulation in T-cell acute lymphoblastic leukemia (T-ALL) that involves posttranslational stabilization of the splicing machinery via deubiquitination. We demonstrate there are extensive exon skipping changes in disease, affecting proteasomal subunits, cell-cycle regulators, and the RNA machinery. We present that the serine/arginine-rich splicing factors (SRSF), controlling exon skipping, are critical for leukemia cell survival. The ubiquitin-specific peptidase 7 (USP7) regulates SRSF6 protein levels via active deubiquitination, and USP7 inhibition alters the exon skipping pattern and blocks T-ALL growth. The splicing inhibitor H3B-8800 affects splicing of proteasomal transcripts and proteasome activity and acts synergistically with proteasome inhibitors in inhibiting T-ALL growth. Our study provides the proof-of-principle for regulation of splicing factors via deubiquitination and suggests new therapeutic modalities in T-ALL.Significance:Our study provides a new proof-of-principle for posttranslational regulation of splicing factors independently of mutations in aggressive T-cell leukemia. It further suggests a new drug combination of splicing and proteasomal inhibitors, a concept that might apply to other diseases with or without mutations affecting the splicing machinery.This article is highlighted in the In This Issue feature, p. 1241

Differential Gene Expression and Enrichment Analysis Between Control and shSRSF6-Treated JURKAT Cells

Supplementary Figures S1-S15 and Legends

Alternative Spliced Genes Between HR and NHR T-ALL Patients and Enrichment Analysis

SRSF Family Proteins Essentiality in Tumor Cell Lines

Motif Analysis of Skip Exon Events in T-ALL Compared to CD3 T Cells

CRISP Screens of RNA Binding Proteins in T-ALL

KEGG Enrichment Analysis of Differential Expressed Genes Between Thymus and T-ALL

Cancer progression and recurrence are linked to a rare population of cancer stem cells (CSC). Here, we hypothesized that interactions with the extracellular matrix drive CSC proliferation and tumor-initiating capacity and investigated the functions of scaffold protein tissue transglutaminase (TG2) in ovarian CSC. Complexes formed by TG2, fibronectin (FN), and integrin β1 were enriched in ovarian CSC and detectable in tumors. A function-inhibiting antibody against the TG2 FN-binding domain suppressed complex formation, CSC proliferation as spheroids, tumor-initiating capacity, and stemness-associated Wnt/β-catenin signaling. Disruption of the interaction between TG2 and FN also blocked spheroid formation and the response to Wnt ligands. TG2 and the Wnt receptor Frizzled 7 (Fzd7) form a complex in cancer cells and tumors, leading to Wnt pathway activation. Protein docking and peptide inhibition demonstrate that the interaction between TG2 and Fzd7 overlaps with the FN-binding domain of TG2. These results support a new function of TG2 in ovarian CSC, linked to spheroid proliferation and tumor-initiating capacity and mediated through direct interactions with Fzd7. We propose this complex as a new stem cell target.Significance: These findings reveal a new mechanism by which ovarian CSCs interact with the tumor microenvironment, promoting cell proliferation and tumor initiation. Cancer Res; 78(11); 2990–3001. ©2018 AACR.

The polymerase-associated factor 1 complex (PAF1C) is a key, post-initiation transcriptional regulator of both promoter-proximal pausing and productive elongation catalyzed by RNA Pol II and is also involved in transcriptional repression of viral gene expression during human immunodeficiency virus–1 (HIV-1) latency. Using a molecular docking–based compound screen in silico and global sequencing–based candidate evaluation in vivo, we identified a first-in-class, small-molecule inhibitor of PAF1C (iPAF1C) that disrupts PAF1 chromatin occupancy and induces global release of promoter-proximal paused RNA Pol II into gene bodies. Transcriptomic analysis revealed that iPAF1C treatment mimics acute PAF1 subunit depletion and impairs RNA Pol II pausing at heat shock–down-regulated genes. Furthermore, iPAF1C enhances the activity of diverse HIV-1 latency reversal agents both in cell line latency models and in primary cells from persons living with HIV-1. In sum, this study demonstrates that efficient disruption of PAF1C by a first-in-class, small-molecule inhibitor may have therapeutic potential for improving current HIV-1 latency reversal strategies.

Many patients with coronavirus disease 2019 in intensive care units suffer from cytokine storm. Although anti-inflammatory therapies are available to treat the problem, very often, these treatments cause immunosuppression. Because angiotensin-converting enzyme 2 (ACE2) on host cells serves as the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), to delineate a SARS-CoV-2–specific anti-inflammatory molecule, we designed a hexapeptide corresponding to the spike S1–interacting domain of ACE2 receptor (SPIDAR) that inhibited the expression of proinflammatory molecules in human A549 lung cells induced by pseudotyped SARS-CoV-2, but not vesicular stomatitis virus. Accordingly, wild-type (wt), but not mutated (m), SPIDAR inhibited SARS-CoV-2 spike S1–induced activation of NF-κB and expression of IL-6 and IL-1β in human lung cells. However, wtSPIDAR remained unable to reduce activation of NF-κB and expression of proinflammatory molecules in lungs cells induced by TNF-α, HIV-1 Tat, and viral dsRNA mimic polyinosinic-polycytidylic acid, indicating the specificity of the effect. The wtSPIDAR, but not mutated SPIDAR, also hindered the association between ACE2 and spike S1 of SARS-CoV-2 and inhibited the entry of pseudotyped SARS-CoV-2, but not vesicular stomatitis virus, into human ACE2-expressing human embryonic kidney 293 cells. Moreover, intranasal treatment with wtSPIDAR, but not mutated SPIDAR, inhibited lung activation of NF-κB, protected lungs, reduced fever, improved heart function, and enhanced locomotor activities in SARS-CoV-2 spike S1–intoxicated mice. Therefore, selective targeting of SARS-CoV-2 spike S1-to-ACE2 interaction by wtSPIDAR may be beneficial for coronavirus disease 2019.

Graphic Abstract Spike S1 of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) binds to angiotensin-converting enzyme 2 (ACE2) on host cells to enter the cell and initiate COVID-19. Since ACE2 is a favorable enzyme, we were interested in finding a molecule capable of binding spike S1, but not ACE2, and inhibiting the interaction between spike S1 and ACE2. Holy basil (Tulsi) has a long history as a medicine for different human disorders. Therefore, we screened different components of Tulsi leaf and found that eugenol, but not other major components (e.g. ursolic acid, oleanolic acid and β-caryophylline), inhibited the interaction between spike S1 and ACE2 in an AlphaScreen-based assay. By in silico analysis and thermal shift assay, we also observed that eugenol associated with spike S1, but not ACE2. Accordingly, eugenol strongly suppressed the entry of pseudotyped SARS-CoV-2, but not vesicular stomatitis virus (VSV), into human ACE2-expressing HEK293 cells. Eugenol also reduced SARS-CoV-2 spike S1-induced activation of NF-κB and the expression of IL-6, IL-1β and TNFα in human A549 lung cells. Moreover, oral treatment with eugenol reduced lung inflammation, decreased fever, improved heart function, and enhanced locomotor activities in SARS-CoV-2 spike S1-intoxicated mice. Therefore, selective targeting of SARS-CoV-2 spike S1, but not ACE2, by eugenol may be beneficial for COVID-19 treatment.

Mutation of p53 is the most common genetic alteration in human cancer. The vast majority of p53 mutations found in cancer are missense mutations, with some single nucleotide point mutations leading to the accumulation of mutant p53 protein with potential gain of oncogenic function. The mechanism for stabilization and accumulation of missense mutant p53 protein in malignant cells is not fully understood. It is thought that DNAJA1 plays a crucial role as a co-chaperone protein by stabilizing mutant p53 and amplifying oncogenic potential. As such, identifying small molecule inhibitors to disrupt the protein-protein interaction between mutant p53 and DNAJA1 may lead to an effective treatment for preventing carcinogenesis. Studying protein-protein interactions and identifying potential druggable hotspots has historically been limited-protein-protein binding sites require more complex characterization than those of single proteins and the crystal structures of many proteins have not been identified. Due to these issues, identifying salient druggable targets in protein-protein interactions through bench research may take years to complete. However, in silico modeling approaches allow for rapid characterization of protein-protein interfaces and the druggable binding sites they contain. In this chapter, we first review the oncogenic potential of mutant p53 and the crucial role of DNAJA1 in stabilizing missense mutant p53. We then detail our methodology for using in silico modeling and molecular biology to identify druggable protein-protein interaction sites/pockets between mutant p53 and DNAJA1. Finally, we discuss screening for and validating the utility of a small molecule inhibitor identified through our in silico framework. Specifically, we describe GY1-22, a unique compound with activity against mutant p53 that demonstrates therapeutic potential to inhibit cancer cell growth both in vivo and in vitro.

Telomerase is a ribonuceloprotein complex responsible for maintaining telomeres and protecting chromosomal integrity. The human telomerase reverse transcriptase (hTERT) is expressed in ~90% of cancer cells where it confers the capacity for limitless proliferation. Along with its established role in telomere lengthening, telomerase also serves non-canonical extra-telomeric roles in oncogenic signaling, resistance to apoptosis, and enhanced DNA damage response. We report a new class of natural product-inspired covalent inhibitors of telomerase that target the catalytic active site.

Identifying and evaluating the right target are the most important factors in early drug discovery phase. Most studies focus on one protein ignoring the multiple splice-variant or protein-isoforms, which might contribute to unexpected therapeutic activity or adverse side effects. Here, we present computational analysis of cancer drug-target interactions affected by alternative splicing. By integrating information from publicly available databases, we curated 883 FDA approved or investigational stage small molecule cancer drugs that target 1,434 different genes, with an average of 5.22 protein isoforms per gene. Of these, 618 genes have ≥5 annotated protein-isoforms. By analyzing the interactions with binding pocket information, we found that 76% of drugs either miss a potential target isoform or target other isoforms with varied expression in multiple normal tissues. We present sequence and structure level alignments at isoform-level and make this information publicly available for all the curated drugs. Structure-level analysis showed ligand binding pocket architectures differences in size, shape and electrostatic parameters between isoforms. Our results emphasize how potentially important isoform-level interactions could be missed by solely focusing on the canonical isoform, and suggest that on- and off-target effects at isoform-level should be investigated to enhance the productivity of drug-discovery research.

MEK4, mitogen-activated protein kinase kinase 4, is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. With advances in both computer and biological high-throughput screening, selective chemical entities can be discovered. Structure-based quantitative structure-activity relationship (QSAR) modeling often fails to generate accurate models due to poor alignment of training sets containing highly diverse compounds. Here we describe a highly predictive, nonalignment based robust QSAR model based on a data set of strikingly diverse MEK4 inhibitors. We computed the electrostatic potential (ESP) charges using a density functional theory (DFT) formalism of the donor and acceptor atoms of the ligands and hinge residues. Novel descriptors were then generated from the perturbation of the charge densities of the donor and acceptor atoms and were used to model a diverse set of 84 compounds, from which we built a robust predictive model.

Mnk kinases (Mnk1 and 2) are downstream effectors of Map kinase pathways and regulate phosphorylation of eukaryotic initiation factor 4E (eIF4E). Engagement of the Mnk pathway is critical in acute myeloid leukemia (AML) leukemogenesis and Mnk inhibitors have potent antileukemic properties in vitro and in vivo, suggesting that targeting Mnk kinases may provide a novel approach for treating AML. Here, we report the development and application of a mutation-based induced-fit (M-IFD) in silico screen to identify novel Mnk inhibitors. The Mnk1 structure was modeled by temporarily mutating an amino acid that obstructs the ATP-binding site in the Mnk1 crystal structure while carrying out docking simulations of known inhibitors. The hit compounds display activity in Mnk biochemical and cellular assays, including acute myeloid leukemia progenitors. This approach will enable further rational structure-based drug design of new Mnk inhibitors and potentially novel ways of therapeutically targeting this kinase.

The sigma-2 receptor has been shown to play important roles in a number of important diseases, including central nervous system (CNS) disorders and cancer. However, mechanisms by which sigma-2 contributes to these diseases remain unclear. The development of new sigma-2 ligands that can be used to probe the function of this protein and potentially as drug discovery leads is therefore of great importance. Herein we report the development of a series of tetrahydroindazole compounds that are highly potent and selective for sigma-2. Structure-activity relationship data were used to generate a pharmacophore model that summarizes the common features present in the potent ligands. Assays for solubility and microsomal stability showed that several members of this compound series possess promising characteristics for further development of useful chemical probes or drug discovery leads.

Major depressive disorder is a critical public health problem with a lifetime prevalence of nearly 17% in the United States. One potential therapeutic target is the interaction between hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and an auxiliary subunit of the channel named tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). HCN channels regulate neuronal excitability in the mammalian hippocampus, and recent work has established that antagonizing HCN function rescues cognitive impairment caused by chronic stress. Here, we utilize a high-throughput virtual screen to find small molecules capable of disrupting the TRIP8b-HCN interaction. We found that the hit compound NUCC-0200590 disrupts the TRIP8b-HCN interaction in vitro and in vivo. These results provide a compelling strategy for developing new small molecules capable of disrupting the TRIP8b-HCN interaction.

Graphical Abstract COVID-19 is an infectious respiratory illness caused by the virus strain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and until now, there is no effective therapy against COVID-19. Since SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) for entering into host cells, to target COVID-19 from therapeutic angle, we engineered a hexapeptide corresponding to the ACE2-interacting domain of SARS-CoV-2 (AIDS) that inhibits the association between receptor-binding domain-containing spike S1 and ACE-2. Accordingly, wild type (wt), but not mutated (m), AIDS peptide inhibited SARS-CoV-2 spike S1-induced activation of NF-κB and expression of IL-6 in human lungs cells. Interestingly, intranasal intoxication of C57/BL6 mice with recombinant SARS-CoV-2 spike S1 led to fever, increase in IL-6 in lungs, infiltration of neutrophils into the lungs, arrhythmias, and impairment in locomotor activities, mimicking some of the important symptoms of COVID-19. However, intranasal treatment with wtAIDS, but not mAIDS, peptide reduced fever, protected lungs, improved heart function, and enhanced locomotor activities in SARS-CoV-2 spike S1-intoxicated mice. Therefore, selective targeting of ACE2-to-SARS-CoV-2 interaction by wtAIDS may be beneficial for COVID-19. Supplementary Information The online version contains supplementary material available at 10.1007/s11481-020-09979-8.

The TP53 gene is the most frequently mutated gene in human cancers, and the majority of TP53 mutations are missense mutations. As a result, these mutant p53 (mutp53) either directly lose wildtype p53 (wtp53) tumor suppressor function or exhibit a dominant negative effect over wtp53. In addition, some mutp53 have acquired new oncogenic function (gain of function). Therefore, targeting mutp53 for its degradation may serve as a promising strategy for cancer prevention and therapy. Based on our previous finding that farnesylated DNAJA1 is a crucial chaperone in maintaining mutp53 stabilization, and by using an in silico approach, we built 3D homology models of human DNAJA1 and mutp53R175H proteins, identified the interacting pocket in the DNAJA1-mutp53R175H complex, and found one critical druggable small molecule binding site in the DNAJA1 glycine/phenylalanine-rich region. We confirmed that the interacting pocket in the DNAJA1-mutp53R175H complex was crucial for stabilizing mutp53R175H using a site-directed mutagenesis approach. We further screened a drug-like library to identify a promising small molecule hit (GY1-22) against the interacting pocket in the DNAJA1-mutp53R175H complex. The GY1-22 compound displayed an effective activity against the DNAJA1-mutp53R175H complex. Treatment with GY1-22 significantly reduced mutp53 protein levels, enhanced Waf1p21 expression, suppressed cyclin D1 expression, and inhibited mutp53-driven pancreatic cancer growth both in vitro and in vivo. Together, our results indicate that the interacting pocket in the DNAJA1-mutp53R175H complex is critical for mutp53's stability and oncogenic function, and DNAJA1 is a robust therapeutic target for developing the efficient small molecule inhibitors against oncogenic mutp53.

Critical aspects of maintaining glucose homeostasis in the face of chronic insulin resistance and type 2 diabetes (T2D) are increased insulin secretion and adaptive expansion of beta cell mass. Nutrient and hormone sensing G protein-coupled receptors are important mediators of these properties. A growing body of evidence now suggests that the G protein-coupled receptor, free fatty acid receptor 2 (FFA2), is capable of contributing to the maintenance of glucose homeostasis by acting at the pancreatic beta cell as well as at other metabolically active tissues. We have previously demonstrated that Gαq/11-biased agonism of FFA2 can potentiate glucose stimulated insulin secretion (GSIS) as well as promote beta cell proliferation. However, the currently available Gαq/11-biased agonists for FFA2 exhibit low potency, making them difficult to examine in vivo. This study sought to identify Gαq/11-biased FFA2-selective agonists with potent GSIS-stimulating effects. To do this, we generated an FFA2 homology model that was used to screen a library of 10 million drug-like compounds. Although FFA2 and the related short chain fatty acid receptor FFA3 share 52% sequence similarity, our virtual screen identified over 50 compounds with predicted selectivity and increased potency for FFA2 over FFA3. Subsequent in vitro calcium mobilization assays and GSIS assays resulted in the identification of a compound that can potentiate GSIS via activation of Gαq/11 with 100-fold increased potency compared with previously described Gαq/11-biased FFA2 agonists. These methods and findings provide a foundation for future discovery efforts to identify biased FFA2 agonists as potential T2D therapeutics.

Influenza A virus envelop protein hemagglutinin (HA) plays important roles in viral entry. We previously have reported that MBX2546, a novel influenza A virus inhibitor, binds to HA and inhibits HA-mediated membrane fusion. In this report, we show that (i) both binding and stabilization of HA by MBX2546 are required for the inhibition of viral infection and (ii) the binding of HA by MBX2546 represses the low-pH-induced conformational change in the HA, which is a prerequisite for membrane fusion. Mutations in MBX2546-resistant influenza A/PR/8/34 (H1N1) viruses are mapped in the HA stem region near the amino terminus of HA2. Finally, we have modeled the binding site of MBX2546 using molecular dynamics and find that the resulting structure is in good agreement with our results. Together, these studies underscore the importance of the HA stem loop region as a potential target for therapeutic intervention.

Myeloproliferative neoplasms are a group of hematopoietic malignancies that are characterized with the activated JAK2-STAT5 signaling and uncontrolled myeloid cell proliferation. Current first line therapies remain suboptimal with ongoing risks for thrombosis, hemorrhage, impaired quality of life, and risk of transformation to acute leukemia. More recent FDA-approved JAK inhibitors are associated with poor tolerance due to the key roles of JAK2 in normal hematopoiesis. There are unmet medical needs to develop novel targeted therapies to treat the disease. Pleckstrin-2 (Plek2) is a widely expressed PH domain-containing protein that binds to phosphoinositide with unclear functions. We previously showed that Plek2 is overexpressed in JAK2V617F mutation positive myeloproliferative neoplasms (MPNs). We identified that Plek2 is a downstream effector of the JAK2-STAT5 pathway. Furthermore, through a mouse genetic approach, we discovered that knockout of Plek2 significantly ameliorated the MPN phenotypes and reverted lethality and thrombosis in JAK2V617F knock-in mice. These studies demonstrate that Plek2 is critical for the pathogenesis of MPNs with the activated JAK2-STAT5 pathway, and form a strong foundation for the development of Plek2 inhibitors for the treatment of MPNs. Importantly, our published study shows that Plek2 knockout mice do not develop anemia or cytopenia, indicating Plek2's proto-oncogenic function is mainly manifested in the disease background, which makes Plek2 inhibitors less likely to cause severe adverse effects compared to JAK inhibitor ruxolitinib. Based on these studies, we used an in silico approach to screen for putative Plek2 binding small molecules and identified hit compounds that bind to the DEP domain of Plek2. Further medicinal chemistry studies identified lead compound NUP-17d that inhibited proliferation of the hyperproliferative hematopoietic cells with potency comparable to ruxolitinib. In addition, biochemical assays showed that NUP-17d inhibited Akt phosphorylation. Further mechanistic studies revealed that Plek2 promoted Akt-PtdIns(3,4)P2 binding via direct interaction with Akt and enhanced Akt activation; while NUP-17d inhibited this process. In addition to the biochemical assays, we also demonstrated Plek2's function through a mouse genetic approach using a Pten hematopoietic specific knockout mouse model. Knockout of Plek2 significantly reverted the myeloproliferative phenotype in these mice and markedly extended their survival. Therefore, NUP-17d blocks hematopoietic cell proliferation through the disruption of the Plek2 complex and inhibition of the PI3K-Akt pathway. Indeed, treatment of erythropoietin-induced myeloproliferative mouse model and JAK2V617F knock-in model with NUP-17d significantly ameliorated MPN phenotypes including increase in CBC and splenomegaly. These studies establish Plek2 as a oncoprotein mediating JAK2-STAT and PI3K-Akt signaling pathways in myeloproliferative neoplasms. In addition to hematologic malignancies, Plek2 is also found to be highly upregulated with an associated worse prognosis in many solid tumors (data from oncomine, kmplot). Therefore, targeting Plek2 could have a broad impact in cancer therapy, especially in cancers with upregulated JAK2-STAT or PI3K-Akt pathway. Disclosures No relevant conflicts of interest to declare.

The sigma-1 and sigma-2 receptors have been shown to play important roles in CNS diseases, cancer, and other disorders. These findings suggest that targeting these proteins with small-molecule modulators may be of important therapeutic value. Here we report the development of a new class of tetrahydroindazoles that are highly potent and selective ligands for sigma-1. Molecular modeling was used to rationalize the observed structure-activity relationships and identify key interactions responsible for increased potency of the optimized compounds. Assays for solubility and microsomal stability showed this series possesses favorable characteristics and is amenable to further therapeutic development. The compounds described herein will be useful in the development of new chemical probes for sigma-1 and to aid in future work therapeutically targeting this protein.

Herein we report the discovery of a novel series of highly potent and selective mitogen-activated protein kinase kinase 4 (MEK4) inhibitors. MEK4 is an upstream kinase in MAPK signalling pathways that phosphorylates p38 MAPK and JNK in response to mitogenic and cellular stress queues. MEK4 is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. Optimization of this series via structure activity relationships and molecular modelling led to the identification of compound 6ff (4-(6-fluoro-2H-indazol-3-yl)benzoic acid), a highly potent and selective MEK4 inhibitor. This series of inhibitors is the first of its kind in both activity and selectivity and will be useful in further defining the role of MEK4 in prostate and other cancers.

Peroxisome proliferator-activated receptor-α (PPARα) regulates hepatic fatty acid catabolism and mediates the metabolic response to starvation. Recently we found that PPARα is constitutively activated in nuclei of hippocampal neurons and controls plasticity via direct transcriptional activation of CREB. Here we report the discovery of three endogenous PPARα ligands-3-hydroxy-(2,2)-dimethyl butyrate, hexadecanamide, and 9-octadecenamide-in mouse brain hippocampus. Mass spectrometric detection of these compounds in mouse hippocampal nuclear extracts, in silico interaction studies, time-resolved FRET analyses, and thermal shift assay results clearly indicated that these three compounds served as ligands of PPARα. Site-directed mutagenesis studies further revealed that PPARα Y464 and Y314 are involved in binding these hippocampal ligands. Moreover, these ligands activated PPARα and upregulated the synaptic function of hippocampal neurons. These results highlight the discovery of hippocampal ligands of PPARα capable of modulating synaptic functions.

The viral envelope protein hemagglutinin (HA) plays a critical role in influenza entry and thus is an attractive target for novel therapeutics. The small molecule tert-butylhydroquinone (TBHQ) has previously been shown to bind to HA and inhibit HA-mediated entry with low micromolar potency. However, enthusiasm for the use of TBHQ has diminished due to the compound's antioxidant properties. In this work we show that the antioxidant properties of TBHQ are not responsible for the inhibition of HA-mediated entry. In addition, we have performed a structure-activity relationship (SAR) analysis of TBHQ derivatives. We find that the most promising compound, 3-Tert-butyl-4-methoxyphenol, exhibits enhanced potency (IC50 = 0.6 μM), decreased toxicity (CC50 = 340 μM), and increased stability (t1/2 < 48 h). Finally, we have characterized the binding properties of 3-Tert-butyl-4-methoxyphenol using NMR and molecular dynamics to guide future efforts for chemical optimization.

Ovarian cancer (OC) is the most lethal gynecologic cancer, characterized by chemoresistance and fatal tumor recurrence after primary treatment. The metastatic progression and post-chemotherapy recurrence of OC is linked to a small population of cancer stem cells (CSCs) detectable as ALDH+/CD133+ phenotype. In OC, the tumor microenvironment (TME) provides a favorable milieu that protects quiescent CSCs during chemotherapy and supports their tumorigenic functions. The multifunctional protein tissue transglutaminase (TG2), with enzymatic and scaffold functions, is an important molecule secreted in the TME where it modulates oncogenic signaling by interacting with extracellular matrix (ECM) components, such as fibronectin (FN) and integrins. Its aberrant expression is correlated with OC progression and with the tumorigenic OCSC phenotype. Here, we hypothesized that the formation of the TG2/FN/integrins ternary complex at the cell membrane promotes survival of CSCs and supports OC spheroid formation by regulating the Wnt/beta-catenin pathway. Our data demonstrate that TG2, FN, and integrin β1 mRNA expression is markedly increased in OCSCs (ALDH+/CD133+) compared with non-CSC (ALDH-/CD133-) and highly enriched in OC cells grown as spheroids compared with monolayers (p Citation Format: Salvatore Condello, Livia Sima, Cristina Ivan, Horacio Cardenas, Gary Schiltz, Rama Mishra, Daniela Matei. Tissue transglutaminase/Frizzled receptor clusters regulate WNT transcriptional activity in ovarian cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B56.

Here we present a virtual docking screen of 1648 commercially available covalent fragments, and identified covalent inhibitors of cysteine protease cathepsin L. These inhibitors did not inhibit closely related protease cathepsin B. Thus, we have established virtual docking of covalent fragments as an approach to discover covalent enzyme inhibitors.

The super elongation complex (SEC) is required for robust and productive transcription through release of RNA polymerase II (Pol II) with its P-TEFb module and promoting transcriptional processivity with its ELL2 subunit. Malfunction of SEC contributes to multiple human diseases including cancer. Here, we identify peptidomimetic lead compounds, KL-1 and its structural homolog KL-2, which disrupt the interaction between the SEC scaffolding protein AFF4 and P-TEFb, resulting in impaired release of Pol II from promoter-proximal pause sites and a reduced average rate of processive transcription elongation. SEC is required for induction of heat-shock genes and treating cells with KL-1 and KL-2 attenuates the heat-shock response from Drosophila to human. SEC inhibition downregulates MYC and MYC-dependent transcriptional programs in mammalian cells and delays tumor progression in a mouse xenograft model of MYC-driven cancer, indicating that small-molecule disruptors of SEC could be used for targeted therapy of MYC-induced cancer.

Cancer progression and recurrence are linked to a rare population of cancer stem cells (CSC). Here, we hypothesized that interactions with the extracellular matrix drive CSC proliferation and tumor-initiating capacity and investigated the functions of scaffold protein tissue transglutaminase (TG2) in ovarian CSC. Complexes formed by TG2, fibronectin (FN), and integrin β1 were enriched in ovarian CSC and detectable in tumors. A function-inhibiting antibody against the TG2 FN-binding domain suppressed complex formation, CSC proliferation as spheroids, tumor-initiating capacity, and stemness-associated Wnt/β-catenin signaling. Disruption of the interaction between TG2 and FN also blocked spheroid formation and the response to Wnt ligands. TG2 and the Wnt receptor Frizzled 7 (Fzd7) form a complex in cancer cells and tumors, leading to Wnt pathway activation. Protein docking and peptide inhibition demonstrate that the interaction between TG2 and Fzd7 overlaps with the FN-binding domain of TG2. These results support a new function of TG2 in ovarian CSC, linked to spheroid proliferation and tumor-initiating capacity and mediated through direct interactions with Fzd7. We propose this complex as a new stem cell target. Significance: These findings reveal a new mechanism by which ovarian CSCs interact with the tumor microenvironment, promoting cell proliferation and tumor initiation. Cancer Res; 78(11); 2990–3001. ©2018 AACR.

In this study, the role of highly structured water molecules present in the active site of the leukotriene A4 hydrolase (LTA4H) enzyme has been critically examined through the docking experiments. It was observed that different experiments were necessary to perform the docking studies. The ligands capable of displacing or interacting with bound-water(s) displayed different binding poses as well as the scores. The docking scores E (CvdW) from Glide and ChemScore (CS) from FlexX, with and without bound-waters, obtained through different docking experiments were used to construct two structure guided bi-parametric linear regression models using the IC50 enzyme activity data. The predictive squared correlation coefficients (Q(2)) obtained for these models, with and without bound waters, were found to be 0.73 and 0.67, respectively. These models were validated using test and validated sets of compounds. Utilizing these QSAR models, 409 proposed structures were docked and their respective predicted IC50 data were generated. From the in-silico evaluation of these 409 proposed structures representing diverse chemotypes, 39 compounds were triaged, synthesized and evaluated in the enzyme inhibition assay. The predicted and experimental biological data (IC50) was correlated and the square of the correlation coefficient (R(2)) between the observed and calculated IC50 was found to be 0.87.

Tumor cells rely on elevated glucose consumption and metabolism for survival and proliferation. Glucose transporters mediating glucose entry are key proximal rate-limiting checkpoints. Unlike GLUT1 that is highly expressed in cancer and more ubiquitously expressed in normal tissues, GLUT4 exhibits more limited normal expression profiles. We have previously determined that insulin-responsive GLUT4 is constitutively localized on the plasma membrane of myeloma cells. Consequently, suppression of GLUT4 or inhibition of glucose transport with the HIV protease inhibitor ritonavir elicited growth arrest and/or apoptosis in multiple myeloma. GLUT4 inhibition also caused sensitization to metformin in multiple myeloma and chronic lymphocytic leukemia and a number of solid tumors suggesting the broader therapeutic utility of targeting GLUT4. This study sought to identify selective inhibitors of GLUT4 to develop a more potent cancer chemotherapeutic with fewer potential off-target effects. Recently, the crystal structure of GLUT1 in an inward open conformation was reported. Although this is an important achievement, a full understanding of the structural biology of facilitative glucose transport remains elusive. To date, there is no three-dimensional structure for GLUT4. We have generated a homology model for GLUT4 that we utilized to screen for drug-like compounds from a library of 18 million compounds. Despite 68% homology between GLUT1 and GLUT4, our virtual screen identified two potent compounds that were shown to target GLUT4 preferentially over GLUT1 and block glucose transport. Our results strongly bolster the utility of developing GLUT4-selective inhibitors as anti-cancer therapeutics.

Cancer cells consume more glucose to fuel metabolic programs fundamental to sustaining their survival, growth and proliferation. Among the fourteen SLC2A family members, GLUTs 1 and 4 are high-affinity glucose transporters. GLUT4 (SLC2A4) is highly expressed in muscle and adipose tissue. Basally retained within the cell, GLUT4 traffics to the plasma membrane (PM) in response to insulin and exercise-stimulation. The plasma cell malignancy multiple myeloma (MM) exhibits increased constitutive expression of GLUT4 on the PM, co-opting use of GLUT4 for survival and proliferation. GLUT4 inhibition by knockdown or treatment with the FDA-approved HIV protease inhibitor ritonavir leads to cytostatic and/or cytotoxic and chemosensitizing effects in tumor cells both in vitro and in vivo. We recently reported our generation of GLUT4 homology models and virtual high-throughput screening (vHTS) to identify multiple series of novel GLUT4 antagonists. In this report, we describe our initial hit-to-lead optimization to synthesize new analogs with improved potency and selectivity for GLUT4, and the biological characterization of these compounds in a variety of assays. We show that our lead compound (compound 20) decreases glucose uptake and cell proliferation as well as inhibits the expression of pro-survival MCL-1 in MM similar to the effect observed via knockdown of GLUT4 expression. Compound 20 is also effective at chemosensitizing multiple myeloma cell lines and patient samples to venetoclax, dexamethasone and melphalan. In sum, we report development of selective GLUT4 inhibitors lacking inhibitory activity against GLUT1 and GLUT8. We show that selective pharmacological inhibition of GLUT4 is feasible and this may represent a novel strategy for the treatment and chemosensitization of multiple myeloma to standard therapeutics.

Induction of TLR2 activation depends on its association with the adapter protein MyD88. We have found that TLR2 and MyD88 levels are elevated in the hippocampus and cortex of patients with Alzheimer's disease (AD) and in a 5XFAD mouse model of AD. Since there is no specific inhibitor of TLR2, to target induced TLR2 from a therapeutic angle, we engineered a peptide corresponding to the TLR2-interacting domain of MyD88 (TIDM) that binds to the BB loop of only TLR2, and not other TLRs. Interestingly, WT TIDM peptide inhibited microglial activation induced by fibrillar Aβ1-42 and lipoteichoic acid, but not 1-methyl-4-phenylpyridinium, dsRNA, bacterial lipopolysaccharide, flagellin, or CpG DNA. After intranasal administration, WT TIDM peptide reached the hippocampus, reduced hippocampal glial activation, lowered Aβ burden, attenuated neuronal apoptosis, and improved memory and learning in 5XFAD mice. However, WT TIDM peptide was not effective in 5XFAD mice lacking TLR2. In addition to its effects in 5XFAD mice, WT TIDM peptide also suppressed the disease process in mice with experimental allergic encephalomyelitis and collagen-induced arthritis. Therefore, selective targeting of the activated status of 1 component of the innate immune system by WT TIDM peptide may be beneficial in AD as well as other disorders in which TLR2/MyD88 signaling plays a role in disease pathogenesis.

Oncogenic mutations in two isocitrate dehydrogenase (IDH)-encoding genes (IDH1 and IDH2) have been identified in acute myelogenous leukemia, low-grade glioma, and secondary glioblastoma (GBM). Our in silico and wet-bench analyses indicate that non-mutated IDH1 mRNA and protein are commonly overexpressed in primary GBMs. We show that genetic and pharmacologic inactivation of IDH1 decreases GBM cell growth, promotes a more differentiated tumor cell state, increases apoptosis in response to targeted therapies, and prolongs the survival of animal subjects bearing patient-derived xenografts (PDXs). On a molecular level, diminished IDH1 activity results in reduced α-ketoglutarate (αKG) and NADPH production, paralleled by deficient carbon flux from glucose or acetate into lipids, exhaustion of reduced glutathione, increased levels of reactive oxygen species (ROS), and enhanced histone methylation and differentiation marker expression. These findings suggest that IDH1 upregulation represents a common metabolic adaptation by GBMs to support macromolecular synthesis, aggressive growth, and therapy resistance.

The NHC-catalyzed transformation of unsaturated aldehydes into saturated esters through an organocatalytic homoenolate process has been thoroughly studied. Leveraging a unique "Umpolung"-mediated β-protonation, this process has evolved from a test bed for homoenolate reactivity to a broader platform for asymmetric catalysis. Inspired by our success in using the β-protonation process to generate enals from ynals with good E/Z selectivity, our early studies found that an asymmetric variation of this reaction was not only feasible, but also adaptable to a kinetic resolution of secondary alcohols through NHC-catalyzed acylation. In-depth analysis of this process determined that careful catalyst and solvent pairing is critical for optimal yield and selectivity; proper choice of nonpolar solvent provided improved yield through suppression of an oxidative side reaction, while employment of a cooperative catalytic approach through inclusion of a hydrogen bond donor cocatalyst significantly improved enantioselectivity.

Influenza outbreaks, particularly the pandemic 1918 H1 and avian H5 strains, are of high concern to public health. The hemagglutinin envelope protein of influenza plays a critical role in viral entry and thus is an attractive target for inhibition of virus entry. The highly conserved stem loop region of hemagglutinin has been shown to undergo critically important conformational changes during the entry process and, moreover, to be a site for inhibition of virus entry by antibodies, small proteins, and small drug-like molecules. In this work we probe the structure-function properties of the H5 hemagglutinin stem loop region by site-directed mutagenesis. We find that most mutations do not disrupt expression, proteolytic processing, incorporation into virus, or receptor binding; however, many of the mutations disrupt the entry process. We further assess the effects of mutations on inhibition of entry by a neutralizing monoclonal antibody (C179) and find examples of increased and decreased sensitivity to the antibody, consistent with the antibody binding site observed by x-ray crystallography. In addition, we tested the sensitivity of the mutants to MBX2329, a small molecule inhibitor of influenza entry. Interestingly, the mutants exhibit increased and decreased sensitivities to MBX2329, which gives further insight into the binding site of the compound on HA and potential mechanisms of escape. Finally, we have modeled the binding site of MBX2329 using molecular dynamics and find that the resulting structure is in good agreement with the mutagenesis results. Together these studies underscore the importance of the stem loop region to HA function and suggest potential sites for therapeutic intervention of influenza entry.

Summary Small molecules that directly target MYC and are also well tolerated in vivo will provide invaluable chemical probes and potential anti-cancer therapeutic agents. We developed a series of small-molecule MYC inhibitors that engage MYC inside cells, disrupt MYC/MAX dimers, and impair MYC-driven gene expression. The compounds enhance MYC phosphorylation on threonine-58, consequently increasing proteasome-mediated MYC degradation. The initial lead, MYC inhibitor 361 (MYCi361), suppressed in vivo tumor growth in mice, increased tumor immune cell infiltration, upregulated PD-L1 on tumors, and sensitized tumors to anti-PD1 immunotherapy. However, 361 demonstrated a narrow therapeutic index. An improved analog, MYCi975 showed better tolerability. These findings suggest the potential of small-molecule MYC inhibitors as chemical probes and possible anti-cancer therapeutic agents.