Prenol, but Not Vitamin C, of Fruit Binds to SARS-CoV-2 Spike S1 to Inhibit Viral Entry: Implications for COVID-19.

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.

Enhancing HIV-1 latency reversal through regulating the elongating RNA Pol II pause-release by a small-molecule disruptor of PAF1C.

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.

Targeting pleckstrin-2/Akt signaling reduces proliferation in myeloproliferative neoplasm models.

Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2/STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2/STAT5 pathway and is overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2-mutated MPNs. The nonessential roles of Plek2 under physiologic conditions make it an ideal target for MPN therapy. Here, we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening approach and cell-based assays, followed by the synthesis of analogs. Plek2-specific small-molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2-signaling complex also includes Hsp72, which protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to the Plek2 dishevelled, Egl-10 and pleckstrin (DEP) domain. The role of Plek2 in activating Akt signaling was further confirmed in vivo using a hematopoietic-specific Pten-knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to that seen with the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34-positive cells from MPN patients. Our studies reveal a Plek2/Akt complex that drives cell proliferation and can be targeted by a class of antiproliferative compounds for MPN therapy.

Druggable sites/pockets of the p53-DNAJA1 protein-protein interaction: In silico modeling and in vitro/in vivo validation.

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.

Discovery of a small-molecule inhibitor of the TRIP8b-HCN interaction with efficacy in neurons.

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.

Selective Inhibition of the Interaction between SARS-CoV-2 Spike S1 and ACE2 by SPIDAR Peptide Induces Anti-Inflammatory Therapeutic Responses.

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-1ß 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.

Eugenol, a Component of Holy Basil (Tulsi) and Common Spice Clove, Inhibits the Interaction Between SARS-CoV-2 Spike S1 and ACE2 to Induce Therapeutic Responses.

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 ß-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-1ß 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.

ACE-2-interacting Domain of SARS-CoV-2 (AIDS) Peptide Suppresses Inflammation to Reduce Fever and Protect Lungs and Heart in Mice: Implications for COVID-19 Therapy.

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.

Identification of a druggable protein-protein interaction site between mutant p53 and its stabilizing chaperone DNAJA1.

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.

The HECT E3 Ligase E6AP/UBE3A as a Therapeutic Target in Cancer and Neurological Disorders.

The HECT (Homologous to the E6-AP Carboxyl Terminus)-family protein E6AP (E6-associated protein), encoded by the UBE3A gene, is a multifaceted ubiquitin ligase that controls diverse signaling pathways involved in cancer and neurological disorders. The oncogenic role of E6AP in papillomavirus-induced cancers is well known, with its action to trigger p53 degradation in complex with the E6 viral oncoprotein. However, the roles of E6AP in non-viral cancers remain poorly defined. It is well established that loss-of-function alterations of the UBE3A gene cause Angelman syndrome, a severe neurodevelopmental disorder with autosomal dominant inheritance modified by genomic imprinting on chromosome 15q. Moreover, excess dosage of the UBE3A gene markedly increases the penetrance of autism spectrum disorders, suggesting that the expression level of UBE3A must be regulated tightly within a physiologically tolerated range during brain development. In this review, current the knowledge about the substrates of E6AP-mediated ubiquitination and their functions in cancer and neurological disorders is discussed, alongside with the ongoing efforts to pharmacologically modulate this ubiquitin ligase as a promising therapeutic target.

Posttranslational Regulation of the Exon Skipping Machinery Controls Aberrant Splicing in Leukemia.

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.

Targeted Covalent Inhibition of Telomerase.

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 noncanonical 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.

In silico analysis of alternative splicing on drug-target gene interactions.

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.

Small-Molecule MYC Inhibitors Suppress Tumor Growth and Enhance Immunotherapy.

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.

Modeling MEK4 Kinase Inhibitors through Perturbed Electrostatic Potential Charges.

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.

Discovery of novel Mnk inhibitors using mutation-based induced-fit virtual high-throughput screening.

Mnk kinases (Mnk1 and 2) are downstream effectors of Map kinase pathways and regulate phosphorylation of eukaryotic initiation factor 4E. 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 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.

Development of Tetrahydroindazole-Based Potent and Selective Sigma-2 Receptor Ligands.

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.

Discovery of a novel class of potent and selective tetrahydroindazole-based sigma-1 receptor ligands.

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.

Synthesis and Biological Evaluation of 3-Arylindazoles as Selective MEK4 Inhibitors.

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 signaling 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 modeling led to the identification of compound 6 ff (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.