Targeting Processive Transcription Elongation via SEC Disruption for MYC-Induced Cancer Therapy.

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.

Identification of regulators of polyploidization presents therapeutic targets for treatment of AMKL.

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.

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.

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.

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.

Discovery of covalent enzyme inhibitors using virtual docking of covalent fragments.

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.

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 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.

Identification and characterization of PPARα ligands in the hippocampus.

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.

Catalytic, Enantioselective ß-Protonation through a Cooperative Activation Strategy.

The NHC-catalyzed transformation of unsaturated aldehydes into saturated esters through an organocatalytic homoenolate process has been thoroughly studied. Leveraging a unique "Umpolung"-mediated ß-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 ß-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.

In Silico Modeling-based Identification of Glucose Transporter 4 (GLUT4)-selective Inhibitors for Cancer Therapy.

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.

Mutagenesis studies of the H5 influenza hemagglutinin stem loop region.

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.

Enantioselective ß-Protonation by a Cooperative Catalysis Strategy.

An enantioselective N-heterocyclic carbene (NHC)-catalyzed ß-protonation through the orchestration of three distinct organocatalysts has been developed. This cooperative catalyst system enhances both yield and selectivity, compared to only the NHC-catalyzed process. This new method allows for the efficient conversion of a large scope of aryl-oxobutenoates to highly enantioenriched succinate derivatives and demonstrates the benefits of combining different activation modes in organocatalysis.

Enantioselective annulations for dihydroquinolones by in situ generation of azolium enolates.

A convergent, catalytic asymmetric formal [4 + 2] annulation for the synthesis of dihydroquinolones has been developed. Carboxylic acids can be employed as precursors to NHC enolates through an in situ activation strategy. Simultaneous generation of a reactive aza-o-quinone methide under the basic conditions employed for NHC generation leads to a dual activation approach.

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.

Knot theory in understanding proteins.

This paper aims to enthuse mathematicians, especially topologists, knot theorists and geometers to examine problems in the study of proteins. We have highlighted those advances and breakthroughs in knot theory that directly and indirectly help in understanding proteins. We have discussed the phenomena of knotting of protein backbone. This paper also provides a few open questions for knot theorists, the answers to which will help in further understanding of proteins.

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.