A small-molecule inhibitor of TopBP1 exerts anti-MYC activity and synergy with PARP inhibitors.

We have previously identified TopBP1 (topoisomerase IIß-binding protein 1) as a promising target for cancer therapy, given its role in the convergence of Rb, PI(3)K/Akt, and p53 pathways. Based on this, we conducted a large-scale molecular docking screening to identify a small-molecule inhibitor that specifically targets the BRCT7/8 domains of TopBP1, which we have named 5D4. Our studies show that 5D4 inhibits TopBP1 interactions with E2F1, mutant p53, and Cancerous Inhibitor of Protein Phosphatase 2A. This leads to the activation of E2F1-mediated apoptosis and the inhibition of mutant p53 gain of function. In addition, 5D4 disrupts the interaction of TopBP1 with MIZ1, which in turn allows MIZ1 to bind to its target gene promoters and repress MYC activity. Moreover, 5D4 inhibits the association of the TopBP1-PLK1 complex and prevents the formation of Rad51 foci. When combined with inhibitors of PARP1/2 or PARP14, 5D4 synergizes to effectively block cancer cell proliferation. Our animal studies have demonstrated the antitumor activity of 5D4 in breast and ovarian cancer xenograft models. Moreover, the effectiveness of 5D4 is further enhanced when combined with a PARP1/2 inhibitor talazoparib. Taken together, our findings strongly support the potential use of TopBP1-BRCT7/8 inhibitors as a targeted cancer therapy.

Integrated Network Analysis to Determine CNN1, MYL9, TAGLN, and SORBS1 as Potential Key Genes Associated with Prostate Cancer.

BACKGROUND: Prostate cancer (PCa) is challenging to treat. It is necessary to screen for related biological markers to accurately predict the prognosis and recurrence of prostate cancer. METHODS: Three data sets, GSE28204, GSE30521, and GSE69223, from the Gene Expression Omnibus (GEO) database were integrated into this study. After the identification of differentially expressed genes (DEGs) between PCa and normal prostate tissues, network analyses including protein-protein interaction (PPI) network, and weighted gene co-expression network analysis (WGCNA) were used to select hub genes. Gene Ontology (GO) term analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to annotate the functions of DEGs and hub modules of the networks. Survival analysis was performed to validate the correlation between the key genes and PCa relapse. RESULTS: In total, 867 DEGs were identified, including 201 upregulated and 666 downregulated genes. Three hub modules of the PPI network and one hub module of the weighted gene co-expression network were determined. Moreover, four key genes (CNN1, MYL9, TAGLN, and SORBS1) were significantly associated with PCa relapse (p < 0.05). CONCLUSIONS: CNN1, MYL9, TAGLN, and SORBS1 may be potential biomarkers for PCa development.

A steroid receptor coactivator stimulator (MCB-613) attenuates adverse remodeling after myocardial infarction.

Progressive remodeling of the heart, resulting in cardiomyocyte (CM) loss and increased inflammation, fibrosis, and a progressive decrease in cardiac function, are hallmarks of myocardial infarction (MI)-induced heart failure. We show that MCB-613, a potent small molecule stimulator of steroid receptor coactivators (SRCs) attenuates pathological remodeling post-MI. MCB-613 decreases infarct size, apoptosis, hypertrophy, and fibrosis while maintaining significant cardiac function. MCB-613, when given within hours post MI, induces lasting protection from adverse remodeling concomitant with: 1) inhibition of macrophage inflammatory signaling and interleukin 1 (IL-1) signaling, which attenuates the acute inflammatory response, 2) attenuation of fibroblast differentiation, and 3) promotion of Tsc22d3-expressing macrophages-all of which may limit inflammatory damage. SRC stimulation with MCB-613 (and derivatives) is a potential therapeutic approach for inhibiting cardiac dysfunction after MI.

Discovery, X-ray Crystallography and Antiviral Activity of Allosteric Inhibitors of Flavivirus NS2B-NS3 Protease.

Flaviviruses, including dengue, West Nile and recently emerged Zika virus, are important human pathogens, but there are no drugs to prevent or treat these viral infections. The highly conserved Flavivirus NS2B-NS3 protease is essential for viral replication and therefore a drug target. Compound screening followed by medicinal chemistry yielded a series of drug-like, broadly active inhibitors of Flavivirus proteases with IC50 as low as 120 nM. The inhibitor exhibited significant antiviral activities in cells (EC68: 300-600 nM) and in a mouse model of Zika virus infection. X-ray studies reveal that the inhibitors bind to an allosteric, mostly hydrophobic pocket of dengue NS3 and hold the protease in an open, catalytically inactive conformation. The inhibitors and their binding structures would be useful for rational drug development targeting Zika, dengue and other Flaviviruses.

DOT1L as a therapeutic target for the treatment of DNMT3A-mutant acute myeloid leukemia.

Mutations in DNA methyltransferase 3A (DNMT3A) are common in acute myeloid leukemia and portend a poor prognosis; thus, new therapeutic strategies are needed. The likely mechanism by which DNMT3A loss contributes to leukemogenesis is altered DNA methylation and the attendant gene expression changes; however, our current understanding is incomplete. We observed that murine hematopoietic stem cells (HSCs) in which Dnmt3a had been conditionally deleted markedly overexpress the histone 3 lysine 79 (H3K79) methyltransferase, Dot1l. We demonstrate that Dnmt3a(-/-) HSCs have increased H3K79 methylation relative to wild-type (WT) HSCs, with the greatest increases noted at DNA methylation canyons, which are regions highly enriched for genes dysregulated in leukemia and prone to DNA methylation loss with Dnmt3a deletion. These findings led us to explore DOT1L as a therapeutic target for the treatment of DNMT3A-mutant AML. We show that pharmacologic inhibition of DOT1L resulted in decreased expression of oncogenic canyon-associated genes and led to dose- and time-dependent inhibition of proliferation, induction of apoptosis, cell-cycle arrest, and terminal differentiation in DNMT3A-mutant cell lines in vitro. We show in vivo efficacy of the DOT1L inhibitor EPZ5676 in a nude rat xenograft model of DNMT3A-mutant AML. DOT1L inhibition was also effective against primary patient DNMT3A-mutant AML samples, reducing colony-forming capacity (CFC) and inducing terminal differentiation in vitro. These studies suggest that DOT1L may play a critical role in DNMT3A-mutant leukemia. With pharmacologic inhibitors of DOT1L already in clinical trials, DOT1L could be an immediately actionable therapeutic target for the treatment of this poor prognosis disease.

GATA2 facilitates steroid receptor coactivator recruitment to the androgen receptor complex.

The androgen receptor (AR) is a key driver of prostate cancer (PC), even in the state of castration-resistant PC (CRPC) and frequently even after treatment with second-line hormonal therapies such as abiraterone and enzalutamide. The persistence of AR activity via both ligand-dependent and ligand-independent mechanisms (including constitutively active AR splice variants) highlights the unmet need for alternative approaches to block AR signaling in CRPC. We investigated the transcription factor GATA-binding protein 2 (GATA2) as a regulator of AR signaling and an actionable therapeutic target in PC. We demonstrate that GATA2 directly promotes expression of both full-length and splice-variant AR, resulting in a strong positive correlation between GATA2 and AR expression in both PC cell lines and patient specimens. Conversely, GATA2 expression is repressed by androgen and AR, suggesting a negative feedback regulatory loop that, upon androgen deprivation, derepresses GATA2 to contribute to AR overexpression in CRPC. Simultaneously, GATA2 is necessary for optimal transcriptional activity of both full-length and splice-variant AR. GATA2 colocalizes with AR and Forkhead box protein A1 on chromatin to enhance recruitment of steroid receptor coactivators and formation of the transcriptional holocomplex. In agreement with these important functions, high GATA2 expression and transcriptional activity predicted worse clinical outcome in PC patients. A GATA2 small molecule inhibitor suppressed the expression and transcriptional function of both full-length and splice-variant AR and exerted potent anticancer activity against PC cell lines. We propose pharmacological inhibition of GATA2 as a first-in-field approach to target AR expression and function and improve outcomes in CRPC.

Structural basis of substrate specificity and protease inhibition in Norwalk virus.

Norwalk virus (NV), the prototype human calicivirus, is the leading cause of nonbacterial acute gastroenteritis. The NV protease cleaves the polyprotein encoded by open reading frame 1 of the viral genome at five nonhomologous sites, releasing six nonstructural proteins that are essential for viral replication. The structural details of how NV protease recognizes multiple substrates are unclear. In our X-ray structure of an NV protease construct, we observed that the C-terminal tail, representing the native substrate positions P5 to P1, is inserted into the active site cleft of the neighboring protease molecule, providing atomic details of how NV protease recognizes a substrate. The crystallographic structure of NV protease with the C-terminal tail redesigned to mimic P4 to P1 of another substrate site provided further structural details on how the active site accommodates sequence variations in the substrates. Based on these structural analyses, substrate-based aldehyde inhibitors were synthesized and screened for inhibition potency. Crystallographic structures of the protease in complex with each of the three most potent inhibitors were determined. These structures showed concerted conformational changes in the S4 and S2 pockets of the protease to accommodate variations in the P4 and P2 residues of the substrate/inhibitor, which could be a mechanism for how the NV protease recognizes multiple sites in the polyprotein with differential affinities during virus replication. These structures further indicate that the mechanism of inhibition by these inhibitors involves covalent bond formation with the side chain of the conserved cysteine in the active site by nucleophilic addition, and such substrate-based aldehydes could be effective protease inhibitors.

Regioselective radical aminofluorination of styrenes.

The copper-catalyzed radical aminofluorination of styrenes with N-fluorobenzenesulfonimide (NFSI) is realized with high regioselectivity, thus affording aminofluorination products with regioselectivities opposite that of the palladium-catalyzed and noncatalyzed processes. Preliminary mechanistic studies suggested the reaction went through a radical pathway and was supported by DFT calculations. In these reactions, NFSI is utilized as both a radical nitrogen source and radical fluorine source, thus rendering it an attractive reagent.

Targeting SARS-CoV-2 nonstructural protein 3: Function, structure, inhibition, and perspective in drug discovery.

As a highly contagious human pathogen, severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) has infected billions of people worldwide with more than 6 million deaths. With several effective vaccines and antiviral drugs now available, the SARS-CoV-2 pandemic been brought under control. However, a new pathogenic coronavirus could emerge in the future, given the zoonotic nature of this virus. Natural evolution and drug-induced mutations of SARS-CoV-2 also require continued efforts for new anti-coronavirus drugs. Nonstructural protein (nsp) 3 of CoVs is a large, multifunctional protein, containing a papain-like protease (PLpro) and a macrodomain (Mac1), which are essential for viral replication. Here, we provide a comprehensive review of the function, structure, and inhibition of SARS-CoV/-CoV-2 PLpro and Mac1. We also discuss advances in, and challenges to, the discovery of drugs against these targets.

Design, Synthesis, X-ray Crystallography, and Biological Activities of Covalent, Non-Peptidic Inhibitors of SARS-CoV-2 Main Protease.

Highly contagious SARS-CoV-2 coronavirus has infected billions of people worldwide with flu-like symptoms since its emergence in 2019. It has caused deaths of several million people. The viral main protease (Mpro) is essential for SARS-CoV-2 replication and therefore a drug target. Several series of covalent inhibitors of Mpro were designed and synthesized. Structure-activity relationship studies show that (1) several chloroacetamide- and epoxide-based compounds targeting Cys145 are potent inhibitors with IC50 values as low as 0.49 µM and (2) Cys44 of Mpro is not nucleophilic for covalent inhibitor design. High-resolution X-ray studies revealed the protein-inhibitor interactions and mechanisms of inhibition. It is of interest that Cys145 preferably attacks the more hindered Cα atom of several epoxide inhibitors. Chloroacetamide inhibitor 13 and epoxide inhibitor 30 were found to inhibit cellular SARS-CoV-2 replication with an EC68 (half-log reduction of virus titer) of 3 and 5 µM. These compounds represent new pharmacological leads for anti-SARS-CoV-2 drug development.

Expression, characterization and inhibition of Toxoplasma gondii 1-deoxy-D-xylulose-5-phosphate reductoisomerase.

The apicomplexan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, is an important human pathogen. 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) in the non-mevalonate isoprene biosynthesis pathway is essential to the organism and therefore a target for developing anti-toxoplasmosis drugs. In order to find potent inhibitors, we expressed and purified recombinant T. gondii DXR (TgDXR). Biochemical properties of this enzyme were characterized and an enzyme activity/inhibition assay was developed. A collection of 11 compounds with a broad structural diversity were tested against TgDXR and several potent inhibitors were identified with Ki values as low as 48 nM. Analysis of the results as well as those of Escherichia coli and Plasmodium falciparum DXR enzymes revealed a different structure-activity relationship profile for the inhibition of TgDXR.

Structure and function of SARS-CoV and SARS-CoV-2 main proteases and their inhibition: A comprehensive review.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) identified in 2003 infected ∼8000 people in 26 countries with 800 deaths, which was soon contained and eradicated by syndromic surveillance and enhanced quarantine. A closely related coronavirus SARS-CoV-2, the causative agent of COVID-19 identified in 2019, has been dramatically more contagious and catastrophic. It has infected and caused various flu-like symptoms of billions of people in >200 countries, including >6 million people died of or with the virus. Despite the availability of several vaccines and antiviral drugs against SARS-CoV-2, finding new therapeutics is needed because of viral evolution and a possible emerging coronavirus in the future. The main protease (Mpro) of these coronaviruses plays important roles in their life cycle and is essential for the viral replication. This article represents a comprehensive review of the function, structure and inhibition of SARS-CoV and -CoV-2 Mpro, including structure-activity relationships, protein-inhibitor interactions and clinical trial status.

Quinazolinone Compounds Have Potent Antiviral Activity against Zika and Dengue Virus.

Dengue (DENV) and Zika (ZIKV) viruses are important human pathogens, causing ∼100 million symptomatic infections each year. These infections carry a 20-fold increased incidence of serious neurological diseases, such as microcephaly in newborns (for ZIKV) and Guillain-Barré syndrome. Moreover, DENV can develop serious and possibly life-threatening dengue hemorrhagic fever in certain patients. Patients recovered from one of the four serotypes of DENV are still susceptible to other serotypes with a higher likelihood of serious disease because of antibody-dependent enhancement. Except for mosquito control, there have been no antiviral drugs to prevent and treat ZIKV/DENV infections. Phenotypic screening found that 2,3,6-trisubstituted quinazolinone compounds are novel inhibitors of ZIKV replication. Fifty-four analogues were synthesized, and their structure-activity relationships are discussed. Additional testing shows that compounds 22, 27, and 47 exhibited broad and potent activities against ZIKV and DENV with EC50 values as low as 86 nM with no significant cytotoxicity to mammalian cells.

Discovery, Structure-Activity Relationship and In Vitro Anticancer Activity of Small-Molecule Inhibitors of the Protein-Protein Interactions between AF9/ENL and AF4 or DOT1L.

Chromosomal translocations involving the mixed lineage leukemia (MLL) gene cause 5-10% acute leukemias with poor clinical outcomes. Protein-protein interactions (PPI) between the most frequent MLL fusion partner proteins AF9/ENL and AF4 or histone methyltransferase DOT1L are drug targets for MLL-rearranged (MLL-r) leukemia. Several benzothiophene-carboxamide compounds were identified as novel inhibitors of these PPIs with IC50 values as low as 1.6 µM. Structure-activity relationship studies of 77 benzothiophene and related indole and benzofuran compounds show that a 4-piperidin-1-ylphenyl or 4-pyrrolidin-1-ylphenyl substituent is essential for the activity. The inhibitors suppressed expression of MLL target genes HoxA9, Meis1 and Myc, and selectively inhibited proliferation of MLL-r and other acute myeloid leukemia cells with EC50 values as low as 4.7 µM. These inhibitors are useful chemical probes for biological studies of AF9/ENL, as well as pharmacological leads for further drug development against MLL-r and other leukemias.

2-Substituted 4,5-dihydrothiazole-4-carboxylic acids are novel inhibitors of metallo-ß-lactamases.

Bacterial resistance to ß-lactam antibiotics caused by class B metallo-ß-lactamases (MBL), especially for certain hospital-acquired, Gram-negative pathogens, poses a significant threat to public health. We report several 2-substituted 4,5-dihydrothiazole-4-carboxylic acids to be novel MBL inhibitors. Structure activity relationship (SAR) and molecular modeling studies were performed and implications for further inhibitor design are discussed.

A proteolysis-targeting chimera molecule selectively degrades ENL and inhibits malignant gene expression and tumor growth.

BACKGROUND: Chromosome translocations involving mixed lineage leukemia 1 (MLL1) cause acute leukemia in most infants and 5-10% children/adults with dismal clinical outcomes. Most frequent MLL1-fusion partners AF4/AFF4, AF9/ENL and ELL, together with CDK9/cyclin-T1, constitute super elongation complexes (SEC), which promote aberrant gene transcription, oncogenesis and maintenance of MLL1-rearranged (MLL1-r) leukemia. Notably, ENL, but not its paralog AF9, is essential for MLL1-r leukemia (and several other cancers) and therefore a drug target. Moreover, recurrent ENL mutations are found in Wilms tumor, the most common pediatric kidney cancer, and play critical roles in oncogenesis. METHODS: Proteolysis-Targeting Chimera (PROTAC) molecules were designed and synthesized to degrade ENL. Biological activities of these compounds were characterized in cell and mouse models of MLL1-r leukemia and other cancers. RESULTS: Compound 1 efficiently degraded ENL with DC50 of 37 nM and almost depleted it at ~ 500 nM in blood and solid tumor cells. AF9 (as well as other proteins in SEC) was not significantly decreased. Compound 1-mediated ENL reduction significantly suppressed malignant gene signatures, selectively inhibited cell proliferation of MLL1-r leukemia and Myc-driven cancer cells with EC50s as low as 320 nM, and induced cell differentiation and apoptosis. It exhibited significant antitumor activity in a mouse model of MLL1-r leukemia. Compound 1 can also degrade a mutant ENL in Wilms tumor and suppress its mediated gene transcription. CONCLUSION: Compound 1 is a novel chemical probe for cellular and in vivo studies of ENL (including its oncogenic mutants) and a lead compound for further anticancer drug development.

Structure-activity relationship and antitumor activity of 1,4-pyrazine-containing inhibitors of histone acetyltransferases P300/CBP.

Acetylation of histone lysine residues by histone acetyltransferase (HAT) p300 and its paralog CBP play important roles in gene regulation in health and diseases. The HAT domain of p300/CBP has been found to be a potential drug target for cancer. Compound screening followed by structure-activity relationship studies yielded a novel series of 1,4-pyrazine-containing inhibitors of p300/CBP HAT with their IC50s as low as 1.4 µM. Enzyme kinetics and other studies support the most potent compound 29 is a competitive inhibitor of p300 HAT against the substrate histone. It exhibited a high selectivity for p300 and CBP, with negligible activity on other classes of HATs in human. Compound 29 inhibited cellular acetylation of several histone lysine residues and showed strong activity against proliferation of a panel of solid and blood cancer cells. These results indicate it is a novel pharmacological lead for drug development targeting these cancers as well as a useful chemical probe for biological studies of p300/CBP.

In vitro and in vivo characterization of erythrosin B and derivatives against Zika virus.

Zika virus (ZIKV) causes significant human diseases without specific therapy. Previously we found erythrosin B, an FDA-approved food additive, inhibited viral NS2B-NS3 interactions, leading to inhibition of ZIKV infection in cell culture. In this study, we performed pharmacokinetic and in vivo studies to demonstrate the efficacy of erythrosin B against ZIKV in 3D mini-brain organoid and mouse models. Our results showed that erythrosin B is very effective in abolishing ZIKV replication in the 3D organoid model. Although pharmacokinetics studies indicated that erythrosin B had a low absorption profile, mice challenged by a lethal dose of ZIKV showed a significantly improved survival rate upon oral administration of erythrosin B, compared to vehicle control. Limited structure-activity relationship studies indicated that most analogs of erythrosin B with modifications on the xanthene ring led to loss or reduction of inhibitory activities towards viral NS2B-NS3 interactions, protease activity and antiviral efficacy. In contrast, introducing chlorine substitutions on the isobenzofuran ring led to slightly increased activities, suggesting that the isobenzofuran ring is well tolerated for modifications. Cytotoxicity studies indicated that all derivatives are nontoxic to human cells. Overall, our studies demonstrated erythrosin B is an effective antiviral against ZIKV both in vitro and in vivo.

Synergistic anti-tumor efficacy of mutant isocitrate dehydrogenase 1 inhibitor SYC-435 with standard therapy in patient-derived xenograft mouse models of glioma.

Clinical outcomes in patients with WHO grade II/III astrocytoma, oligodendroglioma or secondary glioblastoma remain poor. Isocitrate dehydrogenase 1 (IDH1) is mutated in > 70% of these tumors, making it an attractive therapeutic target. To determine the efficacy of our newly developed mutant IDH1 inhibitor, SYC-435 (1-hydroxypyridin-2-one), we treated orthotopic glioma xenograft model (IC-BT142AOA) carrying R132H mutation and our newly established orthotopic patient-derived xenograft (PDX) model of recurrent anaplastic oligoastrocytoma (IC-V0914AOA) bearing R132C mutation. In addition to suppressing IDH1 mutant cell proliferation in vitro, SYC-435 (15 mg/kg, daily x 28 days) synergistically prolonged animal survival times with standard therapies (Temozolomide + fractionated radiation) mediated by reduction of H3K4/H3K9 methylation and expression of mitochondrial DNA (mtDNA)-encoded molecules. Furthermore, RNA-seq of the remnant tumors identified genes (MYO1F, CTC1 and BCL9) and pathways (base excision repair, TCA cycle II, sirtuin signaling, protein kinase A, eukaryotic initiation factor 2 and α-adrenergic signaling) as mediators of therapy resistance. Our data demonstrated the efficacy SYC-435 in targeting IDH1 mutant gliomas when combined with standard therapy and identified a novel set of genes that should be prioritized for future studies to overcome SYC-435 resistance.

Selective inhibitors of histone methyltransferase DOT1L: design, synthesis, and crystallographic studies.

Histone H3-lysine79 (H3K79) methyltransferase DOT1L plays critical roles in normal cell differentiation as well as initiation of acute leukemia. We used structure- and mechanism-based design to discover several potent inhibitors of DOT1L with IC(50) values as low as 38 nM. These inhibitors exhibit only weak or no activities against four other representative histone lysine and arginine methyltransferases, G9a, SUV39H1, PRMT1 and CARM1. The X-ray crystal structure of a DOT1L-inhibitor complex reveals that the N6-methyl group of the inhibitor, located favorably in a predominantly hydrophobic cavity of DOT1L, provides the observed high selectivity. Structural analysis shows that it will disrupt at least one H-bond and/or have steric repulsion for other histone methyltransferases. These compounds represent novel chemical probes for biological function studies of DOT1L in health and disease.