A Genetic Map of the Response to DNA Damage in Human Cells.

The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.

Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery.

Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ ∼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.

TLR7-Induced Mitochondrial Reactive Oxygen Species Production in Monocyte-derived Dendritic Cells Drives IL-12-Dependent NK Cell Activation and Enhances Antitumor Immunity.

Dendritic cell (DC)-based vaccines are promising immunotherapies for cancer. Although DC-based therapies are known to activate tumor-specific T cells, the interplay between DCs and NK cells in this setting is not fully understood. In this study, we demonstrated a novel TLR7/ mitochondrial reactive oxygen species (mROS)/IL-12 axis that drives potent NK cell responses against tumors. We showed that TLR7 activation by imiquimod in peripheral blood monocyte-derived CD11c+ DCs triggered mROS production, leading to enhanced IL-12 secretion and subsequent NK cell activation, as evidenced by increased IFN-γ production and tumor cell cytotoxicity. Notably, mROS neutralization abrogates NK cell-mediated tumor cell lysis, and TLR7-mediated DC activation of NK cells occurs independently of MyD88, suggesting involvement of the noncanonical NF-κB pathway. Our findings provide a rationale for targeting the TLR7/mROS/IL-12 axis to enhance the efficacy of DC-based cancer immunotherapy.

Disruption of XIAP-RIP2 Association Blocks NOD2-Mediated Inflammatory Signaling.

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.

Modulation of dynamic DNA G-quadruplex structures in the hTERT promoter region by ligands.

Small molecules can inhibit cellular processes such as replication and transcription by binding to the promoter regions that are prone to form G-quadruplexes. However, since G-quadruplexes exist throughout the human genome, the G-quadruplex binders suffer from specificity issues. To tackle this problem, a G-quadruplex binder (Pyridostatin, or PDS) is conjugated with a ligand (Polyamide, or PA) that can specifically recognize DNA sequences flanking the G-quadruplex forming region. The binding mechanism of this hybrid ligand to the hTERT promoter region (hTERT 5-12) is then elucidated using optical tweezers. During mechanical unfolding processes, different intermediate structures of hTERT 5-12 in presence of PDS, PA, or PA-PDS conjugate are observed. These intermediate structures are consistent with two folding patterns of G-quadruplexes in the hTERT 5-12 fragment. While the duplex DNA binder PA facilitates the folding of a hairpin-G-quadruplex structure, the PDS assists the formation of two tandem G-quadruplexes. Both replication stop assay in vitro and dual luciferase assay in vivo established the effectiveness of the PA-PDS conjugate for hTERT 5-12 targeting. We expect such a ligand dependent folding dynamics will provide guidelines to the development of drugs that not only target hTERT expressions, but also other oncogenes via interactions with specific G-quadruplex structures formed in their promotor regions.

G-quadruplex in hepatitis B virus pregenomic RNA promotes its translation.

Hepatitis B virus (HBV) is a hepatotropic DNA virus that has a very compact genome. Due to this genomic density, several distinct mechanisms are used to facilitate the viral life cycle. Recently, accumulating evidence show that G-quadruplex (G4) in different viruses play essential regulatory roles in key steps of the viral life cycle. Although G4 structures in the HBV genome have been reported, their function in HBV replication remains elusive. In this study, we treated an HBV replication-competent cell line and HBV-infected cells with the G4 structure stabilizer pyridostatin (PDS) and evaluated different HBV replication markers to better understand the role played by the G4. In both models, we found PDS had no effect on viral precore RNA (pcRNA) or pre-genomic RNA (pgRNA), but treatment did increase HBeAg/HBc ELISA reads and intracellular levels of viral core/capsid protein (HBc) in a dose-dependent manner, suggesting post-transcriptional regulation. To further dissect the mechanism of G4 involvement, we used in vitro-synthesized HBV pcRNA and pgRNA. Interestingly, we found PDS treatment only enhanced HBc expression from pgRNA but not HBeAg expression from pcRNA. Our bioinformatic analysis and CD spectroscopy revealed that pgRNA harbors a conserved G4 structure. Finally, we introduced point mutations in pgRNA to disrupt its G4 structure and observed the resulting mutant failed to respond to PDS treatment and decreased HBc level in in vitro translation assay. Taken together, our data demonstrate that HBV pgRNA contains a G4 structure that plays a vital role in the regulation of viral mRNA translation.

Extended blood stage sensitivity profiles of Plasmodium cynomolgi to doxycycline and tafenoquine, as a model for Plasmodium vivax.

Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which preclude determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax, we determined the physiologically significant delayed death effect induced by doxycycline [IC50(96 h), 1,401 ± 607 nM]. As expected, IC50(96 h) to chloroquine (20.4 nM), piperaquine (12.6 µM), and tafenoquine (1,424 nM) were not affected by extended exposure.

Synthesis and biological evaluation of novel aminoquinolines with an n-octyl linker: Impact of halogen substituents on C(7) or a terminal amino group on anticholinesterase and BACE1 activity.

Alzheimer's disease is age-related multifactorial neurodegenerative disease manifested by gradual loss of memory, cognitive decline and changes in personality. Due to rapid and continuous growth of its prevalence, the treatment of Alzheimer's disease calls for development of new and efficacies drugs, especially those that could be able to simultaneously act on more than one of possible targets of action. Aminoquinolines have proven to be a highly promising structural scaffold in the design of such a drug as cholinesterases and ß-secretase 1 inhibitors. In this study, we synthesised twenty-two new 4-aminoquinolines with different halogen atom and its position in the terminal N-benzyl group or with a trifluoromethyl or a chlorine as C(7)-substituents on the quinoline moiety. All compounds were evaluated as multi-target-directedligands by determining their inhibition potency towards human acetylcholinesterase, butyrylcholinesterase and ß-secretase 1. All of the tested derivatives were very potent inhibitors of human acetylcholinesterase and butyrylcholinesterase with inhibition constants (Ki) in the nM to low µM range. Most were estimated to be able to cross the blood-brain barrier by passive transport and were nontoxic toward cells that represented the main models of individual organs.

Exploring the unexplored chemical space: Rational identification of new Tafenoquine analogs with antimalarial properties.

Patents tend to define a huge chemical space described by the combinatorial nature of Markush structures. However, the optimization of new principal active ingredient is frequently driven by a simple Free Wilson approach. This procedure leads to a highly focused study on the chemical space near a hit compound leaving many unexplored regions that may present highly biological active reservoirs. This study aims to demonstrate that this unveiled chemical space can hide compounds with interesting potential biological activity that would be worth pursuing. This underlines the value and necessity of broadening an approach beyond conventional strategies. Hence, we advocate for an alternative methodology that may be more efficient in the early drug discovery stages. We have selected the case of Tafenoquine, a single-dose treatment for the radical cure of P. vivax malaria approved by the FDA in 2018, as an example to illustrate the process. Through the deep exploration of the Tafenoquine chemical space, seven compounds with potential antimalarial activity have been rationally identified and synthesized. This small set is representative of the chemical diversity unexplored by the 58 analogs reported to date. After biological assessment, results evidence that our approach for rational design has proven to be a very efficient exploratory methodology suitable for the early drug discovery stages.

Tetrahydropyridine appended 8-aminoquinoline derivatives: Design, synthesis, in silico, and in vitro antimalarial studies.

Antimalarial drug resistance is a major obstacle in the ongoing quest against malaria. The disease affects half of the world's population. The majority of them are toddlers and pregnant women. Needed a potent compound to act on drug-resistant Pf at appropriate concentrations without endangering the host. Envisaged solving this issue through rational drug design by creating a novel hybrid drug possessing two pharmacophores that can act on two marvellous and independent aims within the cell. Synthesized a new series of substituted 4-phenyl-1,2,3,6-tetrahydropyridine (THP) 8-Aminoquinoline-based hybrid analogs which have been integrated with quinoline, chloroquine, pamaquine, and primaquine, which exhibited antimalarial activity against Pf. Out of thirteen 4-phenyl-1,2,3,6-THP appended 8-Aminoquinoline derivatives, the compounds 1j, 1e, 1b, and 1l have exhibited good antimalarial activity against chloroquine-sensitive (3D7) and chloroquine-resistant (RKL-9) strain with the minimum inhibitory concentration. Compound 1b was the most effective and showed consistently good potency against the drug-resistant (RKL-9) strain, although all other arrays showed good antimalarial efficacy. Additional docking and molecular dynamics studies were carried out at several targeting sites to quantify the structural parameters necessary for the activity.

Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds.

G-quadruplex (G4)-forming genomic sequences, including telomeres, represent natural replication fork barriers. Stalled replication forks can be stabilized and restarted by homologous recombination (HR), which also repairs DNA double-strand breaks (DSBs) arising at collapsed forks. We have previously shown that HR facilitates telomere replication. Here, we demonstrate that the replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased significantly in cells lacking HR. Treatment with the G4-stabilizing compound pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells, suggesting that G4 formation drives telomere instability. Remarkably, PDS reduces proliferation of HR-defective cells by inducing DSB accumulation, checkpoint activation, and deregulated G2/M progression and by enhancing the replication defect intrinsic to HR deficiency. PDS toxicity extends to HR-defective cells that have acquired olaparib resistance through loss of 53BP1 or REV7. Altogether, these results highlight the therapeutic potential of G4-stabilizing drugs to selectively eliminate HR-compromised cells and tumors, including those resistant to PARP inhibition.

Involvement of nucleus accumbens SERCA2b in methamphetamine-induced conditioned place preference.

Methamphetamine (METH) is a highly addictive psycho-stimulant that induces addictive behaviour by stimulating increased dopamine release in the nucleus accumbens (NAc). The sarco/endoplasmic reticulum calcium ion transport ATPases (SERCA or ATP2A) is a calcium ion (Ca2+) pump in the endoplasmic reticulum (ER) membrane. SERCA2b is a SERCA subtype mainly distributed in the central nervous system. This study used conditioned place preference (CPP), a translational drug reward model, to observe the effects of SERCA and SERCA2b on METH-CPP in mice. Result suggested that the activity of SERCA was significantly decreased in NAc after METH-CPP. Intraperitoneal SERCA agonist CDN1163 injection or bilateral CDN1163 microinjection in the NAc inhibited METH-CPP formation. SERCA2b overexpression by the Adeno-associated virus can reduce the DA release of NAc and inhibit METH-CPP formation. Although microinjection of SERCA inhibitor thapsigargin in the bilateral NAc did not significantly aggravate METH-CPP, interference with SERCA2b expression in NAc by adeno-associated virus increased DA release and promoted METH-CPP formation. METH reduced the SERCA ability to transport Ca2+ into the ER in SHSY5Y cells in vitro, which was reversed by CDN1163. This study revealed that METH dysregulates intracellular calcium balance by downregulating SERCA2b function, increasing DA release in NAc and inducing METH-CPP formation. Drugs that target SERCA2b may have the potential to treat METH addiction.

RAC1 controls progressive movement and competitiveness of mammalian spermatozoa.

Mammalian spermatozoa employ calcium (Ca2+) and cyclic adenosine monophosphate (cAMP) signaling in generating flagellar beat. However, how sperm direct their movement towards the egg cells has remained elusive. Here we show that the Rho small G protein RAC1 plays an important role in controlling progressive motility, in particular average path velocity and linearity. Upon RAC1 inhibition of wild type sperm with the drug NSC23766, progressive movement is impaired. Moreover, sperm from mice homozygous for the genetically variant t-haplotype region (tw5/tw32), which are sterile, show strongly enhanced RAC1 activity in comparison to wild type (+/+) controls, and quickly become immotile in vitro. Sperm from heterozygous (t/+) males, on the other hand, display intermediate RAC1 activity, impaired progressive motility and transmission ratio distortion (TRD) in favor of t-sperm. We show that t/+-derived sperm consist of two subpopulations, highly progressive and less progressive. The majority of highly progressive sperm carry the t-haplotype, while most less progressive sperm contain the wild type (+) chromosome. Dosage-controlled RAC1 inhibition in t/+ sperm by NSC23766 rescues progressive movement of (+)-sperm in vitro, directly demonstrating that impairment of progressive motility in the latter is caused by enhanced RAC1 activity. The combined data show that RAC1 plays a pivotal role in controlling progressive motility in sperm, and that inappropriate, enhanced or reduced RAC1 activity interferes with sperm progressive movement. Differential RAC1 activity within a sperm population impairs the competitiveness of sperm cells expressing suboptimal RAC1 activity and thus their fertilization success, as demonstrated by t/+-derived sperm. In conjunction with t-haplotype triggered TRD, we propose that Rho GTPase signaling is essential for directing sperm towards the egg cells.

Regulation of P2X1 receptors by modulators of the cAMP effectors PKA and EPAC.

P2X1 receptors are adenosine triphosphate (ATP)-gated cation channels that are functionally important for male fertility, bladder contraction, and platelet aggregation. The activity of P2X1 receptors is modulated by lipids and intracellular messengers such as cAMP, which can stimulate protein kinase A (PKA). Exchange protein activated by cAMP (EPAC) is another cAMP effector; however, its effect on P2X1 receptors has not yet been determined. Here, we demonstrate that P2X1 currents, recorded from human embryonic kidney (HEK) cells transiently transfected with P2X1 cDNA, were inhibited by the highly selective EPAC activator 007-AM. In contrast, EPAC activation enhanced P2X2 current amplitude. The PKA activator 6-MB-cAMP did not affect P2X1 currents, but inhibited P2X2 currents. The inhibitory effects of EPAC on P2X1 were prevented by triple mutation of residues 21 to 23 on the amino terminus of P2X1 subunits to the equivalent amino acids on P2X2 receptors. Double mutation of residues 21 and 22 and single mutation of residue 23 also protected P2X1 receptors from inhibition by EPAC activation. Finally, the inhibitory effects of EPAC on P2X1 were also prevented by NSC23766, an inhibitor of Rac1, a member of the Rho family of small GTPases. These data suggest that EPAC is an important regulator of P2X1 and P2X2 receptors.

Tafenoquine and its derivatives as inhibitors for the severe acute respiratory syndrome coronavirus 2.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has severely affected human lives around the world as well as the global economy. Therefore, effective treatments against COVID-19 are urgently needed. Here, we screened a library containing Food and Drug Administration (FDA)-approved compounds to identify drugs that could target the SARS-CoV-2 main protease (Mpro), which is indispensable for viral protein maturation and regard as an important therapeutic target. We identified antimalarial drug tafenoquine (TFQ), which is approved for radical cure of Plasmodium vivax and malaria prophylaxis, as a top candidate to inhibit Mpro protease activity. The crystal structure of SARS-CoV-2 Mpro in complex with TFQ revealed that TFQ noncovalently bound to and reshaped the substrate-binding pocket of Mpro by altering the loop region (residues 139-144) near the catalytic Cys145, which could block the catalysis of its peptide substrates. We also found that TFQ inhibited human transmembrane protease serine 2 (TMPRSS2). Furthermore, one TFQ derivative, compound 7, showed a better therapeutic index than TFQ on TMPRSS2 and may therefore inhibit the infectibility of SARS-CoV-2, including that of several mutant variants. These results suggest new potential strategies to block infection of SARS-CoV-2 and rising variants.

4-Aminoquinolines modulate RNA structure and function: Pharmacophore implications of a conformationally restricted polyamine.

RNA structure plays an important role in regulating cellular function and there is a significant emerging interest in targeting RNA for drug discovery. Here we report the identification of 4-aminoquinolines as modulators of RNA structure and function. Aminoquinolines have a broad range of pharmacological activities, but their specific mechanism of action is often not fully understood. Using electrophoretic mobility shift assays and enzymatic probing we identified 4-aminoquinolines that bind the stem-loop II motif (s2m) of SARS-CoV-2 RNA site-specifically and induce dimerization. Using fluorescence-based RNA binding and T-box riboswitch functional assays we identified that hydroxychloroquine binds the T-box riboswitch antiterminator RNA element and inhibits riboswitch function. Based on its structure and riboswitch dose-response activity we identified that the antagonist activity of hydroxychloroquine is consistent with it being a conformationally restricted analog of the polyamine spermidine. Given the known role that polyamines play in RNA function, the identification of an RNA binding ligand with the pharmacophore of a conformationally restricted polyamine has significant implications for further elucidation of RNA structure-function relationships and RNA-targeted drug discovery.

Activation of the aryl hydrocarbon receptor dampens the severity of inflammatory skin conditions.

Environmental stimuli are known to contribute to psoriasis pathogenesis and that of other autoimmune diseases, but the mechanisms are largely unknown. Here we show that the aryl hydrocarbon receptor (AhR), a transcription factor that senses environmental stimuli, modulates pathology in psoriasis. AhR-activating ligands reduced inflammation in the lesional skin of psoriasis patients, whereas AhR antagonists increased inflammation. Similarly, AhR signaling via the endogenous ligand FICZ reduced the inflammatory response in the imiquimod-induced model of skin inflammation and AhR-deficient mice exhibited a substantial exacerbation of the disease, compared to AhR-sufficient controls. Nonhematopoietic cells, in particular keratinocytes, were responsible for this hyperinflammatory response, which involved upregulation of AP-1 family members of transcription factors. Thus, our data suggest a critical role for AhR in the regulation of inflammatory responses and open the possibility for novel therapeutic strategies in chronic inflammatory disorders.

Inflammatory memory sensitizes skin epithelial stem cells to tissue damage.

The skin barrier is the body's first line of defence against environmental assaults, and is maintained by epithelial stem cells (EpSCs). Despite the vulnerability of EpSCs to inflammatory pressures, neither the primary response to inflammation nor its enduring consequences are well understood. Here we report a prolonged memory to acute inflammation that enables mouse EpSCs to hasten barrier restoration after subsequent tissue damage. This functional adaptation does not require skin-resident macrophages or T cells. Instead, EpSCs maintain chromosomal accessibility at key stress response genes that are activated by the primary stimulus. Upon a secondary challenge, genes governed by these domains are transcribed rapidly. Fuelling this memory is Aim2, which encodes an activator of the inflammasome. The absence of AIM2 or its downstream effectors, caspase-1 and interleukin-1ß, erases the ability of EpSCs to recollect inflammation. Although EpSCs benefit from inflammatory tuning by heightening their responsiveness to subsequent stressors, this enhanced sensitivity probably increases their susceptibility to autoimmune and hyperproliferative disorders, including cancer.

G3BP1 binds to guanine quadruplexes in mRNAs to modulate their stabilities.

RNA guanine quadruplexes (rG4) assume important roles in post-transcriptional regulations of gene expression, which are often modulated by rG4-binding proteins. Hence, understanding the biological functions of rG4s requires the identification and functional characterizations of rG4-recognition proteins. By employing a bioinformatic approach based on the analysis of overlap between peaks obtained from rG4-seq analysis and those detected in >230 eCLIP-seq datasets for RNA-binding proteins generated from the ENCODE project, we identified a large number of candidate rG4-binding proteins. We showed that one of these proteins, G3BP1, is able to bind directly to rG4 structures with high affinity and selectivity, where the binding entails its C-terminal RGG domain and is further enhanced by its RRM domain. Additionally, our seCLIP-Seq data revealed that pyridostatin, a small-molecule rG4 ligand, could displace G3BP1 from mRNA in cells, with the most pronounced effects being observed for the 3'-untranslated regions (3'-UTR) of mRNAs. Moreover, luciferase reporter assay results showed that G3BP1 positively regulates mRNA stability through its binding with rG4 structures. Together, we identified a number of candidate rG4-binding proteins and validated that G3BP1 can bind directly with rG4 structures and regulate the stabilities of mRNAs.

G-quadruplex binders as cytostatic modulators of innate immune genes in cancer cells.

G-quadruplexes (G4s) are non-canonical nucleic acid structures involved in fundamental biological processes. As G4s are promising anticancer targets, in past decades the search for effective anticancer G4 binders aimed at the discovery of more cytotoxic ligands interfering with specific G4 structures at oncogenes or telomeres. Here, we have instead observed a significant activation of innate immune genes by two unrelated ligands at non-cytotoxic concentrations. The studied G4 binders (pyridostatin and PhenDC3) can induce an increase of micronuclei triggering the activation of the cytoplasmic STING (stimulator of interferon response cGAMP interactor 1) signaling pathway in human and murine cancer cells. Ligand activity can then lead to type I interferon production and innate immune gene activation. Moreover, specific gene expression patterns mediated by a G4 binder in cancer cells correlate with immunological hot features and better survival in human TCGA (The Cancer Genome Atlas) breast tumors. The findings open to the development of cytostatic G4 binders as effective immunomodulators for combination immunotherapies in unresponsive tumors.