Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

DNA polymerase θ protects leukemia cells from metabolically induced DNA damage.

Leukemia cells accumulate DNA damage, but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/1-carbon cycle metabolism contributed to the accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases (OTKs: FLT3(internal tandem duplication [ITD]), JAK2(V617F), BCR-ABL1). To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLθ) via ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLθ and its proteasomal degradation. Overexpression of POLθ in OTK-positive cells resulted in the efficient repair of DPC-containing DNA double-strand breaks by POLθ-mediated end-joining. The transforming activities of OTKs and other leukemia-inducing oncogenes, especially of those causing the inhibition of BRCA1/2-mediated homologous recombination with and without concomitant inhibition of DNA-PK-dependent nonhomologous end-joining, was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLθ polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitors or DPC-inducing drug etoposide enhanced the antileukemia effect of POLθ inhibitor in vitro and in vivo. In conclusion, we demonstrated that POLθ plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde, and it can be targeted to achieve a therapeutic effect.

Effects of novel beta-lactam, MC-100093, and ceftriaxone on astrocytic glutamate transporters and neuroinflammatory factors in nucleus accumbens of C57BL/6 mice exposed to escalated doses of morphine.

Chronic exposure to opioids can lead to downregulation of astrocytic glutamate transporter 1 (GLT-1), which regulates the majority of glutamate uptake. Studies from our lab revealed that beta-lactam antibiotic, ceftriaxone, attenuated hydrocodone-induced downregulation of GLT-1 as well as cystine/glutamate antiporter (xCT) expression in central reward brain regions. In this study, we investigated the effects of escalating doses of morphine and tested the efficacy of novel synthetic non-antibiotic drug, MC-100093, and ceftriaxone in attenuating the effects of morphine exposure in the expression of GLT-1, xCT, and neuroinflammatory factors (IL-6 and TGF-ß) in the nucleus accumbens (NAc). This study also investigated the effects of morphine and beta-lactams in locomotor activity, spontaneous alternation percentage (SAP) and number of entries in Y maze since opioids have effects in locomotor sensitization. Mice were exposed to moderate dose of morphine (20 mg/kg, i.p.) on days 1, 3, 5, 7, and a higher dose of morphine (150 mg/kg, i.p.) on day 9, and these mice were then behaviorally tested and euthanized on Day 10. Western blot analysis showed that exposure to morphine downregulated GLT-1 and xCT expression in the NAc, and both MC-100093 and ceftriaxone attenuated these effects. In addition, morphine exposure increased IL-6 mRNA and TGF-ß mRNA expression, and MC-100093 and ceftriaxone attenuated only the effect on IL-6 mRNA expression in the NAc. Furthermore, morphine exposure induced an increase in distance travelled, and MC-100093 and ceftriaxone attenuated this effect. In addition, morphine exposure decreased the SAP and increased the number of arm entries in Y maze, however, neither MC-100093 nor ceftriaxone showed any attenuating effect. Our findings demonstrated for the first time that MC-100093 and ceftriaxone attenuated morphine-induced downregulation of GLT-1 and xCT expression, and morphine-induced increase in neuroinflammatory factor, IL-6, as well as hyperactivity. These findings revealed the beneficial therapeutic effects of MC-100093 and ceftriaxone against the effects of exposure to escalated doses of morphine.

Effects of a Novel Beta Lactam Compound, MC-100093, on the Expression of Glutamate Transporters/Receptors and Ethanol Drinking Behavior of Alcohol-Preferring Rats.

Chronic ethanol exposure affects the glutamatergic system in several brain reward regions including the nucleus accumbens (NAc). Our laboratory has shown that chronic exposure to ethanol reduced the expression of glutamate transporter 1 (GLT-1) and cystine/glutamate exchanger (xCT) and, as a result, increased extracellular glutamate concentrations in the NAc of alcohol-preferring (P) rats. Moreover, previous studies from our laboratory reported that chronic ethanol intake altered the expression of certain metabotropic glutamate receptors in the brain. In addition to central effects, chronic ethanol consumption induced liver injury, which is associated with steatohepatitis. In the present study, we investigated the effects of chronic ethanol consumption in the brain and liver. Male P rats had access to a free choice of ethanol and water bottles for five weeks. Chronic ethanol consumption reduced GLT-1 and xCT expression in the NAc shell but not in the NAc core. Furthermore, chronic ethanol consumption increased fat droplet content as well as peroxisome proliferator-activated receptor alpha (PPAR-α) and GLT-1 expression in the liver. Importantly, treatment with the novel beta-lactam compound, MC-100093, reduced ethanol drinking behavior and normalized the levels of GLT-1 and xCT expression in the NAc shell as well as normalized GLT-1 and PPAR-α expression in the liver. In addition, MC-100093 attenuated ethanol-induced increases in fat droplet content in the liver. These findings suggest that MC-100093 may be a potential lead compound to attenuate ethanol-induced dysfunction in the glutamatergic system and liver injury. SIGNIFICANCE STATEMENT: This study identified a novel beta-lactam, MC-100093, that has demonstrated upregulatory effects on GLT-1. MC-100093 reduced ethanol drinking behavior and normalized levels of GLT-1 and xCT expression in the NAc shell as well as normalized GLT-1 and PPAR-α expression in the liver. In addition, MC-100093 attenuated ethanol-induced increases in fat droplet content in the liver.

MC-100093, a Novel ß-Lactam Glutamate Transporter-1 Enhancer Devoid of Antimicrobial Properties, Attenuates Cocaine Relapse in Rats.

Cocaine use disorder currently lacks Food and Drug Administration-approved treatments. In rodents, the glutamate transporter-1 (GLT-1) is downregulated in the nucleus accumbens after cocaine self-administration, and increasing the expression and function of GLT-1 reduces the reinstatement of cocaine seeking. The ß-lactam antibiotic ceftriaxone upregulates GLT-1 and attenuates cue- and cocaine-induced cocaine seeking without affecting motivation for natural rewards. Although ceftriaxone shows promise for treating cocaine use disorder, it possesses characteristics that limit successful translation from bench to bedside, including poor brain penetration, a lack of oral bioavailability, and a risk of bacterial resistance when used chronically. Thus, we aimed to develop novel molecules that retained the GLT-1-enhancing effects of ceftriaxone but displayed superior drug-like properties. Here, we describe a new monocyclic ß-lactam, MC-100093, as a potent upregulator of GLT-1 that is orally bioavailable and devoid of antimicrobial properties. MC-100093 was synthesized and tested in vitro and in vivo to determine physiochemical, pharmacokinetic, and pharmacodynamic properties. Next, adult male rats underwent cocaine self-administration and extinction training. During extinction training, rats received one of four doses of MC-100093 for 6-8 days prior to a single cue-primed reinstatement test. Separate cohorts of rats were used to assess nucleus accumbens GLT-1 expression and MC-100093 effects on sucrose self-administration. We found that 50 mg/kg MC-100093 attenuated cue-primed reinstatement of cocaine seeking while upregulating GLT-1 expression in the nucleus accumbens core. This dose did not produce sedation, nor did it decrease sucrose consumption or body weight. Thus, MC-100093 represents a potential treatment to reduce cocaine relapse. SIGNIFICANCE STATEMENT: Increasing GLT-1 activity reliably reduces drug-seeking across classes of drugs; however, existing GLT1-enhancers have side effects and lack oral bioavailability. To address this issue, novel GLT-1 enhancers were synthesized, and the compound with the most favorable pharmacokinetic and pharmacodynamic properties, MC-100093, was selected for further testing. MC-100093 attenuated cued cocaine seeking without reducing food seeking or locomotion and upregulated GLT-1 expression in the nucleus accumbens.

Discovery of Novel Small-Molecule Inhibitors of SARS-CoV-2 Main Protease as Potential Leads for COVID-19 Treatment.

The main protease of SARS-CoV-2 virus, Mpro, is an essential element for viral replication, and inhibitors targeting Mpro are currently being investigated in many drug development programs as a possible treatment for COVID-19. An in vitro pilot screen of a highly focused collection of compounds was initiated to identify new lead scaffolds for Mpro. These efforts identified a number of hits. The most effective of these was compound SIMR-2418 having an inhibitory IC50 value of 20.7 µM. Molecular modeling studies were performed to understand the binding characteristics of the identified compounds. The presence of a cyclohexenone warhead group facilitated covalent binding with the Cys145 residue of Mpro. Our results highlight the challenges of targeting Mpro protease and pave the way toward the discovery of potent lead molecules.

Discovery of novel class of histone deacetylase inhibitors as potential anticancer agents.

Selective inhibition of histone deacetylases (HDACs) is an important strategy in the field of anticancer drug discovery. However, lack of inhibitors that possess high selectivity toward certain HDACs isozymes is associated with adverse side effects that limits their clinical applications. We have initiated a collaborative initiatives between multi-institutions aimed at the discovery of novel and selective HDACs inhibitors. To this end, a phenotypic screening of an in-house pilot library of about 70 small molecules against various HDAC isozymes led to the discovery of five compounds that displayed varying degrees of HDAC isozyme selectivity. The anticancer activities of these molecules were validated using various biological assays including transcriptomic studies. Compounds 15, 14, and 19 possessed selective inhibitory activity against HDAC5, while 28 displayed selective inhibition of HDAC1 and HDAC2. Compound 22 was found to be a selective inhibitor for HDAC3 and HDAC9. Importantly, we discovered a none-hydroxamate based HDAC inhibitor, compound 28, representing a distinct chemical probe of HDAC inhibitors. It contains a trifluoromethyloxadiazolyl moiety (TFMO) as a non-chelating metal-binding group. The new compounds showed potent anti-proliferative activity when tested against MCF7 breast cancer cell line, as well as increased acetylation of histones and induce cells apoptosis. The new compounds apoptotic effects were validated through the upregulation of proapoptotic proteins caspases3 and 7 and downregulation of the antiapoptotic biomarkers C-MYC, BCL2, BCL3 and NFĸB genes. Furthermore, the new compounds arrested cell cycle at different phases, which was confirmed through downregulation of the CDK1, 2, 4, 6, E2F1 and RB1 proteins. Taken together, our findings provide the foundation for the development of new chemical probes as potential lead drug candidates for the treatment of cancer.

Novel compounds that reverse the disease phenotype in Type 2 Gaucher disease patient-derived cells.

Gaucher disease (GD) results from inherited mutations in the lysosomal enzyme ß-glucocerobrosidase (GCase). Currently available treatment options for Type 1 GD are not efficacious for treating neuronopathic Type 2 and 3 GD due to their inability to cross the blood-brain barrier. In an effort to identify small molecules which could be optimized for CNS penetration we identified tamoxifen from a high throughput phenotypic screen on Type 2 GD patient-derived fibroblasts which reversed the disease phenotype. Structure activity studies around this scaffold led to novel molecules that displayed improved potency, efficacy and reduced estrogenic/antiestrogenic activity compared to the original hits. Here we present the design, synthesis and structure activity relationships that led to the lead molecule Compound 31.

Novel allosteric PARP1 inhibitors for the treatment of BRCA-deficient leukemia.

The successful use of PARP1 inhibitors like olaparib (Loparza®) in the treatment of BRCA1/2- deficient breast cancer has provided clinical proof of concept for applying personalized medicine based on synthetic lethality to the treatment of cancer. Unfortunately, all marketed PARP1 inhibitors act by competing with the cofactor NAD+ and resistance is already developing to this anti-cancer mechanism. Allosteric PARP1 inhibitors could provide a means of overcoming this resistance. A high throughput screen performed by Tulin et al. identified 5F02 as an allosteric PARP inhibitor that acts by preventing the enzymatic activation of PARP1 by histone H4. 5F02 demonstrated anti-cancer activity in several cancer cell lines and was more potent than olaparib and synergistic with olaparib in these assays. In the present study we explored the structure-activity relationship of 5F02 by preparing analogs that possessed structural variation in four regions of the chemical scaffold. Our efforts led to lead molecule 7, which demonstrated potent anti-clonogenic activity against BRCA-deficient NALM6 leukemia cells in culture and a therapeutic index for the BRCA-deficient cells over their BRCA-proficient isogenic counterparts.

Novel inhibitors of Staphylococcus aureus RnpA that synergize with mupirocin.

We recently discovered RnpA as a promising new drug discovery target for methicillin-resistant S. aureus (MRSA). RnpA is an essential protein that is thought to perform two required cellular processes. As part of the RNA degrasome Rnpa mediates RNA degradation. In combination with rnpB it forms RNase P haloenzymes which are required for tRNA maturation. A high throughput screen identified RNPA2000 as an inhibitor of both RnpA-associated activities that displayed antibacterial activity against clinically relevant strains of S. aureus, including MRSA. Structure-activity studies aimed at improving potency and replacing the potentially metabotoxic furan moiety led to the identification of a number of more potent analogs. Many of these new analogs possessed overt cellular toxicity that precluded their use as antibiotics but two derivatives, including compound 5o, displayed an impressive synergy with mupirocin, an antibiotic used for decolonizing MSRA whose effectiveness has recently been jeopardized by bacterial resistance. Based on our results, compounds like 5o may ultimately find use in resensitizing mupirocin-resistant bacteria to mupirocin.

Design and synthesis of functionalized piperazin-1yl-(E)-stilbenes as inhibitors of 17α-hydroxylase-C17,20-lyase (Cyp17).

The synthesis of steroid hormones is critical to human physiology and improper regulation of either the synthesis of these key molecules or activation of the associated receptors can lead to disease states. This has led to intense interest in developing compounds capable of modulating the synthesis of steroid hormones. Compounds capable of inhibiting Cyp19 (Aromatase), a key enzyme in the synthesis of estrogens, have been successfully employed as breast cancer therapies, while inhibitors of Cyp17 (17α-hydroxylase-17,20-lyase), a key enzyme in the synthesis of glucocorticoids, mineralocorticoids and steroidal sex hormones, are a key component of prostate cancer therapy. Inhibition of CYP17 has also been suggested as a possible target for the treatment of Cushing Syndrome and Metabolic Syndrome. We have identified two novel series of stilbene based CYP17 inhibitors and demonstrated that exemplary compounds in these series have pharmacokinetic properties consistent with orally delivered drugs. These findings suggest that compounds in these classes may be useful for the treatment of diseases and conditions associated with improper regulation of glucocorticoids synthesis and glucocorticoids receptor activation.

Nuclear Magnetic Resonance Structure of the Human Polyoma JC Virus Agnoprotein.

Agnoprotein is an important regulatory protein of the human polyoma JC virus (JCV) and plays critical roles during the viral replication cycle. It forms highly stable dimers and oligomers through its Leu/Ile/Phe-rich domain, which is important for the stability and function of the protein. We recently resolved the partial 3D structure of this protein by NMR using a synthetic peptide encompassing amino acids Thr17 to Gln52, where the Leu/Ile/Phe- rich region was found to adopt a major alpha-helix conformation spanning amino acids 23-39. Here, we report the resolution of the 3D structure of full-length JCV agnoprotein by NMR, which not only confirmed the existence of the previously reported major α-helix domain at the same position but also revealed the presence of an additional minor α-helix region spanning amino acid residues Leu6 to lys13. The remaining regions of the protein adopt an intrinsically unstructured conformation. J. Cell. Biochem. 118: 3268-3280, 2017. © 2017 Wiley Periodicals, Inc.

Design, synthesis and SAR of new-di-substituted pyridopyrimidines as ATP-competitive dual PI3Kα/mTOR inhibitors.

PI3Kα/mTOR ATP-competitive inhibitors are considered as one of the promising molecularly targeted cancer therapeutics. Based on lead compound A from the literature, two similar series of 2-substituted-4-morpholino-pyrido[3,2-d]pyrimidine and pyrido[2,3-d]pyrimidine analogs were designed and synthesized as PI3Kα/mTOR dual inhibitors. Interestingly, most of the series gave excellent inhibition for both enzymes with IC50 values ranging from single to double digit nM. Unlike many PI3Kα/mTOR dual inhibitors, our compounds displayed selectivity for PI3Kα. Based on its potent enzyme inhibitory activity, selectivity for PI3Kα and good therapeutic index in 2D cell culture viability assays, compound 4h was chosen to be evaluated in 3D culture for its IC50 against MCF7 breast cancer cells as well as for docking studies with both enzymes.

Emerging From the Unknown: Structural and Functional Features of Agnoprotein of Polyomaviruses.

Agnoprotein is an important regulatory protein of polyomaviruses, including JCV, BKV, and SV40. In the absence of its expression, these viruses are unable to sustain their productive life cycle. It is a highly basic phosphoprotein that localizes mostly to the perinuclear area of infected cells, although a small amount of the protein is also found in nucleus. Much has been learned about the structure and function of this important regulatory protein in recent years. It forms highly stable dimers/oligomers in vitro and in vivo through its Leu/Ile/Phe-rich domain. Structural NMR studies revealed that this domain adopts an alpha-helix conformation and plays a critical role in the stability of the protein. It associates with cellular proteins, including YB-1, p53, Ku70, FEZ1, HP1α, PP2A, AP-3, PCNA, and α-SNAP; and viral proteins, including small t antigen, large T antigen, HIV-1 Tat, and JCV VP1; and significantly contributes the viral transcription and replication. This review summarizes the recent advances in the structural and functional properties of this important regulatory protein. J. Cell. Physiol. 231: 2115-2127, 2016. © 2016 Wiley Periodicals, Inc.

Ribosome-Templated Azide-Alkyne Cycloadditions: Synthesis of Potent Macrolide Antibiotics by In Situ Click Chemistry.

Over half of all antibiotics target the bacterial ribosome-nature's complex, 2.5 MDa nanomachine responsible for decoding mRNA and synthesizing proteins. Macrolide antibiotics, exemplified by erythromycin, bind the 50S subunit with nM affinity and inhibit protein synthesis by blocking the passage of nascent oligopeptides. Solithromycin (1), a third-generation semisynthetic macrolide discovered by combinatorial copper-catalyzed click chemistry, was synthesized in situ by incubating either E. coli 70S ribosomes or 50S subunits with macrolide-functionalized azide 2 and 3-ethynylaniline (3) precursors. The ribosome-templated in situ click method was expanded from a binary reaction (i.e., one azide and one alkyne) to a six-component reaction (i.e., azide 2 and five alkynes) and ultimately to a 16-component reaction (i.e., azide 2 and 15 alkynes). The extent of triazole formation correlated with ribosome affinity for the anti (1,4)-regioisomers as revealed by measured Kd values. Computational analysis using the site-identification by ligand competitive saturation (SILCS) approach indicated that the relative affinity of the ligands was associated with the alteration of macrolactone+desosamine-ribosome interactions caused by the different alkynes. Protein synthesis inhibition experiments confirmed the mechanism of action. Evaluation of the minimal inhibitory concentrations (MIC) quantified the potency of the in situ click products and demonstrated the efficacy of this method in the triaging and prioritization of potent antibiotics that target the bacterial ribosome. Cell viability assays in human fibroblasts confirmed 2 and four analogues with therapeutic indices for bactericidal activity over in vitro mammalian cytotoxicity as essentially identical to solithromycin (1).

Design, synthesis, and evaluation of (2S,4R)-Ketoconazole sulfonamide analogs as potential treatments for Metabolic Syndrome.

Metabolic Syndrome, also referred to as 'Syndrome X' or 'Insulin Resistance Syndrome,' remains a major, unmet medical need despite over 30years of intense effort. Recent research suggests that there may be a causal link between this condition and abnormal glucocorticoid processing. Specifically, dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) axis leads to increased systemic cortisol concentrations. Cushing' syndrome, a disorder that is also typified by a marked elevation in levels of cortisol, produces clinical symptomology that is similar to those observed in MetS, and they can be alleviated by decreasing circulating cortisol concentrations. As a result, it has been suggested that decreasing systemic cortisol concentration might have a positive impact on the progression of MetS. This could be accomplished through inhibition of enzymes in the cortisol synthetic pathway, 11ß-hydroxylase (Cyp11B1), 17α-hydroxylase-C17,20-lyase (Cyp17), and 21-hydroxylase (Cyp21). We have identified a series of novel sulfonamide analogs of (2S,4R)-Ketoconazole that are potent inhibitors of these enzymes. In addition, selected members of this class of compounds have pharmacokinetic properties consistent with orally delivered drugs, making them well suited to further investigation as potential therapies for MetS.

Small-molecule inhibitors of Staphylococcus aureus RnpA-mediated RNA turnover and tRNA processing.

New agents are urgently needed for the therapeutic treatment of Staphylococcus aureus infections. In that regard, S. aureus RNase RnpA may represent a promising novel dual-function antimicrobial target that participates in two essential cellular processes, RNA degradation and tRNA maturation. Accordingly, we previously used a high-throughput screen to identify small-molecule inhibitors of the RNA-degrading activity of the enzyme and showed that the RnpA inhibitor RNPA1000 is an attractive antimicrobial development candidate. In this study, we used a series of in vitro and cellular assays to characterize a second RnpA inhibitor, RNPA2000, which was identified in our initial screening campaign and is structurally distinct from RNPA1000. In doing so, it was found that S. aureus RnpA does indeed participate in 5'-precursor tRNA processing, as was previously hypothesized. Further, we show that RNPA2000 is a bactericidal agent that inhibits both RnpA-associated RNA degradation and tRNA maturation activities both in vitro and within S. aureus. The compound appears to display specificity for RnpA, as it did not significantly affect the in vitro activities of unrelated bacterial or eukaryotic ribonucleases and did not display measurable human cytotoxicity. Finally, we show that RNPA2000 exhibits antimicrobial activity and inhibits tRNA processing in efflux-deficient Gram-negative pathogens. Taken together, these data support the targeting of RnpA for antimicrobial development purposes, establish that small-molecule inhibitors of both of the functions of the enzyme can be identified, and lend evidence that RnpA inhibitors may have broad-spectrum antimicrobial activities.

Nuclear magnetic resonance structure revealed that the human polyomavirus JC virus agnoprotein contains an α-helix encompassing the Leu/Ile/Phe-rich domain.

UNLABELLED: Agnoprotein is a small multifunctional regulatory protein required for sustaining the productive replication of JC virus (JCV). It is a mostly cytoplasmic protein localizing in the perinuclear area and forms highly stable dimers/oligomers through a Leu/Ile/Phe-rich domain. There have been no three-dimensional structural data available for agnoprotein due to difficulties associated with the dynamic conversion from monomers to oligomers. Here, we report the first nuclear magnetic resonance (NMR) structure of a synthetic agnoprotein peptide spanning amino acids Thr17 to Glu55 where Lys23 to Phe39 encompassing the Leu/Ile/Phe-rich domain forms an amphipathic α-helix. On the basis of these structural data, a number of Ala substitution mutations were made to investigate the role of the α-helix in the structure and function of agnoprotein. Single L29A and L36A mutations exhibited a significant negative effect on both protein stability and viral replication, whereas the L32A mutation did not. In addition, the L29A mutant displayed a highly nuclear localization pattern, in contrast to the pattern for the wild type (WT). Interestingly, a triple mutant, the L29A+L32A+L36A mutant, yielded no detectable agnoprotein expression, and the replication of this JCV mutant was significantly reduced, suggesting that Leu29 and Leu36 are located at the dimer interface, contributing to the structure and stability of agnoprotein. Two other single mutations, L33A and E34A, did not perturb agnoprotein stability as drastically as that observed with the L29A and L36A mutations, but they negatively affected viral replication, suggesting that the role of these residues is functional rather than structural. Thus, the agnoprotein dimerization domain can be targeted for the development of novel drugs active against JCV infection. IMPORTANCE: Agnoprotein is a small regulatory protein of JC virus (JCV) and is required for the successful completion of the viral replication cycle. It forms highly stable dimers and oligomers through its hydrophobic (Leu/Ile/Phe-rich) domain, which has been shown to play essential roles in the stability and function of the protein. In this work, the Leu/Ile/Phe-rich domain has been further characterized by NMR studies using an agnoprotein peptide spanning amino acids T17 to Q54. Those studies revealed that the dimerization domain of the protein forms an amphipathic α-helix. Subsequent NMR structure-based mutational analysis of the region highlighted the critical importance of certain amino acids within the α-helix for the stability and function of agnoprotein. In conclusion, this study provides a solid foundation for developing effective therapeutic approaches against the dimerization domain of the protein to inhibit its critical roles in JCV infection.

Personalized synthetic lethality induced by targeting RAD52 in leukemias identified by gene mutation and expression profile.

Homologous recombination repair (HRR) protects cells from the lethal effect of spontaneous and therapy-induced DNA double-stand breaks. HRR usually depends on BRCA1/2-RAD51, and RAD52-RAD51 serves as back-up. To target HRR in tumor cells, a phenomenon called "synthetic lethality" was applied, which relies on the addiction of cancer cells to a single DNA repair pathway, whereas normal cells operate 2 or more mechanisms. Using mutagenesis and a peptide aptamer approach, we pinpointed phenylalanine 79 in RAD52 DNA binding domain I (RAD52-phenylalanine 79 [F79]) as a valid target to induce synthetic lethality in BRCA1- and/or BRCA2-deficient leukemias and carcinomas without affecting normal cells and tissues. Targeting RAD52-F79 disrupts the RAD52-DNA interaction, resulting in the accumulation of toxic DNA double-stand breaks in malignant cells, but not in normal counterparts. In addition, abrogation of RAD52-DNA interaction enhanced the antileukemia effect of already-approved drugs. BRCA-deficient status predisposing to RAD52-dependent synthetic lethality could be predicted by genetic abnormalities such as oncogenes BCR-ABL1 and PML-RAR, mutations in BRCA1 and/or BRCA2 genes, and gene expression profiles identifying leukemias displaying low levels of BRCA1 and/or BRCA2. We believe this work may initiate a personalized therapeutic approach in numerous patients with tumors displaying encoded and functional BRCA deficiency.

Heterocyclic chalcone activators of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) with improved in vivo efficacy.

Nrf2 activators represent a good drug target for designing agents to treat diseases associated with oxidative stress. Building upon previous work, we designed and prepared a series of heterocyclic chalcone-based Nrf2 activators with reduced lipophilicity and, in some cases, greater in vitro potency compared to the respective carbocyclic scaffold. These changes resulted in enhanced oral bioavailability and a superior pharmacodynamic effect in vivo.