RNA-dependent RNA polymerase, RdRP, a promising therapeutic target for cancer and potentially COVID-19.

A recent outbreak of coronavirus disease (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 has driven a global pandemic with catastrophic consequences. The rapid development of promising therapeutic strategies against COVID-19 is keenly anticipated. Family Coronaviridae comprises positive, single-stranded RNA viruses that use RNA-dependent RNA polymerase (RdRP) for viral replication and transcription. As the RdRP of viruses in this family and others plays a pivotal role in infection, it is a promising therapeutic target for developing antiviral agents against them. A critical genetic driver for many cancers is the catalytic subunit of telomerase: human telomerase reverse transcriptase (hTERT), identified initially as an RNA-dependent DNA polymerase. However, even though hTERT is a DNA polymerase, it has phylogenetic and structural similarities to viral RdRPs. Researchers worldwide, including the authors of this review, are engaged in developing therapeutic strategies targeting hTERT. We have published a series of papers reporting that hTERT has RdRP activity and that this RdRP activity in hTERT is essential for tumor formation. Here, we review the enzymatic function of RdRP in virus proliferation and tumor development, reminding us of how the study of the novel coronavirus has brought us to the unexpected intersection of cancer research and RNA virus research.

The Werner Protein Acts as a Coactivator of Nuclear Factor κB (NF-κB) on HIV-1 and Interleukin-8 (IL-8) Promoters.

The Werner syndrome helicase (WRN) plays a role in maintaining genomic stability. The lack of WRN results in Werner syndrome, a rare autosomal recessive genetic disorder, which causes premature aging accompanied by many complications such as rare forms of cancer and type 2 diabetes. However, the underlying mechanisms of these complications, arising due to the loss of WRN, are poorly understood. In this study, we demonstrated the function of WRN in transcriptional regulation of NF-κB targets. WRN physically interacts via its RecQ C-terminal (RQC) domain with the Rel homology domain of both the RelA (p65) and the p50 subunits of NF-κB. In the steady state, WRN is recruited to HIV-1 long terminal repeat (LTR), a typical NF-κB-responsive promoter, as well as the p50/p50 homodimer, in an NF-κB site-dependent manner. The amount of WRN on LTR increased along with the transactivating RelA/p50 heterodimer in response to TNF-α stimulation. Further, a knockdown of WRN reduced the transactivation of LTR in exogenous RelA/p50-introduced or TNF-α-stimulated cells. Additionally, knockdown of WRN reduced TNF-α stimulation-induced activation of the endogenous promoter of IL-8, an NF-κB-responsive gene, and WRN increased its association with the IL-8 promoter region together with RelA/p50 after TNF-α stimulation. In conjunction with studies that have shown NF-κB to be a key regulator of aging and inflammation, our results indicate a novel role of WRN in transcriptional regulation. Along with NF-κB, the loss of WRN is expected to result in incorrect regulation of downstream targets and leads to immune abnormalities and homeostatic disruption.

Discovery of m(7)G-cap in eukaryotic mRNAs.

Terminal structure analysis of an insect cytoplasmic polyhedrosis virus (CPV) genome RNA in the early 1970s at the National Institute of Genetics in Japan yielded a 2'-O-methylated nucleotide in the 5' end of double-stranded RNA genome. This finding prompted me to add S-adenosyl-L-methionine, a natural methylation donor, to the in vitro transcription reaction of viruses that contain RNA polymerase. This effort resulted in unprecedented mRNA synthesis that generates a unique blocked and methylated 5' terminal structure (referred later to as "cap" or "m(7)G-cap") in the transcription of silkworm CPV and human reovirus and vaccinia viruses that contain RNA polymerase in virus particles. Initial studies with viruses paved the way to discover the 5'-cap m(7)GpppNm structure present generally in cellular mRNAs of eukaryotes. I participated in those studies and was able to explain the pathway of cap synthesis and the significance of the 5' cap (and capping) in gene expression processes, including transcription and protein synthesis. In this review article I concentrate on the description of these initial studies that eventually led us to a new paradigm of mRNA capping.

RECQL4 is essential for the transport of p53 to mitochondria in normal human cells in the absence of exogenous stress.

Mutations in RECQL4 helicase are associated with Rothmund-Thomson syndrome (RTS). A subset of RTS patients is predisposed to cancer and is sensitive to DNA damaging agents. The enhanced sensitivity of cells from RTS patients correlates with the accumulation of transcriptionally active nuclear p53. We found that in untreated normal human cells these two nuclear proteins, p53 and RECQL4, instead colocalize in the mitochondrial nucleoids. RECQL4 accumulates in mitochondria in all phases of the cell cycle except S phase and physically interacts with p53 only in the absence of DNA damage. p53-RECQL4 binding leads to the masking of the nuclear localization signal of p53. The N-terminal 84 amino acids of RECQL4 contain a mitochondrial localization signal, which causes the localization of RECQL4-p53 complex to the mitochondria. RECQL4-p53 interaction is disrupted after stress, allowing p53 translocation to the nucleus. In untreated normal cells RECQL4 optimizes de novo replication of mtDNA, which is consequently decreased in fibroblasts from RTS patients. Wild-type RECQL4-complemented RTS cells show relocalization of both RECQL4 and p53 to the mitochondria, loss of p53 activation, restoration of de novo mtDNA replication and resistance to different types of DNA damage. In cells expressing Δ84 RECQL4, which cannot translocate to mitochondria, all the above functions are compromised. The recruitment of p53 to the sites of de novo mtDNA replication is also regulated by RECQL4. Thus these findings elucidate the mechanism by which p53 is regulated by RECQL4 in unstressed normal cells and also delineates the mitochondrial functions of the helicase.

Antigen itself antibody: an alternative one-step immunoassay for measuring the anti-idiotypic antibody titer.

Immunoassay designs rely on the great specificity of antibodies and a suitable marker that facilitates generation of a quantitative signal. Currently, there is no reliable method for measuring the titers of an anti-idiotypic antibody. Our initial attempt to measure titers of mouse anti-idiotypic antibody after idiotypic vaccination with HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT) failed. Because the injected antigen, nmAb-KT, is a mouse IgG, using a commercial antibody to measure the antibody titer always gave a false positive signal against control mouse serum antibody in parallel with the antigen-treated immunized serum antibodies. To get a reliable and clearly differentiable signal by ELISA, idiotypic antigen was labeled with HRP and HRP-conjugated-nmAb-KT used to measure the antibody titers in the antigen-treated mice. Compared with control mice, signals were found in high anti-nmAb-KT IgG responses in test mice; however, untreated control mice had a significant amount of purified non-specific IgG. This method is amenable to long read lengths and will likely enable anti-idiotypic antibody titer measurement in a more specific and cost effective way without requiring commercial antibody.

The high-osmolarity glycerol- and cell wall integrity-MAP kinase pathways of Saccharomyces cerevisiae are involved in adaptation to the action of killer toxin HM-1.

Fps1p is an aquaglyceroporin important for turgor regulation of Saccharomyces cerevisiae. Previously we reported the involvement of Fps1p in the yeast-killing action of killer toxin HM-1. The fps1 cells showed a high HM-1-resistant phenotype in hypotonic medium and an HM-1-susceptible phenotype in hypertonic medium. This osmotic dependency in HM-1 susceptibility was similar to those observed in Congo red, but different from those observed in other cell wall-disturbing agents. These results indicate that HM-1 exerts fungicidal activity mainly by binding and inserting into the yeast cell wall structure, rather than by inhibiting 1,3-ß-glucan synthase. We next determined HM-1-susceptibility and diphospho-MAP kinase inductions in S. cerevisiae. In the wild-type cell, expressions of diphospho-Hog1p and -Slt2p, and mRNA transcription of CWP1 and HOR2, were induced within 1 h after an addition of HM-1. ssk1 and pbs2 cells, but not sho1 and hkr1 cells, showed HM-1-sensitive phenotypes and lacked inductions of phospho-Hog1p in response to HM-1. mid2, rom2 and bck1 cells showed HM-1-sensitive phenotypes and decreased inductions of phospho-Slt2p in response to HM-1. From these results, we postulated that the Sln1-Ypd1-Ssk1 branch of the high-osmolality glycerol (HOG) pathway and plasma membrane sensors of the cell wall integrity (CWI) pathway detect cell wall stresses caused by HM-1. We further suggested that activations of both HOG and CWI pathways have an important role in the adaptive response to HM-1 toxicity.

New avenues for phage-display library to produce a Cryptococcus-specific anti-idiotypic antibody of HM-1 killer toxin.

Existing antifungal drugs are notable for their inability to act rapidly, as well as their toxicity and limited spectrum. The identification of fungal-specific genes and virulence factors would provide targets for new and influential drugs. The display of repertories of antibody fragments on the surface of filamentous phage offers a new way to produce immunoreagents as defined specificities. Here we report the selection of Cryptococcus-specific targets by using phage-display panning from a cDNA library, where bactericidal antibodies have been developed against conserved surface-exposed antigens. A single-chain variable fragment (scFv) phage library was constructed from splenocyte of an immunized mouse by idiotypic vaccination with HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) that was used for selection against Cryptococcus neoformans membrane fraction (CnMF). Key elements were the selection against antigen (nmAb-KT and CnMF) and the release of bound phages using competitive panning elution with CnMF at neutral pH condition. Isolated scFvs react specifically with C. neoformans and some other pathogenic and non-pathogenic fungal strain's cell wall receptors by exerting strong antifungal activity in vitro. A high affinity clone, designated M1 was selected for detailed characterization and tested anti-cryptococcal activity with IC(50) values at 5.33 × 10(-7) to 5.56 × 10(-7) M against C. neoformans. The method described here is a new technique for the isolation of cell membrane specific immunoreactive phages in the form of scFv using CnMF that contained cell membrane associated proteins.

Genome-wide screen of Saccharomyces cerevisiae for killer toxin HM-1 resistance.

We screened a set of Saccharomyces cerevisiae deletion mutants for resistance to killer toxin HM-1, which kills susceptible yeasts through inhibiting 1,3-beta-glucan synthase. By using HM-1 plate assay, we found that eight gene-deletion mutants had higher HM-1-resistance compared with the wild-type. Among these eight genes, five--ALG3, CAX4, MNS1, OST6 and YBL083C--were associated with N-glycan formation and maturation. The ALG3 gene has been shown before to be highly resistant to HM-1. The YBL083C gene may be a dubious open reading frame that overlaps partially the ALG3 gene. The deletion mutant of the MNS1 gene that encodes 1,2-alpha-mannosidase showed with a 13-fold higher HM-1 resistance compared with the wild-type. By HM-1 binding assay, the yeast plasma membrane fraction of alg3 and mns1 cells had less binding ability compared with wild-type cells. These results indicate that the presence of the terminal 1,3-alpha-linked mannose residue of the B-chain of the N-glycan structure is essential for interaction with HM-1. A deletion mutant of aquaglyceroporin Fps1p also showed increased HM-1 resistance. A deletion mutant of osmoregulatory mitogen-activated protein kinase Hog1p was more sensitive to HM-1, suggesting that high-osmolarity glycerol pathways plays an important role in the compensatory response to HM-1 action.

An altered camelid-like single domain anti-idiotypic antibody fragment of HM-1 killer toxin: acts as an effective antifungal agent.

Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species. The alternative small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data showed that the binding affinity for this single-domain antibody was 272-fold higher (K(d)=1.07×10(-10) M) and antifungal activity was three- to fivefold more efficient (IC(50)=0.46×10(-6) to 1.17×10(-6) M) than that for the conventional antibody (K(d)=2.91×10(-8) M and IC(50)=2.14×10(-6) to 3.78×10(-6) M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain synthetic antibodies will find their way into a number of biotechnological or medical applications.

Peptide derived from anti-idiotypic single-chain antibody is a potent antifungal agent compared to its parent fungicide HM-1 killer toxin peptide.

Based on anti-idiotypic network theory in light of the need for new antifungal drugs, we attempted to identify biologically active fragments from HM-1 yeast killer toxin and its anti-idiotypic antibody and to compare their potency as an antifungal agent. Thirteen overlapping peptides from HM-1 killer toxin and six peptides from its anti-idiotypic single-chain variable fragment (scFv) antibodies representing the complementarity determining regions were synthesized. The binding affinities of these peptides were investigated and measured by Dot blot and surface plasmon resonance analysis and finally their antifungal activities were investigated by inhibition of growth, colony forming unit assay. Peptide P6, containing the potential active site of HM-1 was highly capable of inhibiting the growth of Saccharomyces cerevisiae but was less effective on pathogenic fungi. However, peptide fragments derived from scFv antibody exerted remarkable inhibitory effect on the growth of pathogenic strains of Candida and Cryptococcus species in vitro. One scFv-derived decapeptide (SP6) was selected as the strongest killer peptide for its high binding affinity and antifungal abilities on both Candida and Cryptococcus species with IC(50) values from 2.33 × 10(-7) M to 36.0 × 10(-7) M. SP6 peptide activity was neutralized by laminarin, a ß-1,3-glucan molecule, indicating this peptide derived from scFv anti-idiotypic antibody retains antifungal activity through interaction with cell wall ß-glucan of their target fungal cells. Experimental evidence strongly suggested the possibility of development of anti-idiotypic scFv peptide-based antifungal agents which may lead to improve therapeutics for the management of varieties of fungal infections.

Detection of siRNA administered to cells and animals by using a fluorescence intensity distribution analysis polarization system.

Small interfering RNA (siRNA) has excellent pharmacological features and is expected to be used for therapeutic drug development. To this end, however, new RNA technology needs to be established so that extremely small amounts (less than 1 pmol) of siRNA can be detected in organs of experimental animals and in human blood to facilitate pharmacokinetics studies. An important feature is that this new technology is not dependent on radioisotopes and can detect siRNA molecules identical to those used for drug development in preclinical tests with experimental animals or in clinical tests with humans. We report a convenient method that can detect small amounts of siRNA. The method uses high-power confocal microscopic analysis of fluorescence polarization in DNA probes that are bound to one of the strands of siRNA and directly quantitates the copy number of siRNA molecule after extraction from specimens. A pharmacokinetic study to examine the blood retention time of siRNA/cationic liposomes in mice showed that this straightforward method is consistent with the other reverse transcriptase polymerase chain reaction amplification-based method. We believe that the entire process is simple and applicable for a high-throughput analysis, which provides excellent technical support for fundamental research on RNA interference and development of siRNA drugs.

Purification and functional characterization of a Camelid-like single-domain antimycotic antibody by engineering in affinity tag.

Single-domain single-chain variable fragment (scFv) antibody is sometimes critical for purification using affinity tagging strategy. We failed in our initial effort to purify a prematurely developed Camelid-like E-tagged short scFv-K2 antibody that contained a complete variable region of the heavy chain and partial region of the light chain by using an anti-E-tag affinity column. To expedite the purification of this altered but interesting antimycotic agent, we replaced a long and large E-tag by a short and hydrophilic 6x-Histidine (His(6)) affinity tag by polymerase chain reaction. The short and compact His(6)-tag was placed on the previously constructed expression vector pCANTAB 5 E that contained the large affinity E-tag sequence (13 amino acids) by PCR-based mutagenesis and was expressed in Escherichia coli. The recombinant protein can then be purified by immobilized metal affinity chromatography (IMAC) and be used for biochemical and other functional characterization. This His(6)-tagged short scFv-K2 antibody (20 kDa) had strong cytocidal activity against Saccharomyces and Candida species with a IC(50) value of 0.44x10(-6)M and 1.10 x 10(-6)M, respectively. Tag replacement facilitates the purification of a Camelid-like single-domain scFv antibody and after that meets its different functional characteristics. The present study reflects that the V(H) domain of the scFv antibody is mainly responsible for its biological activity and single-domain scFv antibody may acts as a potent antimicrobial agent.

An improved phage-display panning method to produce an HM-1 killer toxin anti-idiotypic antibody.

BACKGROUND: Phage-display panning is an integral part of biomedical research. Regular panning methods are sometimes complicated by inefficient detachment of the captured phages from the antigen-coated solid supports, which prompted us to modify. Here, we produce an efficient antigen-specific single chain fragment variable (scFv) antibody by using a target-related molecule that favored selection of recombinant antibodies. RESULTS: To produce more selective and specific anti-idiotypic scFv-antibodies from a cDNA library, constructed from HM-1 killer toxin (HM-1)-neutralizing monoclonal antibodies (nmAb-KT), the method was modified by using an elution buffer supplemented with HM-1 that shares structural and functional similarities with the active site of the scFv antibody. Competitive binding of HM-1 to nmAb-KT allowed easy and quick dissociation of scFv-displayed phages from immobilized nmAb-KT to select specific anti-idiotypic scFv antibodies of HM-1. After modified panning, 80% clones (40/50) showed several times higher binding affinity to nmAb-KT than regular panning. The major populations (48%) of these clones (scFv K1) were genotypically same and had strong cytocidal activity against Saccharomyces and Candida species. The scFv K1 (K(d) value = 4.62 x 10(-8) M) had strong reactivity toward nmAb-KT, like HM-1 (K(d) value = 6.74 x 10(-9) M) as judged by SPR analysis. CONCLUSION: The scFv antibodies generated after modified subtractive panning appear to have superior binding properties and cytocidal activity than regular panning. A simple modification of the elution condition in the phage-display panning protocol makes a large difference in determining success. Our method offers an attractive platform to discover potential therapeutic candidates.

Quantitation of full-size small interfering RNA by tailing with terminal deoxynucleotidyl transferase and reverse transcription-polymerase chain reaction analysis.

Accurate estimation of small interfering RNA (siRNA) concentration in cells and blood is increasingly important for pharmacokinetic studies required to develop siRNA drugs. We report a method that detects siRNA having 3'-terminal deoxynucleotide overhangs, such as 3'-dTdT, present in most chemically synthesized siRNAs. Short overhangs were elongated to oligo-dG by incubation with terminal deoxynucleotidyl transferase and dGTP and were used as priming sites for reverse transcription of siRNA to complementary DNA (cDNA). The resultant cDNA was used as a template for quantitation by polymerase chain reaction. This method was reliable for determining the pharmacokinetics of siRNA in blood of injected mice.

Cloning antifungal single chain fragment variable antibodies by phage display and competitive panning elution.

Phage display and two competitive panning elution conditions were used to isolate Candida-specific single chain fragment variable (scFv) antibodies. An scFv phage library constructed from splenic lymphocytes of mice immunized by idiotypic vaccination with an HM-1 killer toxin (HM-1)-neutralizing monoclonal antibody (nmAb-KT) was used for panning against Candidaalbicans membrane fraction (CaMF). Key steps were specific elution conditions to separately release the bound phages with original antigen HM-1+HM-1 peptide 6 and CaMF. The positive phages were screened by using enzyme-linked immunosorbent assay, and after nucleotide sequencing, clone expression, and purification, clone scFv-C1 was selected for detailed characterization. The scFv-C1 showed IC(50) values for cell growth against various Candida species and Saccharomyces cerevisiae as 2.40 to 6.40microM and 2.20microM, respectively. By using surface plasmon resonance analysis, the scFv-C1 had a K(d) value of 3.09x10(-11)M to nmAb-KT, indicating a 260-fold higher affinity than for HM-1. These results showed the generated scFv-C1 mimicking HM-1-binding affinity to nmAb-KT and in vitro antifungal activity. We believe that the effectiveness of the competitive panning elution method and antigen-specific recombinant scFv antibodies obtained in this study are excellent candidates for antimycotic drugs.

Association of human DNA helicase RecQ5beta with RNA polymerase II and its possible role in transcription.

Although RecQ5beta is a ssDNA (single-stranded DNA)-stimulated ATPase and an ATP-dependent DNA helicase with strand-annealing activities, its cellular function remains to be explored. In the present paper, we used immunopurification and MS-based analyses to show that human DNA helicase RecQ5beta is associated with at least four RNAP II (RNA polymerase II) subunits. RecQ5beta was also present in complexes immunoprecipitated using three different antibodies against the large subunit of RNAP II, or in complexes immunoprecipitated using an anti-FLAG antibody against either FLAG-RNAP II 33 kDa subunit or FLAG-Pin1. Different regions of the non-helicase domain of the RecQ5beta molecule were associated with hypophosphorylated and hyperphosphorylated forms of the RNAP II large subunit independently of DNA and RNA. RecQ5beta was also found in nuclear chromatin fractions and associated with the coding regions of the LDL (low-density lipoprotein) receptor and beta-actin genes. Knockdown of the RecQ5beta transcript increased the transcription of those genes. The results of the present study suggest that RecQ5beta has suppressive roles in events associated with RNAP II-dependent transcription.

Role of Werner syndrome gene product helicase in carcinogenesis and in resistance to genotoxins by cancer cells.

Werner syndrome (WS) is an autosomal recessive genetic disorder causing premature aging, and WRN has been identified as the causative gene of WS. The product of the WRN gene (WRN) acts as a DNA helicase with exonuclease activity, and data have accumulated showing that the WRN gene strongly participates in carcinogenesis: (1) the normal WRN gene likely participates in the immortalization of B-lymphoblastoid cell lines through telomeric crisis caused by telomere shortening, (2) a much higher incidence of rare cancers occurs in WS patients than in other kinds of patients, and (3) levels of WRN expressed in virus-transformed cells and cancer cells are usually markedly up-regulated and are inversely correlated with the sensitivity of these cells against various genotoxins, including camptothecin. In this paper, we review the events that show a close correlation of the WRN gene and WRN with carcinogenesis and their underlying molecular mechanisms.

Anticancer activity of RecQL1 helicase siRNA in mouse xenograft models.

Small interfering RNAs (siRNAs) are expected to have a medical application in human therapy as drugs with a high specificity for their molecular target mRNAs. RecQL1 DNA helicase in the human RecQ helicase family participates in DNA repair and recombination pathways in the cell cycle of replication. Silencing the RecQL1 expression by RecQL1-siRNA induces mitotic death in vitro specifically in growing cancer cells. By contrast, the same RecQL1 silencing does not affect the growth of normal cells, emphasizing that RecQL1 helicase is an ideal molecular target for cancer therapy. In this study, we show that local and systemic administration of RecQL1-siRNA mixed with polyethyleneimine polymer or cationic liposomes prevented cancer cell proliferation in vivo in mouse models of cancer without noticeable adverse effects. The results indicate that RecQL1-siRNA in a complex with a cationic polymer is a very promising anticancer drug candidate, and that in particular, RecQL1-siRNA formulated with a cationic liposome has an enormous potential to be used by intravenous injection for therapy specific for liver cancers, including metastasized cancers from the colon and pancreas.

Induction of mitotic cell death in cancer cells by small interference RNA suppressing the expression of RecQL1 helicase.

RecQL1 DNA helicase of the human RecQ helicase family participates in DNA repair and recombination pathways during cell-cycle replication. When we examined the effect of RecQL1 suppression on cell growth, we found that RecQL1 silencing by small interference RNA efficiently prevented proliferation of a wide range of cancer cells by inducing mitotic catastrophe and mitotic cell death. In contrast, such mitotic cell death was not seen in the growing normal fibroblasts used as controls, even if RecQL1 expression was fully downregulated. Our results support the hypothesis that endogenous DNA damage that occurs during DNA replication and remains unrepaired in cancer cells due to RecQL1 silencing induces cancer cell-specific mitotic catastrophe through a less-strict checkpoint in cancer cells than in normal cells. We speculate that normal cells are exempt from such mitotic cell death, despite slow growth, because cell-cycle progression is controlled strictly by a strong checkpoint system that detects DNA damage and arrests progression of the cell cycle until DNA damage is repaired completely. These results suggest that RecQL1 helicase is an excellent molecular target for cancer chemotherapy.

A novel small-molecule inhibitor of NF-kappaB signaling.

The inducible transcription factor NF-kappaB regulates divergent signaling pathways including inflammatory response and cancer development. Selective inhibitors for NF-kappaB signaling are potentially useful for treatment of inflammation and cancer. NF-kappaB is canonically activated by preferential disposal of its inhibitory protein; IkappaB, which suppresses the nuclear translocation of NF-kappaB. IkappaBalpha (a major member of IkappaB family proteins) is phosphorylated with an IkappaB kinase (IKK) and subsequently polyubiquitylated by SCF(betaTrCP1) ubiquitin-ligase in the presence of E1 and E2 prior to proteasomal degradation. Here, we describe a novel inhibitor termed GS143, which suppressed IkappaBalpha ubiquitylation, but not IkappaBalpha phosphorylation, MDM2-directed p53 ubiquitylation, and proteasome activity in vitro. GS143 markedly suppressed the destruction of IkappaBalpha stimulated by TNFalpha and a set of downstream responses coupled to NF-kappaB signaling but not those of p53 and beta-catenin in vivo. Our results indicate that GS143 serves as an effective inhibitor of multiple pathways served by NF-kappaB signaling.