Potency of a small molecule that targets the molluscum contagiosum virus processivity factor increases when conjugated to a tripeptide.

We recently developed compound FC-7269 for targeting the Molluscum contagiosum virus processivity factor (mD4) and demonstrated its ability to inhibit viral processive DNA synthesis in vitro and cellular infection of an mD4-dependent virus (Antiviral Res 211, 2023,105520). However, despite a thorough medicinal chemistry campaign we were unable to generate a potent second analog as a requisite for drug development. We overcame this impasse, by conjugating a short hydrophobic trivaline peptide to FC-7269 to produce FC-TriVal-7269 which significantly increased antiviral potency and reduced cellular toxicity.

A small molecule that targets the processivity factor of molluscum contagiosum virus has therapeutic potential.

Molluscum contagiosum (MC) is an infectious disease that occurs only in humans with a tropism that is narrowly restricted to the outermost epidermal layer of the skin. Molluscum contagiosum virus (MCV) is the causative agent of MC which produces skin lesions that can persist for months to several years. MCV is efficiently transmitted by direct physical contact or by indirect contact with fomites. MC is most prevalent in children and immune compromised patients. The failure to develop a drug that targets MCV replication has been hampered for decades by the inability to propagate MCV in cell culture. To address this dilemma, we recently engineered a surrogate poxvirus expressing the MCV processivity factor (mD4) as the drug target. The mD4 protein is essential for viral replication by keeping the viral polymerase tethered to the DNA template. In this study we have designed and synthesized a lead compound (7269) that is able to prevent mD4 dependent processive DNA synthesis in vitro (IC50 = 6.8 µM) and effectively inhibit propagation of the mD4-VV surrogate virus in BSC-1 cells (EC50 = 13.2 µM) with negligible cytotoxicity. In human liver microsomes, 7269 was shown to be stable for almost 2 h. When tested for penetration into human cadaver skin in a formulated gel, the level of 7269 in the epidermal layer was nearly 100 times the concentration (EC50) needed to inhibit propagation of the mD4-VV surrogate virus in BSC-1 cells. The gel formulated 7269 was scored as a non-irritant on skin and shown to have a shelf-life that was completely stable after several months. In summary, 7269 is a potential Lead for becoming the first MCV anti-viral compound to treat MC and thereby, addresses this unmet medical need that has persisted for many decades.

Debulking different Corona (SARS-CoV-2 delta, omicron, OC43) and Influenza (H1N1, H3N2) virus strains by plant viral trap proteins in chewing gums to decrease infection and transmission.

Because oral transmission of SARS-CoV-2 is 3-5 orders of magnitude higher than nasal transmission, we investigated debulking of oral viruses using viral trap proteins (CTB-ACE2, FRIL) expressed in plant cells, delivered through the chewing gum. In omicron nasopharyngeal (NP) samples, the microbubble count (based on N-antigen) was significantly reduced by 20 µg of FRIL (p < 0.0001) and 0.925 µg of CTB-ACE2 (p = 0.0001). Among 20 delta or omicron NP samples, 17 had virus load reduced below the detection level of spike protein in the RAPID assay, after incubation with the CTB-ACE2 gum powder. A dose-dependent 50% plaque reduction with 50-100 ng FRIL or 600-800 µg FRIL gum against Influenza strains H1N1, H3N2, and Coronavirus HCoV-OC43 was observed with both purified FRIL, lablab bean powder or gum. In electron micrographs, large/densely packed clumps of overlapping influenza particles and FRIL protein were observed. Chewing simulator studies revealed that CTB-ACE2 release was time/dose-dependent and release was linear up to 20 min chewing. Phase I/II placebo-controlled, double-blinded clinical trial (IND 154897) is in progress to evaluate viral load in saliva before or after chewing CTB-ACE2/placebo gum. Collectively, this study advances the concept of chewing gum to deliver proteins to debulk oral viruses and decrease infection/transmission.

Discovery of a potent cytotoxic agent that promotes G2/M phase cell cycle arrest and apoptosis in a malignant human pharyngeal squamous carcinoma cell line.

Squamous cell carcinoma is the major form of malignancy that arises in head and neck cancer. The modest improvement in the 5­year survival rate underpins its complex etiology and provides the impetus for the discovery of new therapeutics. The present study describes the discovery of an indole­based small molecule (24a) that was a potent cytotoxic agent with antiproliferative and pro­apoptotic properties against a pharyngeal carcinoma cell line, Detroit 562, effectively killing the cells at a half­maximal inhibitory concentration of 0.03 µM, as demonstrated using cell proliferation studies. The antiproliferative property of 24a was demonstrated by its ability to promote G2/M blockade, as assessed by cell cycle analysis using flow cytometry and the monitoring of real­time cell cycle progression by the fluorescence ubiquitination­based cell cycle indicator. This pro­apoptotic property is supported by the promotion of TUNEL­staining and increase in the activities of caspases­3/7 and ­6, in addition to the expression of death receptors and the cleavage of poly (ADP­ribose) polymerase 1 protein as demonstrated by western blotting. Given that Detroit 562 lacks functional p53, it is suggested that 24a acts independently of the tumor suppressor.

Herpes Simplex Virus-1 infection in human primary corneal epithelial cells is blocked by a stapled peptide that targets processive DNA synthesis.

PURPOSE: Acyclovir is most commonly used for treating ocular Herpes Keratitis, a leading cause of infectious blindness. However, emerging resistance to Acyclovir resulting from mutations in the thymidine kinase gene of Herpes Simplex Virus -1 (HSV-1), has prompted the need for new therapeutics directed against a different viral protein. One novel target is the HSV-1 Processivity Factor which is essential for tethering HSV-1 Polymerase to the viral genome to enable long-chain DNA synthesis. METHODS: A series of peptides, based on the crystal structure of the C-terminus of HSV-1 Polymerase, were constructed with hydrocarbon staples to retain their alpha-helical conformation. The stapled peptides were tested for blocking both HSV-1 DNA synthesis and infection. The most effective peptide was further optimized by replacing its negative N-terminus with two hydrophobic valine residues. This di-valine stapled peptide was tested for inhibiting HSV-1 infection of human primary corneal epithelial cells. RESULTS: The stapled peptides blocked HSV-1 DNA synthesis and HSV-1 infection. The unstapled control peptide had no inhibitory effects. Specificity of the stapled peptides was confirmed by their inabilities to block infection by an unrelated virus. Significantly, the optimized di-valine stapled peptide effectively blocked HSV-1 infection in human primary corneal epithelial cells with selectivity index of 11.6. CONCLUSIONS: Hydrocarbon stapled peptides that simulate the α-helix from the C-terminus of HSV-1 DNA polymerase can specifically block DNA synthesis and infection of HSV-1 in human primary corneal epithelial cells. These stapled peptides provide a foundation for developing a topical therapeutic for treating human ocular Herpes Keratitis.

Mutation and structure guided discovery of an antiviral small molecule that mimics an essential C-Terminal tripeptide of the vaccinia D4 processivity factor.

The smallpox virus (variola) remains a bioterrorism threat since a majority of the human population has never been vaccinated. In the event of an outbreak, at least two drugs against different targets of variola are critical to circumvent potential viral mutants that acquire resistance. Vaccinia virus (VACV) is the model virus used in the laboratory for studying smallpox. The VACV processivity factor D4 is an ideal therapeutic target since it is both essential and specific for poxvirus replication. Recently, we identified a tripeptide (Gly-Phe-Ile) motif at the C-terminus of D4 that is conserved among poxviruses and is necessary for maintaining protein function. In the current work, a virtual screening for small molecule mimics of the tripeptide identified a thiophene lead that effectively inhibited VACV, cowpox virus, and rabbitpox virus in cell culture (EC50 = 8.4-19.7 µM) and blocked in vitro processive DNA synthesis (IC50 = 13.4 µM). Compound-binding to D4 was demonstrated through various biophysical methods and a dose-dependent retardation of the proteolysis of D4 proteins. This study highlights an inhibitor design strategy that exploits a susceptible region of the protein and identifies a novel scaffold for a broad-spectrum poxvirus inhibitor.

A Conserved Tripeptide Sequence at the C Terminus of the Poxvirus DNA Processivity Factor D4 Is Essential for Protein Integrity and Function.

Vaccinia virus (VACV) is a poxvirus, and the VACV D4 protein serves both as a uracil-DNA glycosylase and as an essential component required for processive DNA synthesis. The VACV A20 protein has no known catalytic function itself but associates with D4 to form the D4-A20 heterodimer that functions as the poxvirus DNA processivity factor. The heterodimer enables the DNA polymerase to efficiently synthesize extended strands of DNA. Upon characterizing the interaction between D4 and A20, we observed that the C terminus of D4 is susceptible to perturbation. Further analysis demonstrated that a conserved hexapeptide stretch at the extreme C terminus of D4 is essential for maintaining protein integrity, as assessed by its requirement for the production of soluble recombinant protein that is functional in processive DNA synthesis. From the known crystal structures of D4, the C-terminal hexapeptide is shown to make intramolecular contact with residues spanning the inner core of the protein. Our mutational analysis revealed that a tripeptide motif (215GFI217) within the hexapeptide comprises apparent residues necessary for the contact. Prediction of protein disorder identified the hexapeptide and several regions upstream of Gly215 that comprise residues of the interface surfaces of the D4-A20 heterodimer. Our study suggests that 215GFI217 anchors these potentially dynamic upstream regions of the protein to maintain protein integrity. Unlike uracil-DNA glycosylases from diverse sources, where the C termini are disordered and do not form comparable intramolecular contacts, this feature may be unique to orthopoxviruses.

Poxvirus uracil-DNA glycosylase-An unusual member of the family I uracil-DNA glycosylases.

Uracil-DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil-DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non-enzymatic function in DNA replication. UNG of vaccinia virus, also known as D4, is the most extensively characterized UNG of the poxvirus family. D4 forms an unusual heterodimeric processivity factor by attaching to a poxvirus-specific protein A20, which also binds to the DNA polymerase E9 and recruits other proteins necessary for replication. D4 is thus integrated in the DNA polymerase complex, and its DNA-binding and DNA scanning abilities couple DNA processivity and DNA base excision repair at the replication fork. The adaptations necessary for taking on the new function are reflected in the amino acid sequence and the three-dimensional structure of D4. An overview of the current state of the knowledge on the structure-function relationship of D4 is provided here.

The processivity factor complex of feline herpes virus-1 is a new drug target.

Feline herpes virus-1 (FHV-1) is ubiquitous in the cat population and is a major cause of blindness for which antiviral drugs, including acyclovir, are not completely effective. Recurrent infections, due to reactivation of latent FHV-1 residing in the trigeminal ganglia, can lead to epithelial keratitis and stromal keratitis and eventually loss of sight. This has prompted the medical need for an antiviral drug that will specifically inhibit FHV-1 infection. A new antiviral target is the DNA polymerase and its associated processivity factor, which forms a complex that is essential for extended DNA strand synthesis. In this study we have cloned and expressed the FHV-1 DNA polymerase (f-UL30) and processivity factor (f-UL42) and demonstrated that both proteins are required to completely synthesize the 7249 nucleotide full-length DNA from the M13 primed-DNA template in vitro. Significantly, a known inhibitor of human herpes simplex virus-1 (HSV-1) processivity complex was shown to inhibit FHV-1 processive DNA synthesis in vitro and block infection of cells. This validates using f-UL42/f-UL30 as a new antiviral drug target to treat feline ocular herpes infection.

A novel target and approach for identifying antivirals against molluscum contagiosum virus.

The dermatological disease molluscum contagiosum (MC) presents as lesions restricted solely to the skin. The poxvirus molluscum contagiosum virus (MCV) is responsible for this skin disease that is easily transmitted through casual contact among all populations, with greater frequency in children and immunosuppressed individuals. In addition, sexual transmission of MCV in adolescents and adults is a health concern. Although the skin lesions ultimately resolve in immunocompetent individuals, they can persist for extended periods, be painful, and result in scarring. Treatment is problematic, and there is no drug that specifically targets MCV. The inability of MCV to propagate in cell culture has impeded drug development. To overcome these barriers, we integrated three new developments. First, we identified a new MCV drug target (mD4) that is essential for processive DNA synthesis in vitro. Second, we discovered a small chemical compound that binds to mD4 and prevents DNA synthesis in vitro. Third, and most significant, we engineered a hybrid vaccinia virus (mD4-VV) in which the natural vaccinia D4 (vD4) gene is replaced by the mD4 target gene. This hybrid virus is dependent on mD4 for viral growth in culture and is inhibited by the small compound. This target system provides, for the first time, a platform and approach for the discovery and evaluation of new therapeutics that can be used to treat MC.

Design of potent poxvirus inhibitors of the heterodimeric processivity factor required for viral replication.

Smallpox constitutes a major bioterrorism threat, which underscores the need to develop antiviral drugs for rapid response in the event of an attack. Viral processivity factors are attractive drug targets in being both specific and essential for their cognate DNA polymerases to synthesize extended strands of DNA. An in silico model of the vacinnia virus processivity factor, comprised of the A20 and D4 heterocomplex, was constructed and used for lead optimization of an indole-based scaffold identified earlier from a high-throughput screening. On the basis of this model, a new class of potent antivirals against vaccinia virus was designed and synthesized, of which two (24a and 24b) exhibited superior improvement over the parent scaffold (IC50 = 42 and 46 vs 82000 nM, respectively). The ability of 24a to suppress vaccinia DNA synthesis is supported by the inhibition of late viral gene expression, as well as by the diminished incorporation of bromodeoxyuridine into viral replication factories.

Tumorigenic adenovirus 12 cells evade NK cell lysis by reducing the expression of NKG2D ligands.

Activation of natural killer (NK) cells depends on a balance between signals received from activation and inhibitory ligands expressed on the surface of target cells. Tumorigenic human adenovirus 12 (Ad12) transformed cells express low levels of the NK cell inhibitory ligand MHC I, but do not exhibit increased sensitivity to NK cell lysis compared to their non-tumorigenic counterparts. Analysis of the expression of activation ligands that bind to the NKG2D receptor revealed that RAE1ß and H60 were reduced on the surface of Ad12 mouse cells as well as at the level of transcription. In accord with these results, RAE1 localization to the synapse and sensitivity to NK cell cytotoxicity were also diminished. The reduced transcription of the rat NKG2D ligands, RAEt1L and RRTL, in tumorigenic rat cells compared to non-tumorigenic counterparts implies that both mouse and rat cell lines share a common mechanism of NKG2D ligand activation subverted by Ad12.

Transformation by E1A oncoprotein involves ubiquitin-mediated proteolysis of the neuronal and tumor repressor REST in the nucleus.

The adenovirus early region 1A (E1A) protein promotes cell immortalization and transformation by mediating the activities of key cellular regulators. The repressor element 1-silencing transcription factor (REST), which is a major neuronal and tumor suppressor, was previously found mainly in the cytoplasm rather than in the nuclei of adenovirus-transformed rodent cells (22). We now demonstrate that the loss of REST in the nucleus is due to its rapid degradation by the ubiquitin-proteasome system. Only nuclear REST, but not its cytoplasmic counterpart, was ubiquitinated and degraded. REST degradation was blocked by the ubiquitination inhibitor PYR-41 and the proteasome inhibitor MG-132 but not by the nuclear export inhibitor leptomycin B. REST degradation required both of its two C-terminal degrons that are recognized by the ubiquitin ligase SCF(ß-TrCP), since deletion or mutation of either degron eliminated degradation. Importantly, E1A was shown to mediate REST ubiquitination and degradation by upregulating ß-TrCP. Knockdown of E1A in virus-transformed cells reduced both ß-TrCP and ubiquitination of nuclear REST. In contrast, when expressed in HeLa cells, E1A enhanced the degradation of nuclear REST. Reconstitution of REST in virus-transformed cells negatively affected E1A-mediated cell proliferation and anchorage-independent growth. These data strongly indicate that E1A stimulates ubiquitination and proteolysis of REST in the nucleus, thereby abolishing the tumor suppressor functions of REST.

Identification of protein-protein interaction inhibitors targeting vaccinia virus processivity factor for development of antiviral agents.

Poxvirus uracil DNA glycosylase D4 in association with A20 and the catalytic subunit of DNA polymerase forms the processive polymerase complex. The binding of D4 and A20 is essential for processive polymerase activity. Using an AlphaScreen assay, we identified compounds that inhibit protein-protein interactions between D4 and A20. Effective interaction inhibitors exhibited both antiviral activity and binding to D4. These results suggest that novel antiviral agents that target the protein-protein interactions between D4 and A20 can be developed for the treatment of infections with poxviruses, including smallpox.

Identification of inhibitors that block vaccinia virus infection by targeting the DNA synthesis processivity factor D4.

Smallpox was globally eradicated 30 years ago by vaccination. The recent threat of bioterrorism demands the development of improved vaccines and novel therapeutics to effectively preclude a reemergence of smallpox. One new therapeutic target is the vaccinia poxvirus processivity complex, comprising D4 and A20 proteins that enable the viral E9 DNA polymerase to synthesize extended strands. Five compounds identified from an AlphaScreen assay designed to disrupt A20:D4 binding were shown to be effective in: (i) blocking vaccinia processive DNA synthesis in vitro, (ii) preventing cellular infection with minimal cytotoxicity, and (iii) binding to D4, as evidenced by ThermoFluor. The EC(50) values for inhibition of viral infectivity ranged from 9.6 to 23 µM with corresponding selectivity indices (cytotoxicity CC(50)/viral infectivity EC(50)) of 3.9 to 17.8. The five compounds are thus potential therapeutics capable of halting smallpox DNA synthesis and infectivity through disruptive action against a component of the vaccinia processivity complex.

Vaccinia virus D4 mutants defective in processive DNA synthesis retain binding to A20 and DNA.

Genome replication is inefficient without processivity factors, which tether DNA polymerases to their templates. The vaccinia virus DNA polymerase E9 requires two viral proteins, A20 and D4, for processive DNA synthesis, yet the mechanism of how this tricomplex functions is unknown. This study confirms that these three proteins are necessary and sufficient for processivity, and it focuses on the role of D4, which also functions as a uracil DNA glycosylase (UDG) repair enzyme. A series of D4 mutants was generated to discover which sites are important for processivity. Three point mutants (K126V, K160V, and R187V) which did not function in processive DNA synthesis, though they retained UDG catalytic activity, were identified. The mutants were able to compete with wild-type D4 in processivity assays and retained binding to both A20 and DNA. The crystal structure of R187V was resolved and revealed that the local charge distribution around the substituted residue is altered. However, the mutant protein was shown to have no major structural distortions. This suggests that the positive charges of residues 126, 160, and 187 are required for D4 to function in processive DNA synthesis. Consistent with this is the ability of the conserved mutant K126R to function in processivity. These mutants may help unlock the mechanism by which D4 contributes to processive DNA synthesis.

Kaposi's sarcoma-associated herpesvirus processivity factor-8 dimerizes in cytoplasm before being translocated to nucleus.

The processivity factor-8 (PF-8) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays an essential role in viral lytic replication. PF-8 forms homodimers in solution and is observed as a dimer on the DNA. Here, we show that PF-8 dimerizes in cells and that amino acid residues 1-21 and residues 277-304 of PF-8 (396R) are required for dimerization in vivo. Importantly, we demonstrate that PF-8 dimerizes in the cytoplasm before being translocated to the nucleus. The significance of PF-8 cytoplasmic dimerization as a possible first step in the formation of a prereplication complex is discussed.

The N terminus of adenovirus type 12 E1A inhibits major histocompatibility complex class I expression by preventing phosphorylation of NF-kappaB p65 Ser276 through direct binding.

The immune-escape strategy employed by human oncogenic adenovirus type 12 (Ad12) involves downregulation of major histocompatibility complex class I (MHC-I) transcription by disabling the transactivator NF-kappaB (p50/p65). This is accomplished by the Ad12 E1A protein (E1A-12), which prevents NF-kappaB from becoming phosphorylated by the protein kinase A catalytic subunit (PKAc). In this study, we examined the interactions between E1A-12 and NF-kappaB. Our data show that an E1A-12 mutant retaining the N-terminal 66 amino acids was as effective as the wild-type E1A-12 protein (266 amino acids) in binding p65, preventing phosphorylation of p65-Ser(276), and inhibiting transactivation. In contrast, the nontumorigenic adenovirus type 5 E1A protein (E1A-5) and other E1A-12 mutants lacking the N-terminal regions were severely defective in these activities. Further studies revealed that an N-terminal peptide consisting of residues 1 to 40 of E1A-12 was able to associate directly with p65 in vitro and prevent PKAc from phosphorylating p65-Ser(276). In the absence of the N terminus, there is an almost complete loss of E1A-12 binding to p65. These findings provide solid evidence for the role of the E1A-12 N terminus as an NF-kappaB binding domain. Significantly, this study indicates that the E1A-12 N terminus prevents PKAc from gaining access to p65 to account for Ser(276) hypophosphorylation. The E1A-12 N terminus interaction with p65 serves as a key explanation of how Ad12 downregulates MHC-I transcription and contributes to oncogenesis by escaping cytotoxic T lymphocytes.

The crystal structure of PF-8, the DNA polymerase accessory subunit from Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi's sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 A. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predicted to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8's mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.

Induction of neuronal and tumor-related genes by adenovirus type 12 E1A.

Adenovirus type 12 (Ad12) E1A protein (E1A-12) contains a unique 20-amino-acid spacer region between the second and third conserved regions. Substitution of a single amino acid in the spacer is able to abrogate Ad12 tumorigenesis. To investigate the function of the spacer, microarray analysis was performed on cells transformed by tumorigenic and nontumorigenic Ad12s that differ only by one amino acid in the spacer. Fewer than 0.8% of approximately 8,000 genes in the microarray exhibited differential expression of threefold and higher. Of these, more than half of the known genes with higher expression in the wild-type Ad12-transformed cells have neuronal-specific functions. Some of the other differentially expressed genes are involved in the regulation of the cell cycle, transcription, cell structure, and tumor invasiveness. Northern blot analyses of a subset of the neuronal genes, including Robo1, N-MYC, and alpha-internexin, confirmed their strong expression in multiple Ad12 tumorigenic cell lines. In contrast, these neuronal genes displayed only minor or negligible expression in cells transformed by spacer-mutated Ad12. Significantly, stable introduction of E1A-12 into nontumorigenic Ad5-transformed cells induced neuronal gene expression. We found that the neuron-restrictive silencer factor, which serves as a master repressor of neuronal genes, was inactivated in both Ad12- and Ad5-transformed cells via cytoplasmic retention, though only Ad12-transformed cells exhibited neuronal gene induction. Mutational analyses of the alpha-internexin promoter demonstrated that E1A-12-mediated neuronal gene induction further required the activation of neuronal promoter E-box elements. These results indicate that the spacer is involved in mediating neuronal and tumor-related genes.