A Novel Pan-RAS Inhibitor with a Unique Mechanism of Action Blocks Tumor Growth in Mouse Models of GI Cancer.

Here, we describe a novel pan-RAS inhibitor, ADT-007, that potently inhibited the growth of RAS mutant cancer cells irrespective of the RAS mutation or isozyme. RAS WT cancer cells with activated RAS from upstream mutations were equally sensitive. Conversely, cells from normal tissues or RAS WT cancer cells harboring downstream BRAF mutations were insensitive. Insensitivity to ADT-007 was attributed to low activated RAS levels and metabolic deactivation by UDP-glucuronosyltransferases expressed in normal cells but repressed in RAS mutant cancer cells. Cellular, biochemical, and biophysical experiments show ADT-007 binds nucleotide-free RAS to block GTP activation of RAS and MAPK/AKT signaling. Local administration of ADT-007 strongly inhibited tumor growth in syngeneic immune-competent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancer while activating innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug inhibited tumor growth, supporting further development of this novel class of pan-RAS inhibitors for treating RAS-driven cancers. SIGNIFICANCE: ADT-007 is a 1 st -in-class pan-RAS inhibitor with ultra-high potency and unique selectivity for cancer cells with mutant or activated RAS capable of circumventing resistance and activating antitumor immunity. Further development of ADT-007 analogs or prodrugs with oral bioavailability as a generalizable monotherapy or combined with immunotherapy is warranted.

PDE5 and PDE10 inhibition activates cGMP/PKG signaling to block Wnt/ß-catenin transcription, cancer cell growth, and tumor immunity.

Although numerous reports conclude that nonsteroidal anti-inflammatory drugs (NSAIDs) have anticancer activity, this common drug class is not recommended for long-term use because of potentially fatal toxicities from cyclooxygenase (COX) inhibition. Studies suggest the mechanism responsible for the anticancer activity of the NSAID sulindac is unrelated to COX inhibition but instead involves an off-target, phosphodiesterase (PDE). Thus, it might be feasible develop safer and more efficacious drugs for cancer indications by targeting PDE5 and PDE10, which are overexpressed in various tumors and essential for cancer cell growth. In this review, we describe the rationale for using the sulindac scaffold to design-out COX inhibitory activity, while improving potency and selectivity to inhibit PDE5 and PDE10 that activate cGMP/PKG signaling to suppress Wnt/ß-catenin transcription, cancer cell growth, and tumor immunity.

Influenza A (H1N1) virus resistance to cyanovirin-N arises naturally during adaptation to mice and by passage in cell culture in the presence of the inhibitor.

Influenza A/New Caledonia/20/99 (H1N1) virus was studied for development of resistance to cyanovirin-N (CVN). CVN neutralizes virus infectivity by binding to specific high-mannose oligosaccharides on the viral haemagglutinin 1 (HA1) subunit. During virus adaptation to mice in the absence of CVN treatment the virus became resistant to CVN (CVN-MR virus), as did virus passaged in cell culture in the presence of CVN (CVN-R virus). The CVN-R virus possessed a single amino acid change at position 94a (Asn94aAsp) of HA1 that eliminated this glycosylation site. The CVN-MR virus at mouse passage 7 was a mixture of clones, consisting of a single mutation (Asp225Gly) and double mutations (Asn63Ser+Asp225Gly or Asn94a+Asp225Gly), eliminating glycosylation sites. CVN did not bind well to the CVN-R and CVN-MR viruses. Propagating these viruses in cells treated with 1 mM deoxymannojirimycin (dMJ, mannosidase inhibitor) increased sensitivity to CVN, suggesting that glycans attached at other sites on HA1 that typically are not high-mannosidic became so due to dMJ treatment. Further evaluation showed that the Asp225Gly mutant virus was sensitive to the inhibitor and did not kill mice or induce weight loss. The CVN-R virus was also avirulent to mice. The double-mutant CVN-MR viruses were resistant to CVN and caused deaths and severe weight loss in mice. CVN-R virus subjected to mouse adaptation acquired the 225 mutation and a lethal phenotype. Thus, the 225 mutation in the HA receptor-binding site in combination with a loss of glycan at Asn (63 or 94a) are important for mouse adaptation in this virus. The mutations reported here causing resistance to CVN are consistent with its known mode of action.

The domain-swapped dimer of cyanovirin-N is in a metastable folded state: reconciliation of X-ray and NMR structures.

The structure of the potent HIV-inactivating protein cyanovirin-N was previously found by NMR to be a monomer in solution and a domain-swapped dimer by X-ray crystallography. Here we demonstrate that, in solution, CV-N can exist both in monomeric and in domain-swapped dimeric form. The dimer is a metastable, kinetically trapped structure at neutral pH and room temperature. Based on orientational NMR constraints, we show that the domain-swapped solution dimer is similar to structures in two different crystal forms, exhibiting solely a small reorientation around the hinge region. Mutation of the single proline residue in the hinge to glycine significantly stabilizes the protein in both its monomeric and dimeric forms. By contrast, mutation of the neighboring serine to proline results in an exclusively dimeric protein, caused by a drastic destabilization of the monomer.

Thioltransferase (glutaredoxin) mediates recovery of motor neurons from excitotoxic mitochondrial injury.

Mitochondrial dysfunction involving electron transport components is implicated in the pathogenesis of several neurodegenerative disorders and is a critical event in excitotoxicity. Excitatory amino acid L-beta-N-oxalylamino-L-alanine (L-BOAA), causes progressive corticospinal neurodegeneration in humans. In mice, L-BOAA triggers glutathione loss and protein thiol oxidation that disrupts mitochondrial complex I selectively in motor cortex and lumbosacral cord, the regions affected in humans. We examined the factors regulating postinjury recovery of complex I in CNS regions after a single dose of L-BOAA. The expression of thioltransferase (glutaredoxin), a protein disulfide oxidoreductase regulated through AP1 transcription factor was upregulated within 30 min of L-BOAA administration, providing the first evidence for functional regulation of thioltransferase during restoration of mitochondrial function. Regeneration of complex I activity in motor cortex was concurrent with increase in thioltransferase protein and activity, 1 hr after the excitotoxic insult. Pretreatment with alpha-lipoic acid, a thiol delivery agent that protects motor neurons from L-BOAA-mediated toxicity prevented the upregulation of thioltransferase and AP1 activation, presumably by maintaining thiol homeostasis. Downregulation of thioltransferase using antisense oligonucleotides prevented the recovery of complex I in motor cortex and exacerbated the mitochondrial dysfunction in lumbosacral cord, providing support for the critical role for thioltransferase in maintenance of mitochondrial function in the CNS.

Multisite and multivalent binding between cyanovirin-N and branched oligomannosides: calorimetric and NMR characterization.

Binding of the protein cyanovirin-N to oligomannose-8 and oligomannose-9 of gp120 is crucially involved in its potent virucidal activity against the human immunodeficiency virus (HIV). The interaction between cyanovirin-N and these oligosaccharides has not been thoroughly characterized due to aggregation of the oligosaccharide-protein complexes. Here, cyanovirin-N's interaction with a nonamannoside, a structural analog of oligomannose-9, has been studied by nuclear magnetic resonance and isothermal titration calorimetry. The nonamannoside interacts with cyanovirin-N in a multivalent fashion, resulting in tight complexes with an average 1:1 stoichiometry. Like the nonamannoside, an alpha1-->2-linked trimannoside substructure interacts with cyanovirin-N at two distinct protein subsites. The chitobiose and internal core trimannoside substructures of oligomannose-9 are not recognized by cyanovirin-N, and binding of the core hexamannoside occurs at only one of the sites on the protein. This is the first detailed analysis of a biologically relevant interaction between cyanovirin-N and high-mannose oligosaccharides of HIV-1 gp120.

The microbicide cyanovirin-N expressed on the surface of commensal bacterium Streptococcus gordonii captures HIV-1.

OBJECTIVE: To explore the feasibility of expressing the potent HIV-inactivating protein, cyanovirin-N (CV-N), in the human commensal bacterium Streptococcus gordonii, as a possible approach for local delivery of CV-N to prevent sexual transmission of HIV-1. DESIGN AND METHODS: To express CV-N in S. gordonii, we used the host-vector system we had previously developed. CV-N was expressed as a fusion protein both attached to the bacterial surface and secreted in soluble form in the supernatant of liquid cultures. The soluble form of recombinant CV-N was tested for gp120-binding activity in an enzyme-linked immunosorbent assay, whereas S. gordonii strain expressing CV-N on the surface was analyzed in an in vitro HIV capturing assay. RESULTS: Two recombinant S. gordonii strains secreting or displaying CV-N on the bacterial surface were constructed and the expression of CV-N was confirmed by immunoblot and flow-cytometric analysis. The secreted form of recombinant CV-N exhibited a concentration-dependent binding to the envelope glycoprotein gp120 of HIV-1, whereas CV-N displayed on the bacterial surface was able to capture HIV virions efficiently. CONCLUSION: The anti-HIV protein CV-N in S. gordonii was expressed in a biologically active form. This represents a first step in the development of a system to deliver and maintain an effective concentration of a microbicide in the vaginal mucosa.

High throughput screening for cyanovirin-N mimetics binding to HIV-1 gp41.

The human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp41 is an important mediator of viral entry into host cells. Previous studies showed that the virucidal protein cyanovirin-N (CV-N) bound to both gp120 and gp41, and that this binding was associated with its antiviral activity. We constructed an HTS assay based on the interaction of europium-labeled CV-N with recombinant glycosylated gp41 ectodomain to support identification of small-molecule mimetics of CV-N that might be developed as antiviral drug leads. Primary screening of over 107,000 natural product extracts in the assay yielded 347 confirmed hits. Secondary assays eliminated extracts that bound directly to labeled CV-N or for which the simple sugars mannose and N-acetylglucosamine blocked the interaction with gp41 (lectin activity). Extracts were further prioritized based on anti-HIV activity and other biological, biochemical, and chemical criteria. The distribution of source organism taxonomy of active extracts was analyzed, as was the cross-correlation of activity between the CV-N-gp41 binding competition assay and the previously reported CV-N-gp120 binding competition assay. A limited set of extracts was selected for bioassay-guided fractionation.

Cyanovirin-N inhibits AIDS virus infections in vaginal transmission models.

The cyanobacterial protein cyanovirin-N (CV-N) potently inactivates diverse strains of HIV-1 and other lentiviruses due to irreversible binding of CV-N to the viral envelope glycoprotein gp120. In this study, we show that recombinant CV-N effectively blocks HIV-1(Ba-L) infection of human ectocervical explants. Furthermore, we demonstrate the in vivo efficacy of CV-N gel in a vaginal challenge model by exposing CV-N-treated female macaques (Macaca fascicularis) to a pathogenic chimeric SIV/HIV-1 virus, SHIV89.6P. All of the placebo-treated and untreated control macaques (8 of 8) became infected. In contrast, 15 of 18 CV-N-treated macaques showed no evidence of SHIV infection. Further, CV-N produced no cytotoxic or clinical adverse effects in either the in vitro or in vivo model systems. Together these studies suggest that CV-N is a good candidate for testing in humans as an anti-HIV topical microbicide.

Cyanovirin-N gel as a topical microbicide prevents rectal transmission of SHIV89.6P in macaques.

Cyanovirin-N (CV-N), an 11-kDa cyanobacterial protein, potently inactivates diverse strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and also prevents virus-to-cell fusion, virus entry, and infection of cells in vitro. These properties make CV-N an attractive candidate for use as a topical microbicide to prevent the sexual transmission of HIV. We evaluated the efficacy of gel-formulated, recombinant CV-N gel asa topical microbicide in male macaques (Macaca fascicularis) that were rectally challenged with a chimeric SIV/HIV-1 virus known as SHIV89.6P. All of the untreated macaques were infected and experienced CD4+T cell depletion. In contrast, none of the macaques that received either 1% or 2% CV-N gel showed evidence of SHIV89.6P infection. Neither CV-N nor placebo gels produced any adverse effects in any macaque following the rectal application. These results indicate that CV-N gel as a topical microbicide can prevent rectal transmission of SHIV in macaques. These studies encourage clinical evaluation of CV-N as a topical microbicide to prevent sexual transmission of HIV in humans.

Constitutive expression and localization of the major drug metabolizing enzyme, cytochrome P4502D in human brain.

Cytochrome P4502D6, an important isoform of cytochrome P450, mediates the metabolism of several psychoactive drugs in liver. Quantitatively, liver is the major drug metabolizing organ, however metabolism of drugs in brain could modulate pharmacological and pharmacodynamic effects of psychoactive drugs at their site of action and explain some of the variation typically seen in patient population. We have measured cytochrome P450 content and examined constitutive expression of CYP2D mRNA and protein in human brain regions by reverse transcription polymerase chain reaction, Northern and immunoblotting and localized it by in situ hybridization and immunohistochemistry. CYP2D mRNA was expressed constitutively in neurons of cerebral cortex, Purkinje and granule cell layers of cerebellum, reticular neurons of midbrain and pyramidal neurons of CA1, CA2 and CA3 subfields of hippocampus. Immunoblot studies demonstrated the presence of cytochrome P4502D protein in cortex, cerebellum, midbrain, striatum and thalamus of human brain. Immunohistochemical localization showed the predominant presence of cytochrome P4502D not only in neuronal soma but also in dendrites of Purkinje and cortical neurons. These studies demonstrate constitutive expression of cytochrome P4502D in neuronal cell population in human brain, indicating its possible role in metabolism of psychoactive drugs directly at or near their site of action, in neurons, in human brain.

Cyanovirin-N binds to the viral surface glycoprotein, GP1,2 and inhibits infectivity of Ebola virus.

Ebola virus (Ebo) causes severe hemorrhagic fever and high mortality in humans. There are currently no effective therapies. Here, we have explored potential anti-Ebo activity of the human immunodeficiency virus (HIV)-inactivating protein cyanovirin-N (CV-N). CV-N is known to potently inhibit the infectivity of a broad spectrum of HIV strains at the level of viral entry. This involves CV-N binding to N-linked high-mannose oligossacharides on the viral glycoprotein gp120. The Ebola envelope contains somewhat similar oligosaccharide constituents, suggesting possible susceptibility to inhibition by CV-N. Our initial results revealed that CV-N had both in vitro and in vivo antiviral activity against the Zaire strain of the Ebola virus (Ebo-Z). Addition of CV-N to the cell culture medium at the time of Ebo-Z infection inhibited the development of viral cytopathic effects (CPEs). CV-N also delayed the death of Ebo-Z-infected mice, both when given as a series of daily subcutaneous injections and when the virus was incubated ex vivo together with CV-N before inoculation into the mice. Furthermore, similar to earlier results with HIV gp120, CV-N bound with considerable affinity to the Ebola surface envelope glycoprotein, GP(1,2). Competition experiments with free oligosaccharides were consistent with the view that carbohydrate-mediated CV-N/GP(1,2) interactions involve oligosaccharides residing on the Ebola viral envelope. Overall, these studies broaden the range of viruses known to be inhibited by CV-N, and further implicate carbohydrate moieties on viral surface proteins as common viral molecular targets for this novel protein.

Application of high-field NMR and cryogenic probe technologies in the structural elucidation of poecillastrin a, a new antitumor macrolide lactam from the sponge poecillastra species.

Poecillastrin A (1), a new polyketide-derived macrolide lactam, was isolated from a deep-water collection of the marine sponge Poecillastra species. The structure of poecillastrin A (1) was assigned using NMR data acquired at 500 MHz with an inverse-detection cryogenic probe and at 800 MHz with a room-temperature probe.

Salicylihalamides A and B, Novel Cytotoxic Macrolides from the Marine Sponge Haliclona sp.

Two novel, highly potent, cytotoxic macrolides, salicylihalamides A (1) and B (2), were isolated from the sponge Haliclona sp. This new macrolide class incorporates salicylic acid, a 12-membered lactone ring, and an enamide side chain. COMPARE pattern-recognition analyses of the NCI 60-cell mean graph screening profiles of 1 did not reveal any significant correlations to the profiles of known antitumor compounds in the NCI's "standard agent database", thus supporting the conclusion that the salicylihalamides represent a potentially important new class for antitumor lead optimization and in vivo investigations.

Cytotoxic alkaloids from the marine sponge Thorectandra sp.

Three beta-carboline alkaloids, Compound 1, 1-deoxysecofascaplysin A (2), and fascaplysin (3), were isolated from the aqueous and organic extracts of the marine sponge Thorectandra sp. The structures of 1 and 2 were determined on the basis of spectral data. Compound 1 inhibited the growth of MCF-7 (breast) with an IC50 of 5.9 microg/mL while Compound 2 inhibited the growth of MCF-7 as well as OVCAR-3 (ovarian) human tumor cell lines with IC50s of 1.5 and 2.2 microg/mL, respectively.

Candidaspongiolides, distinctive analogues of tedanolide from sponges of the genus Candidaspongia.

Fractionation of cytotoxic extracts of specimens of a newly described sponge genus, Candidaspongia, has yielded the candidaspongiolides (3), a complex mixture of acyl esters of a macrolide related to tedanolide. The general structure of the candidaspongiolides was determined by analyses of various 2D NMR and MS data sets. The acyl ester components were identified by GC-MS analysis of the derived fatty acid methyl esters. The mixture could be selectively converted to the deacylated macrolide core (4) by enzymolysis with immobilized porcine lipase, with the structure of the candidaspongiolide core then secured by NMR and MS analysis. The candidaspongiolide mixture was potently cytotoxic, exhibiting a mean panel 50% growth inhibition (GI50) of 14 ng/mL in the National Cancer Institute's 60-cell-line in vitro antitumor screen.

Bioengineering lactic acid bacteria to secrete the HIV-1 virucide cyanovirin.

An urgent need exists to prevent the sexual transmission of HIV-1. With prevalence rates exceeding 35% in parts of sub-Saharan Africa, increasing attention has been placed on developing and testing microbicidal agents capable of preventing virus transmission at mucosal sites. HIV-1 microbicides must meet several requirements before their widespread use. The drugs must be able to neutralize a diversity of HIV-1 strains, not induce mucosal inflammation, be associated with minimal side effects, and be effective for a prolonged period after a single application. Recent work has demonstrated the utility of recombinant lactic acid bacteria (LAB) as agents of mucosal drug delivery. Here, we describe the bioengineering of strains of LAB to secrete the prototypic virucidal compound cyanovirin (CV-N) and demonstrate the anti-HIV-1 activity of secreted CV-N. Our results suggest that recombinant LAB may serve as effective microbicidal compounds and deserve in vivo testing in simian immunodeficiency virus models of mucosal virus transmission.

Antitumor activity and distribution of pyrroloiminoquinones in the sponge genus Zyzzya.

A detailed analysis of four different collections of the sponge genus Zyzzya yielded nine pyrroloiminoquinones of the makaluvamine, batzelline, and isobatzelline/damirone classes. Dereplication analyses of additional Zyzzya extracts did not disclose more potent or additional new compounds. Comparative testing of these compounds in the National Cancer Institute's 60 cell line human tumor screen revealed varying levels of potency and differential cytotoxicity, apparently related to the unsaturation levels in and substitution patterns on the core ring system. Further studies on the topoisomerase II-mediated DNA cleavage were conducted. Reductive activation of the pyrroloiminoquinones led to DNA damage in vitro, which correlated with half wave potentials and reversibility parameters. DNA damage could be abrogated by ascorbate. Fluorescence displacement was used to measure intercalation with DNA; intercalation efficiency did not correlate with DNA-damaging proficiency. Makaluvamine H (5) emerged as the most potent and differential of our isolates, roughly comparable to makaluvamines C (in vitro) and I (in vivo). 3,7-Dimethyl guanine was isolated from one of the Zyzzya collections and from the sponge Latrunculia purpurea.

Isolation and characterization of griffithsin, a novel HIV-inactivating protein, from the red alga Griffithsia sp.

Griffithsin (GRFT), a novel anti-HIV protein, was isolated from an aqueous extract of the red alga Griffithsia sp. The 121-amino acid sequence of GRFT has been determined, and biologically active GRFT was subsequently produced by expression of a corresponding DNA sequence in Escherichia coli. Both native and recombinant GRFT displayed potent antiviral activity against laboratory strains and primary isolates of T- and M- tropic HIV-1 with EC50 values ranging from 0.043 to 0.63 nM. GRFT also aborted cell-to-cell fusion and transmission of HIV-1 infection at similar concentrations. High concentrations (e.g. 783 nM) of GRFT were not lethal to any tested host cell types. GRFT blocked CD4-dependent glycoprotein (gp) 120 binding to receptor-expressing cells and bound to viral coat glycoproteins (gp120, gp41, and gp160) in a glycosylation-dependent manner. GRFT preferentially inhibited gp120 binding of the monoclonal antibody (mAb) 2G12, which recognizes a carbohydrate-dependent motif, and the (mAb) 48d, which binds to CD4-induced epitope. In addition, GRFT moderately interfered with the binding of gp120 to sCD4. Further data showed that the binding of GRFT to soluble gp120 was inhibited by the monosaccharides glucose, mannose, and N-acetylglucosamine but not by galactose, xylose, fucose, N-acetylgalactosamine, or sialic acid-containing glycoproteins. Taken together these data suggest that GRFT is a new type of lectin that binds to various viral glycoproteins in a monosaccharide-dependent manner. GRFT could be a potential candidate microbicide to prevent the sexual transmission of HIV and AIDS.

Discovery of a stable dimeric mutant of cyanovirin-N (CV-N) from a T7 phage-displayed CV-N mutant library.

Mutant proteins with altered properties can be useful probes for investigating structure, ligand binding sites, mechanisms of action, and physicochemical attributes of the corresponding wild-type proteins of interest. In this report, we illuminate properties of mutants of the potent HIV-inactivating protein, cyanovirin-N (CV-N), selected by construction of a mutant library by error-prone polymerase chain reaction and affinity-based screening using T7 phage display technology. After three rounds of biopanning, two phage-displayed, one-point mutants of CV-N, Ser52Pro and Ala77Thr, were isolated. After the elucidation of biological activities of the mutants displayed on phage as well as the Escherichia coli-expressed, purified mutant proteins, we subsequently subjected the mutants to analyses by native PAGE and size-exclusion chromatography. We found that the Ser52Pro mutant not only was active against HIV but also existed exclusively as a dimer in solution. This was in marked contrast to the wild-type CV-N, which exists in solution predominantly as the monomer. The Ser52Pro mutant provides a novel model for further investigations of the folding mechanism as well as structure-activity requirements for CV-N's antiviral properties.