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

Here we characterize a novel pan-RAS inhibitor, ADT-007, that potently and selectively inhibited the growth of histologically diverse cancer cell lines with mutant or activated RAS irrespective of the RAS mutation or isozyme. Growth inhibition was dependent on activated RAS and associated with reduced GTP-RAS levels and MAPK/AKT signaling. ADT-007 bound RAS in lysates from sensitive cells with sub-nanomolar EC 50 values but did not bind RAS in lysates from insensitive cells with low activated RAS. Insensitivity to ADT-007 was attributed to metabolic deactivation by UGT-mediated glucuronidation, providing a detoxification mechanism to protect normal cells from pan-RAS inhibition. Molecular modeling and experiments using recombinant RAS revealed that ADT-007 binds RAS in a nucleotide-free conformation to block GTP activation. Local injection of ADT-007 strongly inhibited tumor growth in syngeneic immune competent and xenogeneic immune deficient mouse models of colorectal and pancreatic cancer and activated innate and adaptive immunity in the tumor microenvironment. SIGNIFICANCE: ADT-007 is a novel pan-RAS inhibitor with a unique mechanism of action having potential to circumvent resistance to mutant-specific KRAS inhibitors and activate antitumor immunity. The findings support further development of ADT-007 analogs and/or prodrugs with oral bioavailability as a generalizable monotherapy or combined with immunotherapy for RAS mutant cancers. BACKGROUND: It is projected that colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDA) will cause 52,580 and 49,830 deaths in the US in 2023, respectively (1). The 5-year survival rates for CRC and PDA are 65% and 12%, respectively (1). Over 50% of CRC and 90% of PDA patients harbor mutations in KRAS genes that are associated with poor prognosis, making the development of novel KRAS inhibitors an urgent unmet medical need (2).

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.

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.

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.

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.

Development of a fluorescent microplate assay for determining cyanovirin-N levels in plasma.

A sensitive immunosorbent competition assay was developed for quantitation of the anti-HIV protein cyanovirin-N (CV-N) in plasma using a 96-well plate format and a fluorescent endpoint. The assay is based on the binding of CV-N in plasma to plate-bound anti-CV-N antibodies, followed by removal of the plasma and addition of europium-labeled CV-N (Eu3+ -CV-N) to compete for the remaining antibody sites. Detection by addition of a dissociative fluorescence enhancement solution and time-resolved fluorescence measurements allowed correlation to the concentration of the native CV-N in plasma. A linear detection range of 1-100 nM (r2>0.99) was obtained for CV-N in mouse plasma. This assay was then utilized for analysis of plasma levels of CV-N samples following subcutaneous injection of CV-N into mice. The results of these studies confirmed the reliability and sensitivity of this assay and the feasibility of its use for pharmacokinetic studies in a variety of species.

Neamphamide A, a new HIV-inhibitory depsipeptide from the Papua New Guinea marine sponge Neamphius huxleyi.

A new HIV-inhibitory cyclic depsipeptide, neamphamide A (2), was isolated from a Papua New Guinea collection of the marine sponge Neamphius huxleyi. Its structure was established through interpretation of spectroscopic data and by acid hydrolysis, derivatization of the free amino acids, and LC-MS analysis of the derivatives. Neamphamide A (2) contains 11 amino acid residues and an amide-linked 3-hydroxy-2,4,6-trimethylheptanoic acid moiety. The amino acid constituents were identified as L-Leu, L-NMeGln, D-Arg, D- and L-Asn, two residues of D-allo-Thr, L-homoproline, (3S,4R)-3,4-dimethyl-L-glutamine, beta-methoxytyrosine, and 4-amino-7-guanidino-2,3-dihydroxyheptanoic acid. In a cell-based XTT assay, 2 exhibited potent cytoprotective activity against HIV-1 infection with an EC50 of approximately 28 nM.

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.

Gymnangiamide, a cytotoxic pentapeptide from the marine hydroid Gymnangium regae.

A cytotoxic aqueous extract from the marine hydroid Gymnangium regae provided a novel linear pentapeptide, designated gymnangiamide (1). The planar structure of 1 was elucidated by interpretation of spectral data as well as chemical degradation and derivatization studies. In addition to the amino acids isoleucine and phenylserine, this peptide contained N-desmethyldolaisoleuine, O-desmethyldolaproine, and alpha-guanidino serine, three residues that have not previously been reported in a natural product. The absolute configurations of the constituent amino/guanidino acids were determined by chemical degradation and derivatization, followed by HPLC and LC-MS comparison with authentic standards. Gymnangiamide (1) was moderately cytotoxic against a number of human tumor cell lines in vitro.

A frameshift mutation and alternate splicing in human brain generate a functional form of the pseudogene cytochrome P4502D7 that demethylates codeine to morphine.

A frameshift mutation 138delT generates an open reading frame in the pseudogene, cytochrome P4502D7 (CYP2D7), and an alternate spliced functional transcript of CYP2D7 containing partial inclusion of intron 6 was identified in human brain but not in liver or kidney from the same individual. mRNA and protein of the brain variant CYP2D7 were detected in 6 of 12 human autopsy brains. Genotyping revealed the presence of the frameshift mutation 138delT only in those human subjects who expressed the brain variant CYP2D7. Genomic DNA analysis in normal volunteers revealed the presence of functional CYP2D7 in 4 of 8 individuals. In liver, the major organ involved in drug metabolism, a minor metabolic pathway mediated by CYP2D6 metabolizes codeine (pro-drug) to morphine (active drug), whereas norcodeine is the major metabolite. In contrast, when expressed in Neuro2a cells, brain variant CYP2D7 metabolized codeine to morphine with greater efficiency compared with the corresponding activity in cells expressing CYP2D6. Morphine binds to micro-opioid receptors in certain regions of the central nervous system, such as periaqueductal gray, and produces pain relief. The brain variant CYP2D7 and micro-opioid receptor colocalize in neurons of the periaqueductal gray area in human brain, indicating that metabolism of codeine to morphine could occur at the site of opioid action. Histio-specific isoforms of P450 generated by alternate splicing, which mediate selective metabolism of pro-drugs within tissues, particularly the brain, to generate active drugs may play an important role in drug action and provide newer insights into the genetics of metabolism.

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.

Cyclonellin, a new cyclic octapeptide from the marine sponge Axinella carteri.

Cyclonellin (1), a new cyclic octapeptide, was isolated from an aqueous extract of the marine sponge Axinella carteri. Its structure was elucidated by interpretation of NMR spectral data of the intact compound and N-terminal Edman sequencing of linear peptide fragments obtained by partial hydrolysis of 1. The absolute configurations of the constituent amino acids were determined by acid hydrolysis, derivitization with FDAA, and LC-MS analyses.

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.

Identification of a new chondropsin class of antitumor compound that selectively inhibits V-ATPases.

We identify a new naturally occurring class of inhibitor of vacuolar H+-ATPases (V-ATPases) isolated from vacuolar membranes of Neurospora crassa and from chromaffin granule membranes of Bos taurus. To date, the new class includes six chondropsins and poecillastrin A, large polyketide-derived macrolide lactams with 33-37 membered rings. In the National Cancer Institute's 60-cell screen the chondropsin class showed a tumor cell growth inhibitory fingerprint essentially indistinguishable from that of the bafilomycin/concanamycin and the salicylihalamide/lobatamide classes of well-established V-ATPase inhibitors. Half-maximal inhibition of V-ATPase activity in vitro occurred at 0.04-0.7 microM for the fungal vacuolar V-ATPase and at 0.4 to >10 microM for the chromaffin granule V-ATPase. Thus, the new inhibitors are somewhat less potent than the other two classes, which typically have Ki values of <10 nM for V-ATPases, and the new inhibitors differ from the other two classes in their specificity. The bafilomycin class inhibits all eucaryotic V-ATPases, the salicylihalamide class inhibits mammalian V-ATPases but not fungal V-ATPases, and the new chondropsin class inhibits the N. crassa V-ATPase better than the chromaffin granule V-ATPase. Two mutations in the N. crassa V-ATPase that affect the binding of bafilomycin had small but reproducible effects on the affinity of chondropsins for the V-ATPase, suggesting the possibility of a similar mechanism of inhibition.

Potent anti-influenza activity of cyanovirin-N and interactions with viral hemagglutinin.

The novel antiviral protein cyanovirin-N (CV-N) was initially discovered based on its potent activity against the human immunodeficiency virus (HIV). Subsequent studies identified the HIV envelope glycoproteins gp120 and gp41 as molecular targets of CV-N. More recently, mechanistic studies have shown that certain high-mannose oligosaccharides (oligomannose-8 and oligomannose-9) found on the HIV envelope glycoproteins comprise the specific sites to which CV-N binds. Such selective, carbohydrate-dependent interactions may account, at least in part, for the unusual and unexpected spectrum of antiviral activity of CV-N described herein. We screened CV-N against a broad range of respiratory and enteric viruses, as well as flaviviruses and herpesviruses. CV-N was inactive against rhinoviruses, human parainfluenza virus, respiratory syncytial virus, and enteric viruses but was moderately active against some herpesvirus and hepatitis virus (bovine viral diarrhea virus) strains (50% effective concentration [EC(50)] = approximately 1 micro g/ml) while inactive against others. Remarkably, however, CV-N and related homologs showed highly potent antiviral activity against almost all strains of influenza A and B virus, including clinical isolates and a neuraminidase inhibitor-resistant strain (EC(50) = 0.004 to 0.04 micro g/ml). When influenza virus particles were pretreated with CV-N, viral titers were lowered significantly (>1,000-fold). Further studies identified influenza virus hemagglutinin as a target for CV-N, showed that antiviral activity and hemagglutinin binding were correlated, and indicated that CV-N's interactions with hemagglutinin involved oligosaccharides. These results further reveal new potential avenues for antiviral therapeutics and prophylaxis targeting specific oligosaccharide-comprised sites on certain enveloped viruses, including HIV, influenza virus, and possibly others.

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.

A potent novel anti-HIV protein from the cultured cyanobacterium Scytonema varium.

A new anti-HIV protein, scytovirin, was isolated from aqueous extracts of the cultured cyanobacterium Scytonema varium. The protein displayed potent anticytopathic activity against laboratory strains and primary isolates of HIV-1 with EC50 values ranging from 0.3 to 22 nM. Scytovirin binds to viral coat proteins gp120, gp160, and gp41 but not to cellular receptor CD4 or other tested proteins. This unique protein consists of a single 95-amino acid chain with significant internal sequence duplication and 10 cysteines forming five intrachain disulfide bonds.

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.