A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.

Protein Kinase Activity and Identification of a Toxic Effector Domain of the Target of Rapamycin TOR Proteins in Yeast

In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM.

Synthesis and biological activity of DNA damaging agents that form decoy binding sites for the estrogen receptor

It is a goal of cancer chemotherapy to achieve the selective killing of tumor cells while minimizing toxicity to normal tissues. We describe the design of selective toxins forming DNA adducts that attract the estrogen receptor (ER), a transcription factor that is overexpressed in many human breast and ovarian tumors. The compounds consist of 4-(3-aminopropyl)-N,N-(2-chloroethyl)-aniline linked to 2-(4′-hydroxyphenyl)-3-methyl-5-hydroxy-indole. The former moiety is a DNA damaging nitrogen mustard and the latter is a ligand for the ER. The connection between these groups was refined to permit DNA adducts formed by the mustard portion of the molecule to present the ligand domain so that it was able to interact efficiently with the ER. By using 16-mers containing specific DNA adducts, it was determined that monoadducts and putative intrastrand crosslinks were preferred targets for the ER over interstrand crosslinks. A series of structurally related 2-phenylindole mustards was prepared, some of which were selectively toxic to the ER-positive breast cancer cell line MCF-7, as compared with the ER(−) negative line MDA-MB231. The ability both to bind to DNA and to interact significantly with the ER were essential to achieve selective lethality toward ER(+) cells. Compounds forming DNA adducts without the ability to bind receptor showed similar toxicities in the two cell lines. Several models could explain the selective toxicity of the mustard–phenylindole compounds toward ER(+) cells. The favored model suggests that a mustard–DNA adduct is shielded by the ER from DNA repair enzymes and hence cells possessing an abundance of the ER selectively retain the adduct and are killed.

Gonadotropin-releasing hormone analogue conjugates with strong selective antitumor activity

Conjugation of gonadotropin-releasing hormone (GnRH) analogues GnRH-III, MI-1544, and MI-1892 through lysyl side chains and a tetrapeptide spacer, Gly-Phe-Leu-Gly (X) to a copolymer, poly(N-vinylpyrrolidone-co-maleic acid) (P) caused increased antiproliferative activity toward MCF-7 and MDA-MB-231 breast, PC3 and LNCaP prostate, and Ishikawa endometrial cancer cell lines in culture and against tumor development by xenografts of the breast cancer cells in immunodeficient mice. MCF-7 cells treated with P-X-1544 and P-X-1892 displayed characteristic signs of apoptosis, including vacuoles in the cytoplasm, rounding up, apoptotic bodies, bleb formation, and DNA fragmentation. Conjugates, but not free peptides, inhibited cdc25 phosphatase and caused accumulation of Ishikawa and PC3 cells in the G2/M phase of the cell cycle after 24 h at lower doses and in the G1 and G2 phases after 48 h. Since P-X-peptides appear to be internalized, the increased cytotoxicity of the conjugates is attributed to protection of peptides from proteolysis, enhanced interaction of the peptides with the GnRH receptors, and/or internalization of P-X-peptide receptor complexes so that P can exert toxic effects inside, possibly by inhibiting enzymes involved in the cell cycle. The additional specificity of P-X-peptides compared with free peptides for direct antiproliferative effects on the cancer cells but not for interactions in the pituitary indicates the therapeutic potential of the conjugates.

In vivo activity in a catalytic antibody-prodrug system: Antibody catalyzed etoposide prodrug activation for selective chemotherapy

Effective chemotherapy remains a key issue for successful cancer treatment in general and neuroblastoma in particular. Here we report a chemotherapeutic strategy based on catalytic antibody-mediated prodrug activation. To study this approach in an animal model of neuroblastoma, we have synthesized prodrugs of etoposide, a drug widely used to treat this cancer in humans. The prodrug incorporates a trigger portion designed to be released by sequential retro-aldol/retro-Michael reactions catalyzed by aldolase antibody 38C2. This unique prodrug was greater than 102-fold less toxic than etoposide itself in in vitro assays against the NXS2 neuroblastoma cell line. Drug activity was restored after activation by antibody 38C2. Proof of principle for local antibody-catalyzed prodrug activation in vivo was established in a syngeneic model of murine neuroblastoma. Mice with established 100-mm3 s.c. tumors who received one intratumoral injection of antibody 38C2 followed by systemic i.p. injections with the etoposide prodrug showed a 75% reduction in s.c. tumor growth. In contrast, injection of either antibody or prodrug alone had no antitumor effect. Systemic injections of etoposide at the maximum tolerated dose were significantly less effective than the intratumoral antibody 38C2 and systemic etoposide prodrug combination. Significantly, mice treated with the prodrug at 30-fold the maximum tolerated dose of etoposide showed no signs of prodrug toxicity, indicating that the prodrug is not activated by endogenous enzymes. These results suggest that this strategy may provide a new and potentially nonimmunogenic approach for targeted cancer chemotherapy.

Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo.

Insertion of introns into cloned cDNA of two isolates of the plant potyvirus pea seedborne mosaic virus facilitated plasmid amplification in Escherichia coli. Multiple stop codons in the inserted introns interrupted the open reading frame of the virus cDNA, thereby terminating undesired translation of virus proteins in E. coli. Plasmids containing the full-length virus sequences, placed under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase termination signal, were stable and easy to amplify in E. coli if one or more introns were inserted into the virus sequence. These plasmids were infectious when inoculated mechanically onto Pisum sativum leaves. Examination of the cDNA-derived viruses confirmed that intron splicing of in vivo transcribed pre-mRNA had occurred as predicted, reestablishing the virus genome sequences. Symptom development and virus accumulation of the cDNA derived viruses and parental viruses were identical. It is proposed that intron insertion can be used to facilitate manipulation and amplification of cloned DNA fragments that are unstable in, or toxic to, E. coli. When transcribed in vivo in eukaryotic cells, the introns will be eliminated from the sequence and will not interfere with further analysis of protein expression or virus infection.

A tumor-selective somatostatin analog (TT-232) with strong in vitro and in vivo antitumor activity.

We report a series of new in vitro and in vivo data proving the selective antitumor activity of our somatostatin structural derivative, TT-232. In vitro, it inhibited the proliferation of 20 different human tumor cell lines in the range of 50-95% and induced a very strong apoptosis. In vivo TT-232 was effective on transplanted animal tumors (Colon 26, B16 melanoma, and S180 sarcoma) and on human tumor xenografts. Treatment of MDA-MB-231 human breast cancer xenografted in mice with low submaximal doses of TT-232 [0.25 and 0.5 mg/kg of body weight (b.w.)] caused an average 80% decrease in the tumor volume resulting in 30% tumor-free animals surviving for longer than 200 days. Treatment of prostate tumor (PC-3) xenografted animals with 20 mg/kg of b.w. of TT-232 for 3 weeks resulted in 60% decrease in tumor volume and 100% survival even after 60 days, while 80% of nontreated animals perished. We have demonstrated that TT-232 did not bind to the membrane preparation of rat pituitary and cortex and had no antisecretory activity. TT-232 was not toxic at a dose of 120 mg/kg of b.w. in mice. Long-term incubation (24 h) of tumor cells with TT-232 caused significant inhibition of tyrosine kinases in good correlation with the apoptosis-inducing effect. The level of p53 or KU86 did not change following TT-232 treatment, suggesting a p53-independent apoptotic effect. Preincubation of human breast cancer cells (MDA-MB-453) with TT-232 for 2 h decreased the growth factor receptor autophosphorylation. All of these data suggest that TT-232 is a promising and selective antitumor agent.

PR-39, a proline-rich antibacterial peptide that inhibits phagocyte NADPH oxidase activity by binding to Src homology 3 domains of p47 phox.

Reactive oxygen intermediates generated by the phagocyte NADPH oxidase are critically important components of host defense. However, these highly toxic oxidants can cause significant tissue injury during inflammation; thus, it is essential that their generation and inactivation are tightly regulated. We show here that an endogenous proline-arginine (PR)-rich antibacterial peptide, PR-39, inhibits NADPH oxidase activity by blocking assembly of this enzyme through interactions with Src homology 3 domains of a cytosolic component. This neutrophil-derived peptide inhibited oxygen-dependent microbicidal activity of neutrophils in whole cells and in a cell-free assay of NADPH oxidase. Both oxidase inhibitory and direct antimicrobial activities were defined within the amino-terminal 26 residues of PR-39. Oxidase inhibition was attributed to binding of PR-39 to the p47phox cytosolic oxidase component. Its effects involve both a polybasic amino-terminal segment and a proline-rich core region of PR-39 that binds to the p47phox Src homology 3 domains and, thereby, inhibits interaction with the small subunit of cytochrome b558, p22phox. These findings suggest that PR-39, which has been shown to be involved in tissue repair processes, is a multifunctional peptide that can regulate NADPH oxidase production of superoxide anion O2-. thus limiting excessive tissue damage during inflammation.

Identification of haptoglobin as a natural inhibitor of trypanocidal activity in human serum.

Trypanosomes are protozoan parasites of medical and veterinary importance. Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense infect humans, causing African sleeping sickness. However, Trypanosoma brucei brucei can only infect animals, causing the disease Nagana in cattle. Man is protected from this subspecies of trypanosomes by a toxic subtype of high density lipoproteins (HDLs) called the trypanosome lytic factor (TLF). The toxic molecule in TLF is believed to be the haptoglobin-related protein that when bound to hemoglobin kills the trypanosome via oxidative damage initiated by its peroxidase activity. The amount of lytic activity in serum varies widely between different individuals with up to a 60-fold difference in activity. In addition, an increase in the total amount of lytic activity occurs during the purification of TLF, suggesting that an inhibitor of TLF (ITLF) exists in human serum. We now show that the individual variation in trypanosome lytic activity in serum correlates to variations in the amount of ITLF. Immunoblots of ITLF probed with antiserum against haptoglobin recognize a 120-kDa protein, indicating that haptoglobin is present in partially purified ITLF. Haptoglobin involvement is further shown in that it inhibits TLF in a manner similar to ITLF. Using an anti-haptoglobin column to remove haptoglobin from ITLF, we show that the loss of haptoglobin coincides with the loss of inhibitor activity. Addition of purified haptoglobin restores inhibitor activity. This indicates that haptoglobin is the molecule responsible for inhibition and therefore causing the individual variation in serum lytic activity.

The benzene metabolite p-benzoquinone forms adducts with DNA bases that are excised by a repair activity from human cells that differs from an ethenoadenine glycosylase.

Benzene is a ubitiquous human environment mental carcinogen. One of the major metabolites is hydroquinone, which is oxidized in vivo to give p-benzoquinone (p-BQ). Both metabolites are toxic to human cells. p-BQ reacts with DNA to form benzetheno adducts with deoxycytidine, deoxyadenosine, and deoxyguanosine. In this study we have synthesized the exocyclic compounds 3-hydroxy-3-N4-benzetheno-2'-deoxycytidine (p-BQ-dCyd) and 9-hydroxy-1,N6-benzetheno-2'-deoxyadenosine (p-BQ-dAdo), respectively, by reacting deoxycytidine and deoxyadenosine with p-BQ. These were converted to the phosphoamidites, which were then used to prepare site-specific oligonucleotides with either the p-BQ-dCyd or p-BQ-dAdo adduct (pbqC or pbqA in sequences) at two different defined positions. These oligonucleotides were efficiently nicked 5' to the adduct by partially purified HeLa cell extracts--the pbqC-containing oligomer more rapidly than the pbqA-containing oligomer. In contrast to the enzyme binding to derivatives produced by the vinyl chloride metabolite chloroacetaldehyde, the oligonucleotides up to 60-mer containing p-BQ adducts did not bind measurably to the same enzyme preparation in a gel retardation assay. Furthermore, there was no competition for the binding observed between oligonucleotides containing 1,N6-etheno A deoxyadenosine (1,N6-etheno-dAdo; epsilon A in sequences) and these oligomers containing either of the p-BQ adducts, even at 120-fold excess. When highly purified fast protein liquid chromatography (FPLC) enzyme fractions were obtained, there appeared to be two closely eluting nicking activities. One of these enzymes bound and cleaved the epsilon A-containing deoxyoligonucleotide. The other enzyme cleaved the pbqA- and pbqC-containing deoxyoligonucleotides. One additional unexpected fact was that bulk p-BQ-treated salmon sperm DNA did compete effectively with the epsilon A-containing oligonucleotide for protein binding. This raises the possibility that such DNA contains other, as-yet-uncharacterized adducts that are recognized by the same enzyme that recognizes the etheno adducts. In summary, we describe a previously undescribed human DNA repair activity, possibly a glycosylase, that excises from DNA pbqC and pbqA, exocyclic adducts resulting from reaction of deoxycytidine and deoxyadenosine with the benzene metabolite, p-BQ. This glycosylase activity is not identical to the one previously reported from this laboratory as excising the four etheno bases from DNA.