Identification of the primary caspase 3 cleavage site in alpha II-spectrin during apoptosis.

Alpha II-spectrin is one of the major proteins responsible for maintaining the cytoskeletal integrity of the cell. The caspase 3-mediated cleavage of alpha II-spectrin during apoptotic cell death may play an important role in altering membrane stability and the formation of apoptotic bodies. In this study, we identified the primary caspase 3 cleavage site in alpha II-spectrin. We found that the transcriptional inhibitor, actinomycin D, induced caspase 3 activation and that caspase 3 activation is coincident with the cleavage of alpha II-spectrin protein at a primary cleavage site. Deletion analysis and site directed mutagenesis identified the primary cleavage site in alpha II spectrin at amino acid 1185 (DETD). The primary caspase 3 cleavage site in alpha II spectrin is conserved in immature and mature B cells. Our results indicate that alpha II-spectrin is initially cleaved at a caspase 3 consensus site and this primary event likely alters the structural conformation of the protein exposing subsequent cleavage sites and altering cytoskeletal integrity. Identification of the primary cleavage site for caspase 3 may help to elucidate the role of alpha II-spectrin in membrane stability and apoptosis as well as provide new insights into alpha II-spectrin autoantibody formation associated with the autoimmune disease, Sjögren's syndrome.

An approach to the identification of potent inhibitors of influenza virus fusion using parallel synthesis methodology.

Structure-activity studies associated with the salicylic acid-derived inhibitor of influenza fusion, BMY-27709, were examined using a parallel synthesis approach. This SAR survey led to the discovery of potent influenza inhibitory activity in a series of aromatic amides and thioamides derived from 1,3,3-trimethyl-5-hydroxycyclohexylmethylamine. Select compounds were characterized as inhibitors of the H1 subtype of influenza A viruses that act by preventing the pH-induced fusion process, thereby blocking viral entry into host cells. In a plaque-reduction assay, the most potent inhibitors displayed EC(50) values of 0.02-0.14 microg/mL.

Spectrin oligomerization is cooperatively coupled to membrane assembly: a linkage targeted by many hereditary hemolytic anemias?

In the erythrocyte, ankyrin is the major adapter protein linking tetramers of band 3 to the spectrin-actin cytoskeleton. This linkage involves a direct interaction between ankyrin and the 14th-15th repeat unit of beta-spectrin. The spectrin cytoskeleton itself is stabilized by the self-association of spectrin heterodimers into tetramers and larger oligomers, a process mediated by the 17th repeat unit of beta-spectrin and a short NH(2) -terminal sequence in alpha-spectrin. The self-association of spectrin and its ankyrin-mediated membrane binding have generally been considered independent events. We now demonstrate that spectrin self-association, the binding of spectrin to ankyrin, and the binding of ankyrin to the 43-kDa cytoplasmic domain of band 3 (cdb3) are coupled in a positively cooperative way. In solution, [(125)I]-labeled ankyrin was found by ND-PAGE3 to enhance the affinity of spectrin self-association by 10-fold. The reciprocal process was also true, in that spectrin tetramers and oligomers bound ankyrin with enhanced affinity relative to dimer spectrin. Saturation of the beta-spectrin self-association site by an NH(2) -terminal 80-kDa alpha-spectrin peptide enhanced the affinity of spectrin dimer for ankyrin, indicating a direct relationship between ankyrin binding and the occupancy of the beta-spectrin self-association site. cdb3 accentuated these cooperative interactions. Several inherited spectrin mutations that cause hemolytic disease but that do not directly destabilize the self-association or ankyrin-binding sites can be explained by these results. Three classes of mutations appear to disrupt cooperative coupling between self-association and ankyrin binding: (i) mutation of the linker sequences that join helices C and A in repeat units that intervene between the two functional sites, mutations that presumably block repeat-to-repeat transfer of conformational information; (ii) mutations in alpha-spectrin repeats 4 to 6 that disrupt the ability of this region to trans-regulate ankyrin binding by the adjacent beta-spectrin repeats 14-15; and (iii) exon-skipping mutations that shorten alpha-spectrin and force repeats 4 to 6 to fall out-of-register with the ankyrin-binding motif in beta-spectrin. Collectively, these results demonstrate a molecular mechanism whereby a membrane receptor can directly promote cytoskeletal assembly.

Treatment options in patients with recurrent ovarian cancer.

The majority of patients with advanced ovarian cancer need a second-line treatment for recurrent disease after surgical cytoreduction and first-line chemotherapy. In these patients, treatment planning is mainly dependent on the platinum-free-interval. The patients may be distinguished as platinum-refractory (progression under platinum-based therapy), platinum-resistant (relapse within 6 months), or platinum-sensitive (relapse after 6 months). Patients with platinum-refractory or -resistant disease should be encouraged to enter clinical trials. Alternatively, these patients could receive tamoxifen or a non-platinum single-agent therapy. Since response rate and duration to different single-agents are similar, patient convenience, toxicities from prior treatment, side-effects and costs play a role in the drug selection for salvage chemotherapy. Patients with platinum-sensitive disease should receive carboplatin based or carboplatin-plus paclitaxel-based regimens. Secondary surgical cytoreduction may have a role in highly selected patients with good performance status, with long disease-free interval and without extra-abdominal or liver metastases.

Salicylamide inhibitors of influenza virus fusion.

Structural variation of the quinolizidine heterocycle of the influenza fusion inhibitor BMY-27709 was examined by several topological dissections in order to illuminate the critical features of the ring system. This exercise resulted in the identification of a series of synthetically more accessible decahydroquinolines that retained the structural elements of BMY-27709 important for antiviral activity. The 2-methyl-cis-decahydroquinoline 6f was the most potent influenza inhibitor identified that demonstrated an EC50 of 90 ng/mL in a plaque reduction assay.

p53 status is neither a predictive nor a prognostic variable in patients with advanced ovarian cancer treated with a paclitaxel-based regimen.

BACKGROUND: The aim of this study was to assess the relationship between p53 status and the clinical outcome of patients with advanced ovarian cancer treated with a paclitaxel-based regimen. PATIENTS AND METHODS: The investigation was conducted on 38 patients with FIGO stage III-IV ovarian cancer from whom tumor tissue samples for p53 protein immunostaining were obtained during initial cytoreductive surgery. All these patients subsequently received six cycles of first-line combination chemotherapy with paclitaxel 175 mg/m2 (3-hour infusion) plus carboplatin AUC 6 with or without epidoxorubicin 75 mg/m2. RESULTS: Positive p53 immunostaining was detected in tissue samples collected from 24 (63.2%) ovarian cancers. A clinical complete response was obtained in 14 (58.3%) of the 24 patients with positive p53 immunostaining compared to 9 (64.3%) of the 14 patients with negative p53 immunostaining (p = 0.717). A pathological complete response was found in 6 (25.0%) of the former compared to 4 (28.6%) of the latter (p = 0.956). Similarly, survival did not correlate with p53 status (p = 0.1271). DISCUSSION: p53 status seems to be neither a predictive nor a prognostic variable in patients with advanced ovarian cancer treated with a paclitaxel-based regimen. These results are consistent with experimental data showing that paclitaxel cytotoxicity in ovarian cancer is likely to be mediated by a p53-independent pathway.

Transforming growth factor beta induces caspase 3-independent cleavage of alphaII-spectrin (alpha-fodrin) coincident with apoptosis.

Transforming growth factor beta (TGF-beta) is a potent growth inhibitor and inducer of cell death in B-lymphocytes and is essential for immune regulation and maintenance of self-tolerance. In this report the mouse immature B cell line, WEHI 231, was used to examine the mechanisms involved in TGF-beta-mediated apoptosis. Induction of apoptosis is detected as early as 8 h after TGF-beta administration. Coincident with the onset of apoptosis, the cytoskeletal actin-binding protein, alphaII-spectrin (alpha-fodrin) is cleaved into 150-, 115-, and 110-kDa fragments. The broad spectrum caspase inhibitor (Boc-D-fmk (BD-fmk)) completely abolished TGF-beta-induced apoptosis and alphaII-spectrin cleavage. Caspase 3, although present in WEH1 231 cells, was not activated by TGF-beta, nor was its substrate, poly(ADP-ribose) polymerase. These results identify alphaII-spectrin as a novel substrate that is cleaved during TGF-beta-induced apoptosis. Our data provide the first evidence of calpain and caspase 3-independent cleavage of alphaII-spectrin during apoptosis and suggests that TGF-beta induces apoptosis and alphaII-spectrin cleavage via a potentially novel caspase. This report also provides the first direct evidence of caspase 3 activation in WEH1 231 cells and indicates that at least two distinct apoptotic pathways exist.

Novel quinolizidine salicylamide influenza fusion inhibitors.

A novel series of quinolizidine salicylamides was synthesized as specific inhibitors of the H1 subtype of influenza A viruses. These inhibitors inhibit the pH-induced fusion process, thereby blocking viral entry into host cells. Compound 16 was the most active inhibitor in this series with an EC50 of 0.25 microg/mL in plaque reduction assay. The synthesis and the SAR of these compounds are discussed.

Protracted continuous infusion of 5-fluorouracil and low-dose leucovorin in patients with metastatic colorectal cancer resistant to 5-fluorouracil bolus-based chemotherapy: a phase II study.

Continuous-infusion (c.i.) 5-fluorouracil (5-FU) can overcome resistance to bolus 5-FU, and leucovorin (LV) enhances the cytotoxic effects of 5-FU, mainly when the duration of exposure to the latter is prolonged. The main objective of this study was therefore to determine the activity of a prolonged infusion schedule of 5-FU + LV in patients with metastatic colorectal cancer resistant to a 5-FU bolus-based chemotherapy. Only patients with metastatic measurable disease in progression during or within 2 months of the end of a 5-FU bolus +/- LV-based chemotherapy were eligible for the study. 5-FU and l-LV were given as a 14-day c.i. every 28 days, the 5-FU dose being 200 mg/m2 per day and the l-LV dose being 5 mg/m2 per day. A total of 59 patients entered the study, of which 48 were resistant to 5-FU + LV and 11, to 5-FU + levamisole. Treatment was well tolerated, and WHO grade 3-4 toxicities were uncommon (11% of patients developed stomatitis and 7%, diarrhea). According to an intent-to-treat analysis, 10 of 59 patients obtained an objective response (1 complete response, 9 partial responses), for an objective response rate of 16% (95% confidence interval 8-25%). The median progression-free survival and overall survival were 4 and 9 months, respectively. The protracted 5-FU + LV c.i. schedule used in the present study is a well-tolerated and moderately active regimen in metastatic colorectal cancer patients resistant to 5-FU bolus +/- LV. Only randomized studies can determine whether this palliative treatment has advantages in comparison with other second-line therapies such as 5-FU c.i. without LV, irinotecan, or oxaliplatin.

pH-dependent changes in photoaffinity labeling patterns of the H1 influenza virus hemagglutinin by using an inhibitor of viral fusion.

The hemagglutinin (HA) protein undergoes a low-pH-induced conformational change in the acidic milieu of the endosome, resulting in fusion of viral and cellular membranes. A class of compounds that specifically interact with the HA protein of H1 and H2 subtype viruses and inhibit this conformational change was recently described (G. X. Luo et al., Virology 226:66-76, 1996, and J. Virol. 71:4062-4070, 1997). In this study, purified HA trimers (bromelain-cleaved HA [BHA]) are used to examine the properties and binding characteristics of these inhibitors. Compounds were able to inhibit the low-pH-induced change of isolated trimers, as detected by resistance to digestion with trypsin. Protection from digestion was extremely stable, as BHA-inhibitor complexes could be incubated for 24 h in low pH with almost no change in BHA structure. One inhibitor was prepared as a radiolabeled photoaffinity analog and used to probe for specific drug interactions with the HA protein. Analysis of BHA after photoaffinity analog binding and UV cross-linking revealed that the HA2 subunit of the HA was specifically radiolabeled. Cross-linking of the photoaffinity analog to BHA under neutral (native) pH conditions identified a stretch of amino acids within the alpha-helix of HA2 that interact with the inhibitor. Interestingly, cross-linking of the analog under acidic conditions identified a different region within the HA2 N terminus which interacts with the photoaffinity compound. These attachment sites help to delineate a potential binding pocket and suggest a model whereby the BHA is able to undergo a partial, reversible structural change in the presence of inhibitor compound.

Brain and muscle express a unique alternative transcript of alphaII spectrin.

Alternative splicing of pre-mRNA transcripts of alpha and beta spectrin has emerged as an important generator of diversity in this gene family, yet the functional consequences and extent of this diversity remains unknown. We have cloned and characterized full-length alphaII spectrin cDNA from human fetal brain (GenBank and ). On the basis of the predicted amino acid sequence, 11 amino acid substitutions, presumably representing polymorphisms, have been identified that distinguish this alphaII spectrin from human lung fibroblast alphaII spectrin. In addition, human fetal brain spectrin displays a novel five amino acid insertion in repeat 15 that arises from alternative mRNA splicing and that distinguishes this spectrin from lung fibroblast alphaII++ spectrin. This discovery, together with two previously identified regions of alternative mRNA splicing in alphaII spectrin suggest that as many as eight different splice forms of the mature protein might exist if all combinations (at inserts 1, 2, and 3) of alternative mRNA splicing are utilized. To assess this possibility, the tissue distribution of alternative exon usage was investigated by semiquantitative PCR with intron-jumping primer sets. Tissues examined were from mouse and included heart, kidney, lung, liver, thymus, spleen, brain, ovary, testis, and skeletal muscle, as well as mouse embryonic tissue. Transcripts both with and without insert 1, representing a 60 bp insertion within alphaII spectrin repeat 10, were identified in all tissues. In contrast, transcripts with insert 2, the novel 15 bp insertion reported here, were only expressed in brain, heart, skeletal muscle, and embryonic tissue. In all tissues examined only transcripts positive for insert 3, an 18 bp insertion in repeat 21, were amplified, even under conditions in which a 30% level of insert 3 negative transcript could be easily detected in artificially prepared control samples. All combinations of insert 1 and insert 2 were identified together in individual transcripts, verifying at least four distinct isoforms of alphaII spectrin. These have been named alphaIISigma1 through alphaIISigma4, in accord with current spectrin naming conventions. Dynamic molecular modeling of the 15th repeat unit incorporating insert 2 predicts that the spliced sequence forms a loop between helices A and B, and suggests that this insert might constitute a novel protein interaction site. The presence of this sequence in alphaIISigma3 and alphaIISigma4 spectrin suggests a specialized and heretofore unanticipated function for the 15th repeat of this molecule.

Conquering influenza: recent advances in anti-influenza drug discovery.

Morbidity and mortality associated with influenza virus infection have cut a wide swathe through medical history. Even with the development of killed virus vaccines, illness due to influenza virus infection continues to be a major health problem throughout the world. This may change over the next few years with the development of a new class of antiviral agents targeting the catalytic site of the viral neuraminidase enzyme. These agents have been demonstrated to be both efficacious and clinically beneficial. In addition, investigation into alternative anti-influenza targets continues. These include inhibitors of hemagglutinin-mediated functions, the viral polymerase and new inhibitors of M2 ion channel activity.

Development of antivirals against influenza.

Morbidity and mortality due to influenza virus infections remain a major problem throughout the world. Yearly, medical costs and loss of productivity resulting from influenza infection are estimated to be in the range of 12 dollars bn in the USA. The predicted increases in the elderly and immune-deficient populations will make influenza an even greater threat in the future. Despite the availability of vaccines, they have been least effective in these high-risk populations. Coupled with the requirement for routine revaccination, the need for effective antiviral agents is illustrated. The currently approved drugs, amantadine, rimantadine and ribavirin (in some countries), have limitations. They are only inhibitory against influenza A viruses, are prone to adverse reactions and quickly give rise to resistant virus. This review examines current drug therapies, antivirals in development and possible future opportunities for anti-influenza drugs.

Differential effect of modified capped RNA substrates on influenza virus transcription.

The RNA-dependent RNA polymerase of influenza virus transcribes messenger RNA through a unique cap scavenging mechanism. Viral enzyme binds to the cap structure of host mRNA, cleaves the molecule 9-15 bases downstream of the cap, and uses the short capped oligonucleotide as a primer for mRNA synthesis. Previously, we have shown that the viral polymerase can efficiently bind capped RNAs shorter than 9 nucleotides in length, but the viral enzyme can not utilize these RNAs as primers. For this reason, these short capped oligonucleotides are potent inhibitors of influenza virus transcription. In these studies, it is now shown that short capped oligomers inhibit capped-RNA dependent transcription at the initial step of cap binding. In contrast, low concentrations of these short capped RNAs can actually stimulate viral transcription primed with high concentrations of the dinucleotide ApG. Another capped RNA derivative containing phosphorothioate oligonucleotides was also investigated as a potential polymerase inhibitor. This longer capped RNA was able to bind to the polymerase, but could not be cleaved to primer length by the enzyme associated endonuclease. Thus, the capped phosphorothioate RNA inhibited cap-primed transcription at the step of cap binding. However, in contrast to the short capped oligonucleotide, it also inhibited ApG primed viral transcription.

Molecular mechanism underlying the action of a novel fusion inhibitor of influenza A virus.

In the initial stages of influenza virus infection, the hemagglutinin (HA) protein of influenza virus mediates both adsorption and penetration of the virus into the host cell. Recently, we identified and characterized BMY-27709 as an inhibitor of the H1 and H2 subtypes of influenza A virus that specifically inhibits the HA function necessary for virus-cell membrane fusion (G.-X. Luo, R. Colonno, and M. Krystal, Virology 226:66-76, 1996). Studies presented herein show that the inhibition is mediated through specific interaction with the HA protein. This binding represses the low-pH-induced conformational change of the HA protein which is a prerequisite for membrane fusion. In an attempt to define the binding pocket within the HA molecule, a number of drug-resistant viruses have been isolated and characterized. Sequence analyses of the HA gene of these drug-resistant viruses mapped amino acid changes responsible for drug resistance to a region located near the amino terminus of HA2. In addition, we have identified inactive analogs of BMY-27709 which are able to compete out the inhibitory activity of BMY-27709. This finding suggests that inhibition of the HA-mediated membrane fusion by this class of compounds is not solely the result of binding within the HA molecule but requires specific interactions.

Site-directed mutagenesis of alpha II spectrin at codon 1175 modulates its mu-calpain susceptibility.

Intracellular proteolysis by the calpains, a family of Ca2+ activated cysteine proteases, is a ubiquitous yet poorly understood process. Their action is implicated in an array of cellular and pathologic processes, including long-term potentiation, synaptic remodeling, protein kinase C and steroid receptor activation, ischemic cellular injury, and apoptosis. Unlike most proteases, the calpains display unusually strict substrate specificity, often cleaving only one or two bonds in proteins with hundreds of potential sites. Studies of synthetic peptides have defined sequences that modulate their specificity, but little data exist in the context of a bona fide protein. A prominent substrate for mu-calpain is alpha II spectrin (fodrin, brain spectrin), which is cleaved between Tyr1176 and Gly1177 within spectrin's 11th structural repeat unit. We have cloned and characterized human fetal brain alpha II spectrin (GenBank no. U26396) and identified a new Thr1300 to Ile polymorphism. From this clone, recombinant GST-fusion proteins representing repeat units 8-14 have been prepared and used to systematically explore the in vitro determinants of mu-calpain sensitivity. Twenty different amino acids were substituted by site-directed mutagenesis for wild-type Val1175, the penultimate (P2) residue flanking the major calpain cleavage site in alpha II spectrin. Gly, Pro, and Asp, and to a lesser extent Phe and Glu, substantively inhibited the susceptibility of this site to mu-calpain; other substitutions yielded lesser effects. Dynamic molecular modeling of the 11th structural repeat of human alpha II spectrin incorporating the various mutations suggests that the calpain cleavage site with its flanking calmodulin binding domain interrupts helix C of alpha II spectrin's 11th repetitive unit without significantly disrupting the repeat's triple-helical motif. This model predicts that the critical Tyr1176-Gly1177 bond occurs in a highly exposed loop juxtaposed between helix C and the calmodulin binding domain and that mutations at the P2 position subtly alter the conformation about this site. We conclude that secondary and tertiary conformational features surrounding the cleavage site, rather than the linear sequence itself, dominate the determinants that define alpha II spectrin's mu-calpain susceptibility.

Lobucavir is phosphorylated in human cytomegalovirus-infected and -uninfected cells and inhibits the viral DNA polymerase.

Lobucavir (LBV) is a deoxyguanine nucleoside analog with broad-spectrum antiviral activity. LBV was previously shown to inhibit herpes simplex virus (HSV) DNA polymerase after phosphorylation by the HSV thymidine kinase. Here we determined the mechanism of action of LBV against human cytomegalovirus (HCMV). LBV inhibited HCMV DNA synthesis to a degree comparable to that of ganciclovir (GCV), a drug known to target the viral DNA polymerase. The expression of late proteins and RNA, dependent on viral DNA synthesis, was also inhibited by LBV. Immediate-early and early HCMV gene expression was unaffected, suggesting that LBV acts temporally coincident with HCMV DNA synthesis and not through cytotoxicity. In vitro, the triphosphate of LBV was a potent inhibitor of HCMV DNA polymerase with a Ki of 5 nM. LBV was phosphorylated to its triphosphate form intracellularly in both infected and uninfected cells, with phosphorylated metabolite levels two- to threefold higher in infected cells. GCV-resistant HCMV isolates, with deficient GCV phosphorylation due to mutations in the UL97 protein kinase, remained sensitive to LBV. Overall, these results suggest that LBV-triphosphate halts HCMV DNA replication by inhibiting the viral DNA polymerase and that LBV phosphorylation can occur in the absence of viral factors including the UL97 protein kinase. Furthermore, LBV may be effective in the treatment of GCV-resistant HCMV.