Stable analogues of geranylgeranyl diphosphate possessing improved geranylgeranyl versus farnesyl protein transferase inhibitory selectivity

Phosphonoacetamido(oxy) groups have proven to be good mimics of the diphosphate portion in geranylgeranyl protein transferase I (GGTase I) inhibitors. The introduction of small alkyl groups (Me, Et) into the diphosphate mimic moiety caused a further decrease in collateral farnesyl protein transferase (FTase) inhibitory activity, thereby improving GGTase I over FTase selectivity.     

Design and synthesis of factor Xa inhibitors and their prodrugs

In addition to our previously reported fluoro acrylamides Xa inhibitors 2 and 3, a series of potent and novel cyclic diimide amidine compounds has been identified. In efforts to improve their oral bioavailability, replacement of the amidine group with methyl amidrazone gives compounds of moderate potency (14, IC(50)=0.028 microM). In the amidoxime prodrug approach, the amidoxime compounds show good oral bioavailability in rats and dogs. High plasma level of prodrug 26 and significant concentration of active drug 26a were obtained upon oral administration of prodrug 26 in rats.     

Bisphosphonates derived from fatty acids are potent inhibitors of Trypanosoma cruzi farnesyl pyrophosphate synthase

Studies on the mode of action of a series of bisphosphonates derived from fatty acids, which had previously proved to be potent inhibitors against Trypanosoma cruzi proliferation in in vitro assays, have been performed. Some of these drugs proved to be potent inhibitors against the intracellular form of the parasite, exhibiting IC(50) values at the low micromolar level. As bisphosphonates are FDA clinically approved for treatment of bone resorption disorders, their potential innocuousness makes them good candidates to control tropical diseases.     

Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping

Alternative splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 is regulated by a combination of cis-acting elements distributed through the exon and both flanking introns (IVS8 and IVS9). Several studies have identified in the IVS8 intron 3' splice site a regulatory element that is composed of a polymorphic (TG)m(T)n repeated sequence. At present, no cellular factors have been identified that recognize this element. We have identified TDP-43, a nuclear protein not previously described to bind RNA, as the factor binding specifically to the (TG)m sequence. Transient TDP-43 overexpression in Hep3B cells results in an increase in exon 9 skipping. This effect is more pronounced with concomitant overexpression of SR proteins. Antisense inhibition of endogenous TDP-43 expression results in increased inclusion of exon 9, providing a new therapeutic target to correct aberrant splicing of exon 9 in CF patients. The clinical and biological relevance of this finding in vivo is demonstrated by our characterization of a CF patient carrying a TG10T9(DeltaF508)/TG13T3(wt) genotype leading to a disease-causing high proportion of exon 9 skipping.     

Insulin receptor substrate-1, p70S6K, and cell size in transformation and differentiation of hemopoietic cells

After an initial burst of cell proliferation, the type 1 insulin-like growth factor receptor (IGF-IR) induces granulocytic differentiation of 32D IGF-IR cells, an interleukin-3-dependent murine hemopoietic cell line devoid of insulin receptor substrate-1 (IRS-1). The combined expression of the IGF-IR and IRS-1 (32D IGF-IR/IRS-1 cells) inhibits IGF-I-mediated differentiation, and causes malignant transformation of 32D cells. Because of the role of IRS-1 in changing the fate of 32D IGF-IR cells from differentiation (and subsequent cell death) to malignant transformation, we have looked for differences in IGF-IR signaling between 32D IGF-IR and 32D IGF-IR/IRS-1 cells. In this report, we have focused on p70(S6K), which is activated by the IRS-1 pathway. We find that the ectopic expression of IRS-1 and the inhibition of differentiation correlated with a sustained activation of p70(S6K) and an increase in cell size. Phosphorylation in vivo of threonine 389 and, to a lesser extent, of threonine 421/serine 424 of p70(S6K) seemed to be a requirement for inhibition of differentiation. A role of IRS-1 and p70(S6K) in the alternative between transformation or differentiation of 32D IGF-IR cells was confirmed by findings that inhibition of p70(S6K) activation or IRS-1 signaling, by rapamycin or okadaic acid, induced differentiation of 32D IGF-IR/IRS-1 cells. We have also found that the expression of myeloperoxidase mRNA (a marker of differentiation, which sharply increases in 32D IGF-IR cells), does not increase in 32D IGF-IR/IRS-1 cells, suggesting that the expression of IRS-1 in 32D IGF-IR cells causes the extinction of the differentiation program initiated by the IGF-IR, while leaving intact its proliferation program.     

Nitric oxide synthase inhibition in human sperm affects sperm-oocyte fusion but not zona pellucida binding

There is recent evidence that mouse and human spermatozoa contain constitutive nitric oxide synthase (cNOS) and can synthesize nitric oxide. The aim of this study was to investigate whether the inhibition of human sperm cNOS could affect sperm-oocyte fusion and sperm binding to the zona pellucida (ZP). N(G)-nitro-L-arginine methyl ester (L-NAME) was used as cNOS inhibitor. Sperm-oocyte fusion was evaluated using the hamster egg penetration test (HEPT). The ZP binding was evaluated using the hemizona assay. L-NAME added from the onset of capacitation strongly inhibited sperm-oocyte fusion. This inhibitory effect was dose dependent, stereospecific, and suppressed by L-arginine in a dose-dependent manner. L-NAME also inhibited sperm-oocyte fusion in the HEPT enhanced with progesterone (P), where P (5 microM) was added for 15 min to capacitated sperm. A lesser but significant inhibition was also observed when sperm suspensions were exposed to L-NAME following capacitation in both versions of HEPT. On the contrary, L-NAME did not affect ZP binding. In conclusion, the present study provides the evidence that cNOS plays a role in the human sperm's capacity to fuse with oocyte but not in the ZP binding.     

Differential effects of N-acetyl-2-aminofluorene and N-2-aminofluorene adducts on the conformational change in the structure of DNA polymerase I (Klenow fragment)

The carcinogen N-acetyl-2-aminofluorene forms two major DNA adducts: the N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene adduct (dG-C8-AAF) and its deacetylated derivative, the N-(2'-deoxyguanosin-8-yl)-2-aminofluorene adduct (dG-C8-AF). It is well established that the AAF adduct is a very strong block for DNA synthesis in vitro while the AF adduct is more easily bypassed. In an effort to understand the molecular mechanism of this phenomenon, the structure of the complex of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) bound to primer-templates containing either an AF or AAF adduct in or near the active site was probed by nuclease and protease digestion analyses. The results of these experiments suggest that positioning the AAF adduct in the polymerase active site strongly inhibits the conformational change that is required for the insertion of a nucleotide. Similar experiments with AF-modified primer-templates shows a much less pronounced effect. The inhibition of the conformational change by either adduct is not detected if they are positioned in the single-stranded part of the template just one nucleotide before the active site. These findings may explain the different abilities of these lesions to block DNA synthesis.     

Critical Temporal Modulation of Neuronal Programmed Cell Injury

1. As a free radical, nitric oxide (NO) may be toxic to neurons through mechanisms that directly involve DNA damage. Lubeluzole, a novel benzothiazole compound, has recently been demonstrated to be neuroprotective through the signal transduction pathways of NO. We therefore examined whether neuroprotection by lubeluzole was dependent upon the molecular pathways of programmed cell death (PCD).2. In primary hippocampal neurons, evidence of PCD was determined by hematoxylin and eosin (H&E) stain, transmission electron microscopy, and annexin-V binding. NO administration with the NO generators sodium nitroprusside (300 M) or SIN-1 (300 M) directly induced PCD.3. Neurons positive for PCD increased from 22 ± 3% (untreated) to 72 ± 3% (NO) over a 24-hr period. Coadministration of NO and lubeluzole (750 nM), a neuroprotective concentration, actively decreased PCD expression on H&E stain from 72 ± 3% (NO only) to 25 ± 3% (NO and lubeluzole). Significant reduction in DNA fragmentation by lubeluzole also was evident on electron microscopy. Application of lubeluzole in concentrations that were not neuroprotective or administration of the biologically inactive R-isomer did not significantly alter NO-induced PCD, suggesting that neuroprotection by lubeluzole was intimately linked to the modulation of PCD. Lubeluzole also was able to prevent the initial stages of cellular membrane inversion labeled with annexin-V binding, an early and sensitive indicator of PCD. Interestingly, the critical period for lubeluzole to reverse PCD induction appeared to be within the first 4 hr following NO exposure.4. Further investigation into the neuroprotective pathways that alter PCD may provide greater insight into the molecular mechanisms that ultimately determine neuronal injury.     

Compound heterozygosity for two different amino-acid substitution mutations in the thrombopoietin receptor (c-mpl gene) in congenital amegakaryocytic thrombocytopenia (CAMT)

Congenital amegakaryocytic thrombocytopenia (CAMT) without physical anomalies is a rare disease, presenting isolated thrombocytopenia and megakaryocytopenia in infancy, which can evolve into aplastic anemia and leukemia. Recently, two heterozygous truncating mutations of the thrombopoietin (TPO) receptor MPL, coded by the c-mpl gene, were identified in a 10-year-old Japanese patient with CAMT transmitted in an autosomal recessive manner. Here, we report for the first time two different MPL amino-acid substitutions in a 2-year-old Italian boy with CAMT and compound heterozygosis for two (c-mpl point mutations. C-to-T transitions were detected on exons 5 and 12 at the 769 and 1904 cDNA nucleotide positions, respectively. The mutation in exon 5 substitutes an arginine with a cysteine (R257C) in the extracellular domain, 11 amino acids distant from the WSXWS motif conserved in the cytokine-receptor superfamily. The mutation in exon 12 substitutes a proline with a leucine (P635L) in the last amino acid of the C-terminal intracellular domain, responsible for signal transduction. As in the Japanese family, the mutations were both transmitted from the parents. TPO plasma levels were highly increased in the patient. The patient's 7-year-old brother, who was a candidate donor for allografting, turned out to be an asymptomatic heterozygous carrier of P635L and showed defective megakaryocyte colony formation from bone-marrow progenitor cells. The present study provides important confirmation that CAMT can be associated with (c-mpl) mutations.     

Behaviour of interleukin-2 serum levels in advanced non-small-cell lung cancer patients: relationship with response to therapy and survival

 Interleukin(IL)-2 is a T helper (Th) 1 type cytokine that has been shown to play an important role in antitumour immune responses. In this study, the prognostic significance of serum IL-2 levels was investigated in 60 advanced non-small-cell lung cancer (NSCLC) patients. IL-2 serum levels were determined before chemotherapy, at the end of chemotherapy and during follow-up, using a commercially available enzyme-linked immunoadsorbent assay kit. The results were analysed according to the response to therapy and were used to generate a model predicting overall survival and time to treatment failure. All 60 patients were shown to have higher IL-2 serum levels than controls (P < 0.0001). Stage IV patients had significantly lower IL-2 levels than stage III patients (P < 0.0001), although they were still significantly higher than controls (P < 0.0001). It is interesting that, when patients were divided into responders and non-responders according to the response to therapy, the former were shown to have significantly higher pre-chemotherapy levels than the latter (P < 0.0001). Moreover, a further significant increase in IL-2 serum levels (P=0.004) and a significant decrease (P < 0.0001) were shown in responders and non-responders, respectively at the end of the therapy. Using univariate and multivariate analyses, both overall survival and time to treatment failure were shown to be affected by the mean pathological levels of IL-2. Furthermore, the prognostic significance of the serum level of IL-2 was confirmed by the stepwise regression analysis. In conclusion, determination of pre-treatment IL-2 serum levels was shown to be of independent prognostic utility in patients with advanced NSCLC; therefore, its possible use for prediction of outcome is proposed. Received: 16 March 2000 / Accepted: 27 July 2000