Protein prenylation such as farnesylation and geranylgeranylation is associated with various diseases. Thus, many inhibitors of prenyltransferase have been developed. We report novel inhibitors of farnesyltransferase with a zinc-site recognition moiety and a farnesyl/dodecyl group. Molecular docking analysis showed that both parts of the inhibitor fit well into the catalytic domain of farnesyltransferase. The synthesized inhibitors showed activity against farnesyltransferase in vitro and inhibited proliferation of the pancreatic cell line AsPC-1. Among the compounds with farnesyl and dodecyl groups, the inhibitor with a farnesyl group was found to have stronger and more selective activity.Download high-res image (67KB)Download full-size image

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G, A3G), is a human anti-virus restriction protein which works deaminase-dependently and -independently. A3G is known to be ubiquitinated by HIV-1 viral infectivity factor (Vif) protein, leading to proteasomal degradation. A3G contains two zinc ions at the N-terminal domain and the C-terminal domain. Four lysine residues, K297, K301, K303, and K334, are known to be required for Vif-mediated A3G ubiquitination and degradation. Previously, we reported compound SN-1, a zinc chelator that increases steady-state expression level of A3G in the presence of Vif. In this study, we prepared Biotin-SN-1, a biotinylated derivative of SN-1, to study the SN-1–A3G interaction. A pull-down assay revealed that Biotin-SN-1 bound A3G. A zinc-abstraction experiment indicated that SN-1 binds to the zinc site of A3G. We carried out a SN-1–A3G docking study using molecular operating environment. The calculations revealed that SN-1 binds to the C-terminal domain through Zn2+, H216, P247, C288, and Y315. Notably, SN-1-binding covers the H257, E259, C288, and C291 residues that participate in zinc-mediated deamination, and the ubiquitination regions of A3G. The binding of SN-1 presumably perturbs the secondary structure between C288 and Y315, leading to less efficient ubiquitination.Abnormal ubiquitination leads to inhibition of proteasome degradation of A3G, suppressing reverse transcription of HIV-1 deaminase-independently in a target cell.

BMMP [2-(benzothiazol-2-ylmethylthio)-4-methylpyrimidine], an inhibitor of HIV-1 replication, was linked to biotin to study the interaction with the presumed target, HIV-1 Pr55Gag or CA, by means of surface plasmon resonance. The synthesized Biotin–BMMP inhibited HIV-1 replication to a similar extent as BMMP alone, but did not interact with Pr55Gag or CA, suggesting that BMMP exerts its activity by a different mechanism.

Previously we have reported a metal chelating histidine–pyridine–histidine system possessing a trityl group on the histidine imidazole, namely HPH-2Trt, which induces apoptosis in human pancreatic adenocarcinoma AsPC-1 cells. Herein the influence of the imidazole substitution of HPH-2Trt was examined. Five related compounds, HPH-1Trt, HPH-2Bzl, HPH-1Bzl, HPH-2Me, and HPH-1Me were newly synthesized and screened for their activity against AsPC-1 and brain tumor cells U87 and U251. HPH-1Trt and HPH-2Trt were highly active among the tested HPH compounds. In vitro DNA cleavage assay showed both HPH-1Trt and HPH-2Trt completely disintegrate pUC19 DNA. The introduction of trityl group decisively potentiated the activity.

•A tetrapeptide, l-histidyl-glycyl-glycyl-l-histidine (HGGH), was synthesized.•The pUC19 plasmid DNA cleaving activity of Cu(II)−HGGH was investigated.•Cu(II)−HGGH (50 nM) converted a supercoiled DNA efficiently to a linear DNA.•The DNA cleavage took place in the presence of 500 μM H2O2/sodium ascorbate.A tetrapeptide, l-histidyl-glycyl-glycyl-l-histidine (HGGH), was synthesized and the pUC19 plasmid DNA cleaving activity by copper(II) complex of HGGH (Cu(II)−HGGH) was investigated. Cu(II)−HGGH showed bleomycin-like DNA cleaving activity and, at 50 nM, converted a supercoiled DNA efficiently to a linear DNA in the presence of 500 μM H2O2/sodium ascorbate through an oxidative pathway.

The precursor of Gag protein (Pr55Gag) of human immunodeficiency virus, the principal structural component required for virus assembly, is known to bind D-myo-phosphatidylinositol 4,5-bisphosphate (PIP2). The N-terminus of Pr55Gag, the MA domain, plays a critical role in the binding of Pr55Gag to the plasma membrane. Herein, we designed and synthesized myo-phosphatidylinositol 2,3,4,5,6-pentakisphosphate (PIP5) derivatives comprising highly phosphorylated inositol and variously modified diacylglycerol to examine the MA-binding properties. The inositol moiety was synthesized starting with myo-inositol and assembled with a hydrophobic glycerol moiety through a phosphate linkage. The Kd value for MA-binding of the PIP5 derivative 2 (Kd = 0.25 μM) was the lowest (i.e., highest affinity) of all derivatives, i.e., 70-fold lower than the Kd for the PIP2 derivative 1 (Kd = 16.9 μM) and 100-fold lower than the Kd for IP6 (Kd = 25.7 μM), suggesting the possibility that the PIP5 derivative blocks Pr55Gag membrane binding by competing with PIP2 in MA-binding.

A bifunctional molecule containing biotin and d-myo-inositol 1,3,4,5-tetrakisphosphate was synthesized. This molecule was designed on the basis of X-ray structure of the complex of d-myo-inositol 1,3,4,5-tetrakisphosphates, Ins(1,3,4,5)P4, and Grp1 PH (general receptor of phosphoinositides pleckstrin homology) domain for the application to the widely employed biotin–avidin techniques. The building block of inositol moiety was synthesized starting with myo-inositol and assembled with the biotin-linker moiety through a phosphate linkage. The equilibrium dissociation constant KD of biotinylated Ins(1,3,4,5)P4 binding of original Grp1 PH domain was 0.14 μM in pull-down analysis, which was comparable to that of unmodified Ins(1,3,4,5)P4. Furthermore, biotinylated Ins(1,3,4,5)P4 had an ability to distinguish Grp1 PH domain from PLCδ1 PH domain. Thus, biotinylated Ins(1,3,4,5)P4 retained the binding affinity and selectivity of original Grp1 PH domain, and realized the intracellular Ins(1,3,4,5)P4 despite a tethering at the 1-phosphate group of inositol.

Human immunodeficiency virus type 1 (HIV-1) Gag protein is the principal structural component of the HIV particle. Localization of the Pr55Gag protein to the plasma membrane initiates virus assembly. Recent studies indicated that d-myo-phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) regulates Pr55Gag localization and assembly. We determined the binding affinity between Pr55Gag or its N-terminal MA domain and various phosphoinositide derivatives using a highly sensitive surface plasmon resonance (SPR) sensor and biotinylated inositol phosphate. The equilibrium dissociation constants obtained using this approach reflected the distinct magnitude of acyl group-based and phosphate group-based interactions. The dissociation constant (KD) for Pr55Gag complexed with 1,4,5-IP3 (an inositol with divalent phosphate groups and devoid of lipid groups) was 2170 μM, while the KD for di-C8-PI (a lipid-containing inositol devoid of divalent phosphate groups) was 186 μM, and the KD for di-C8-PI(4,5)P2 (an inositol with both lipid and divalent phosphate groups) was 47.4 μM. The same trend in affinity was observed when these phosphoinositides were complexed with MA. Our results suggest that the contribution of hydrophobic acyl chains is greater than negatively charged inositol phosphates in Pr55Gag/MA binding. Furthermore, each inositol phosphate (devoid of lipid groups) tested showed a distinct Pr55Gag-binding affinity depending on the position and number of phosphate groups. However, the position and number of phosphate groups had no effect on MA-binding affinity.

Six bifunctional molecules containing biotin and various inositol phosphates were synthesized. These compounds were designed on the basis of X-ray structures of the complexes of D-myo-inositol 1,4,5-triphosphates (IP3) and phospholipase C δ pleckstrin homology domain (PLCδ PH) considering the application to the biotin–avidin techniques. The building blocks of the inositol moiety were synthesized starting with optically resolved myo-inositol derivatives and assembled to the biotin linker through a phosphate linkage.

The inhibitory effect of 7,8-dihydroxy-4-methylcoumarin (7,8-DHMC), 5,7-dihydroxy-4-methylcoumarin (5,7-DHMC), and gallic acid on the DNA binding of recombinant p50 protein and their interaction with zinc ion were studied. Electrophoretic mobility shift assay (EMSA) using p50 and biotin labeled DNA has shown that gallic acid is more effective than the dihydroxycoumarins in inhibiting the p50–DNA binding. Molecular modeling studies suggest an explanation for these observations. Effect of the addition of zinc after p50–DNA-binding inhibition by gallic acid was also studied. Chemical speciation and formation constant studies show that gallic acid forms a more stable 1:1 complex with zinc ion in comparison to the dihydroxycoumarins.

A novel metal chelator comprising a 4-(naphthalen-1-yl)pyridine and 2-aminoethanethiol was synthesized. This showed inhibitory activity against human protein farnesyltransferase with IC50 1.9 μM, induced morphological change in K-ras-NRK cells at 0.5 μg/mL and showed growth inhibition of K-ras-NRK cells with IC50 0.32 μg/mL.A metal-chelating compound 2 was designed and synthesized. Compound 2 showed inhibitory activity against farnesyltransrferase and induced morphological change in K-ras-NRK cells.

A compound named HPH–Pep, a peptide constructed from pyridine and histidine units, showed sensitizing effect on the hyperthermic treatment of L-1210, Molt-4, and HL60 cells. The survival rate of these cells was greatly reduced by combined treatment with heating at 44 °C and HPH–pep. The treatment of L-1210 and Molt-4 cells with HPH–Pep resulted in a significant breakdown of the survival rate at 44 °C. The cell death induced by HPH–Pep under hyperthermic condition seemed to involve iron and peroxide.A synthetic peptide HPH–Pep showed sensitising effect on the hyperthermic treatment of L-1210, Molt-4, and HL60 cells.

The metal-interaction of aurine tricarboxylic acid (ATA) and its inhibitory effect on the DNA binding of NFκB were studied. Chemical speciation and spectroscopic studies have shown the strong interaction of ATA with metal ions present in the biological systems. EPR, FTIR and electronic spectral studies indicated the square planar structure of the metal-binding carboxylic and hydroxyl groups of ATA indicating the ground state 2B1g. Electrophoretic mobility shift assay using NFκB and 32P labeled DNA has shown that ATA was inhibitory against the DNA-NFκB binding at 30 μM. This activity was the strongest among the metal-chelating inhibitors of NFκB-DNA binding reported so far.

Six bifunctional molecules containing biotin and various inositol phosphates were synthesized. These compounds were designed on the basis of X-ray structures of the complexes of D-myo-inositol 1,4,5-triphosphates (IP3) and phospholipase C δ pleckstrin homology domain (PLCδ PH) considering the application to the biotin–avidin techniques. The building blocks of the inositol moiety were synthesized starting with optically resolved myo-inositol derivatives and assembled to the biotin linker through a phosphate linkage.

The precursor of Gag protein (Pr55Gag) of human immunodeficiency virus, the principal structural component required for virus assembly, is known to bind D-myo-phosphatidylinositol 4,5-bisphosphate (PIP2). The N-terminus of Pr55Gag, the MA domain, plays a critical role in the binding of Pr55Gag to the plasma membrane. Herein, we designed and synthesized myo-phosphatidylinositol 2,3,4,5,6-pentakisphosphate (PIP5) derivatives comprising highly phosphorylated inositol and variously modified diacylglycerol to examine the MA-binding properties. The inositol moiety was synthesized starting with myo-inositol and assembled with a hydrophobic glycerol moiety through a phosphate linkage. The Kd value for MA-binding of the PIP5 derivative 2 (Kd = 0.25 μM) was the lowest (i.e., highest affinity) of all derivatives, i.e., 70-fold lower than the Kd for the PIP2 derivative 1 (Kd = 16.9 μM) and 100-fold lower than the Kd for IP6 (Kd = 25.7 μM), suggesting the possibility that the PIP5 derivative blocks Pr55Gag membrane binding by competing with PIP2 in MA-binding.