Binding efficacy of aminobenzoic acid derivatives with DNA duplex: drug binding sites and DNA structure and dynamics.

Communicated by Ramaswamy H. Sarma.

Biomolecular study and conjugation of two para-aminobenzoic acid derivatives with serum proteins: drug binding efficacy and protein structural analysis.

Two aminobenzoic acid derivatives DAB-0 and DAB-1 showed distinct biological properties on murine bladder cancer (BCa) cell line MB49-I. In contrast to DAB-1, DAB-0 does not possess any anti-inflammatory activity and is less toxic. Furthermore, DAB-0 does not interfere with INFγ-induced STAT1 activation and TNFα-induced IκB phosphorylation, while DAB-1 does. In order to rationalize these results, the binding efficacy of DAB-0 and DAB-1 with serum proteins such a human serum albumin (HSA), bovine serum albumin (BSA) and beta-lactoglobulin (ß-LG) was investigated in aqueous solution at physiological pH. Multiple spectroscopic methods and thermodynamic analysis were used to determine the binding efficacy of DAB-0 and DAB-1 with serum proteins. Drug-protein conjugation was observed via through ionic contacts. DAB-1 forms stronger adducts than DAB-0, while ß-LG shows more affinity with the order of stability ß-LG > BSA > HSA. The stronger complexation of DAB-1 with serum proteins might account for its biological potential and transport in the blood. The binding efficacy ranged from 40 to 60%. Major alterations of protein secondary structures were detected upon drug complexation. Serum proteins are capable of delivering DAB-1 in vitro.Communicated by Ramaswamy H. Sarma.

Complexation of cis-Pt and trans-Pt(NH3)2Cl2 with serum proteins: A potential application for drug delivery.

AbbreviationsHAShuman serum albuminBSAbovine serum albuminß-LGbeta-lactoglobulincis-Pt and trans-PtPt(NH3)2Cl2FTIRFourier transform infraredCommunicated by Ramaswamy H. Sarma.

Location of multiple binding sites for testo and testo-Pt(II) with tRNA.

We report the binding of testo and testo-Pt(II) complexes (testosterone derivatives) with tRNA in aqueous solution at physiological pH. Thermodynamic parameter ΔH0 -8 to -3 (kJ mol-1), ΔS0 35 to 18 (J mol-1K-1) and ΔG0 -14 to -13 (kJ mol-1) and other spectroscopic results showed drug-tRNA binding occurs via ionic contacts with testo-Pt(II) forming more stable tRNA complexes in comparison to testo: Ktesto-Pt(II)-tRNA= 3.2 (± 0.9) × 105 M-1 > Ktesto-tRNA= 2.1 (± 0.7) × 105 M-1. Molecular modeling showed multiple binding sites for testo and testo-Pt(II) on tRNA molecule. Some of the useful molecular descriptors are calculated. Major structural changes were observed for biopolymers upon drug complexation, while tRNA remains in the A-family structures.

Testo and testo-Pt(II) bind DNA at different locations.

The development of new targeted anticancer agents able to efficiently and specifically destroy cancer cells with minimal toxic side effects is nowadays a subject of intensive research endeavors. We report the conjugation of testo and testo-Pt(II) (two semi-synthetic testosterone derivatives) with calf thymus DNA in aqueous solution at physiological pH. Multiple spectroscopic methods, thermodynamic analysis and modeling were used to determine the binding efficacy of these drugs to DNA duplex. Thermodynamic parameters showed drug-DNA conjugation occurs via ionic interactions with testo-Pt(II) forming more stable DNA adducts than testo with Ktesto-DNA = 1.80 (±0.5) x 105 M-1 and Ktesto-Pt(II)-DNA = 2.3 (±0.8) x 105 M-1. Molecular modeling shows that testo and testo-Pt(II) bind DNA at different locations.

Conjugation of testo and testo-Pt(II) with serum proteins: Loading efficacy and protein conformation.

The potential application of hybrid anticancer molecules requires further investigation. There is a great interest in developing new site-specific anticancer agents able to efficiently destroy cancer cells with minimal toxic side effects. Serum proteins are known to play an important role as drug delivery system with important clinical applications. Hence, the conjugation of testo and testo-Pt(II) (two semi-synthetic testosterone derivatives) with human serum albumin HSA), bovine serum albumin (BSA) and beta-lactoglobulin (ß-LG) was investigated in aqueous solution at physiological pH. Multiple spectroscopic methods, thermodynamic analysis and modeling were used to determine the binding efficacy of these bioactive compounds to serum proteins. Drug-protein conjugation occurred via ionic contacts. BSA forms more stable conjugates than HSA and ß-LG with the order of stability testo > testo-Pt(II). Major alterations of protein secondary structures were observed upon drug complexation. Serum proteins can be used to deliver these bioactive materials in vitro.

Testosterone and its dimers alter tRNA morphology.

The morphology of tRNA was studied upon conjugation with testosterone and its aliphatic and aromatic dimers, using multiple spectroscopic methods, transmission electron microscopy (TEM) and molecular modeling. Structural analysis showed that testosterone binds tRNA through A62, A64, C60, C61, C63, G51, U50 and U59 bases. The binding affinity was testosterone dimer-aromatic>testosterone dimer-aliphatic>testosterone. The steroid loading efficacy was 35-45%. Transmission electron microscopy showed major changes in tRNA morphology upon testosterone interaction with an increase in the diameter of the tRNA aggregate, indicating encapsulation of testosterone by tRNA.

Effect of testosterone and its aliphatic and aromatic dimers on DNA morphology.

Conjugation of DNA with testosterone and it aliphatic dimer (alip) and aromatic dimer (arom) was investigated in aqueous solution at pH 7.4. Multiple spectroscopic methods, transmission electron microscopy (TEM) and molecular modeling were used to characterize steroid-DNA binding and DNA morphology. Spectroscopic analysis showed that testosterone binds DNA via A7, A16, A17, T8, T15 and T18 nucleobases with overall binding constants Ktest-DNA=1.8 (±0.4)×104M-1, Ktest-dimeralip-DNA=5.7 (±0.7)×104M-1 and Ktest-dimer-arom-DNA=7.3 (±0.9)×104M-1. The binding affinity increases in this order: testosterone dimer-aromatic>testosterone dimer-aliphatic>testosterone. The steroid loading efficacy was 40-50%. Transmission electron microscopy showed major changes in DNA morphology as testosterone-DNA interaction occurred with increase in the diameter of the DNA aggregate, indicating encapsulation of testosterone by DNA. Modeling showed the presence of several nucleobases attached to testosterone with the free binding energy of -4.93Kcal/mol.

Review on the delivery of steroids by carrier proteins.

Due to the poor solubility of steroids in aqueous solution, delivery of these biomaterials is of major biomedical importance. We have reviewed the conjugation of testosterone and it aliphatic dimer and aromatic dimer with several carrier proteins, human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (b-LG) in aqueous solution at physiological pH. The results of multiple spectroscopic methods, transmission electron microscopy (TEM) and molecular modeling were compared here. Steroid-protein bindings are via hydrophilic and H-bonding contacts. HSA forms more stable conjugate than BSA and b-LG. The stability of steroid-protein conjugates is testosterone>dimer-aromatic>dimer-aliphatic. Encapsulation of steroids by protein is shown by TEM images. Modeling showed the presence of H-bonding, which stabilized testosterone-protein complexes with the free binding energy of -12.95 for HSA and -11.55 for BSA and -8.92kcal/mol for b-LG conjugates. Steroid conjugation induced major perturbations of serum protein conformations. Serum proteins can transport steroids to the target molecules.

An overview on the delivery of antitumor drug doxorubicin by carrier proteins.

Serum proteins play an increasing role as drug carriers in the clinical settings. In this review, we have compared the binding modalities of anticancer drug doxorubicin (DOX) to three model carrier proteins, human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (ß-LG) in order to determine the potential application of these model proteins in DOX delivery. Molecular modeling studies showed stronger binding of DOX with HSA than BSA and ß-LG with the free binding energies of -10.75 (DOX-HSA), -9.31 (DOX-BSA) and -8.12kcal/mol (DOX-ß-LG). Extensive H-boding network stabilizes DOX-protein conjugation and played a major role in drug-protein complex formation. DOX complexation induced major alterations of HSA and BSA conformations, while did not alter ß-LG secondary structure. The literature review shows that these proteins can potentially be used for delivery of DOX in vitro and in vivo.

Review on the binding of anticancer drug doxorubicin with DNA and tRNA: Structural models and antitumor activity.

In this review, we have compared the results of multiple spectroscopic studies and molecular modeling of anticancer drug doxorubicin (DOX) bindings to DNA and tRNA. DOX was intercalated into DNA duplex, while tRNA binding is via major and minor grooves. DOX-DNA intercalation is close to A-7, C-5, *C-19 (H-bonding with DOX NH2 group), G-6, T-8 and T-18 with the free binding energy of -4.99kcal/mol. DOX-tRNA groove bindings are near A-29, A-31, A-38, C-25, C-27, C-28, *G-30 (H-bonding) and U-41 with the free binding energy of -4.44kcal/mol. Drug intercalation induced a partial B to A-DNA transition, while tRNA remained in A-family structure. The structural differences observed between DOX bindings to DNA and tRNA can be the main reasons for drug antitumor activity. The results of in vitro MTT assay on SKC01 colon carcinoma are consistent with the observed DNA structural changes. Future research should be focused on finding suitable nanocarriers for delivery of DOX in vivo in order to exploit the full capacity of this very important anticancer drug.

Transporting testosterone and its dimers by serum proteins.

A substantial part of steroids is bound to serum proteins in vivo. We report the association of testosterone and it aliphatic dimer (alip) and aromatic dimer (arom) with human serum albumin (HSA) and bovine serum albumin (BSA) in aqueous solution at physiological pH. Multiple spectroscopic methods, transmission electron microscopy (TEM) and molecular modeling were used to characterize steroid-protein binding and protein aggregation process. Spectroscopic analysis showed that steroids bind protein via hydrophobic, hydrophilic and H-bonding interactions. HSA forms more stable complexes than BSA. The binding affinity of steroid-protein adducts is testosterone>dimer-aromatic>dimer-aliphatic. Transmission electron microscopy showed major changes in protein morphology as steroid-protein complexation occurred with increase in the diameter of the protein aggregate indicating encapsulation of steroids by serum proteins. Modeling showed the presence of H-bonding stabilized testosterone-protein complexes with the free binding energy of -12.95 for HSA and -11.55 kcal/mol for BSA, indicating that the interaction process is spontaneous at room temperature. Steroid complexation induced more perturbations of BSA conformation than HSA.

Encapsulation of testosterone and its aliphatic and aromatic dimers by milk beta-lactoglobulin.

The encapsulation of testosterone and it aliphatic dimer (alip) and aromatic dimer (arom) with milk ß-lactoglobulin (ß-LG) was studied in aqueous solution at pH 7.4. Multiple spectroscopic methods, transmission electron microscopy (TEM) and molecular modeling were used to characterize testosterone-ß-LG binding and protein aggregation process. Spectroscopic analysis showed that steroids bind ß-LG via hydrophobic and H-bonding interactions with overall binding constants K test-ß-LG = 5.6 (± 0.6) × 10(4)M(-1), K test-dimeralip-ß-LG = 4.8 (± 0.5) × 10(3)M(-1) and K test-dimer-arom-ß-LG = 2.9 (± 0.4) × 10(4)M(-1). The binding affinity was testosterone > testosterone dimer-aromatic > testosterone dimer-aliphatic. Transmission electron microscopy showed major changes in protein morphology as testosterone-protein complexation occurred with increase in the diameter of the protein aggregate indicating encapsulation of steroids by ß-LG. Modeling showed the presence of H-bonding stabilized testosterone-ß-LG complexes with the free binding energy of -9.82 Kcal/mol indicating that the interaction process is spontaneous at room temperature.

ERα-targeted therapy in ovarian cancer cells by a novel estradiol-platinum(II) hybrid.

As we previously showed, we have synthesized a new family of 17ß-estradiol-platinum(II) hybrids. Earlier studies revealed the VP-128 hybrid to show high efficiency compared with cisplatin toward hormone-dependent breast cancer cells. In the present research, we have studied the antitumor activity of VP-128 in vitro and in vivo against ovarian cancer. In nude mice with ovarian xenografts, VP-128 displayed selective activity toward hormone-dependent tumors and showed higher efficiency than cisplatin to inhibit tumor growth. Similarly, in vitro, transient transfection of estrogen receptor (ER)-α in ERα-negative A2780 cells increased their sensitivity to VP-128-induced apoptosis, confirming the selectivity of VP-128 toward hormone-dependent tumor cells. In agreement, Western blot analysis revealed that VP-128 induced higher caspase-9, caspase-3, and poly (ADP-ribose) polymerase cleavage compared with cisplatin. The activation of caspase-independent apoptosis was also observed in ERα-negative A2780 cells, in which VP-128 rapidly induced the translocation of apoptosis-inducing factor to the nucleus. Conversely, subcellular localization of apoptosis-inducing factor was not modified in ERα-positive Ovcar-3 cells. We also discovered that VP-128 induces autophagy in ovarian cancer cells because of the formation of acidic vesicular organelles (AVOs) and increase of Light Chain 3B-II protein responsible for the formation of autophagosomes; pathways related to autophagy (AKT and mammalian target of rapamycin) were also down-regulated, supporting this mechanism. Finally, the inhibition of autophagy using chloroquine increased VP-128 efficiency, indicating a possible combination therapy. Altogether these results highlight the beneficial value of VP-128 for the treatment of hormone-dependent ovarian cancers and provide preliminary proof of concept for the efficient targeting of ERα- by 17ß-estradiol-Pt(II)-linked chemotherapeutic hybrids in these tumors.

Phosphorylation of the CCAAT displacement protein (CDP)/Cux transcription factor by cyclin A-Cdk1 modulates its DNA binding activity in G(2).

Stable DNA binding by the mammalian CCAAT displacement protein (CDP)/Cux transcription factor was previously found to be up-regulated at the G(1)/S transition as the result of two events, dephosphorylation by the Cdc25A phosphatase and proteolytic processing, to generate an amino-truncated isoform of 110 kDa. In S phase, CDP/Cux was shown to interact with and repress the core promoter of the p21(WAF1) gene. Here we demonstrate that DNA binding by p110 CDP/Cux is down-modulated as cells progress into G(2). Accordingly, cyclin A-Cdk1 was found to bind to CDP/Cux and modulate its DNA binding activity in vitro and in vivo. Interaction with CDP/Cux required the presence of both cyclin A and a cyclin-dependent kinase (Cdk)-activating kinase-activated Cdk1 and involved the Cut homeodomain and a downstream Cy motif. Phosphorylation of serines 1237 and 1270 caused inhibition of DNA binding in vitro. In cotransfection studies, cyclin A-Cdk1 inhibited CDP/Cux stable DNA binding and prevented repression of the p21(WAF1) reporter. In contrast, mutant CDP/Cux proteins in which serines 1237 and 1270 were replaced with alanines were not affected by cyclin A-Cdk1. In summary, our results suggest that the phosphorylation of CDP/Cux by cyclin A-Cdk1 contributes to down-modulate CDP/Cux activity as cells progress into the G(2) phase of the cell cycle.

S phase-specific proteolytic cleavage is required to activate stable DNA binding by the CDP/Cut homeodomain protein.

The CCAAT displacement protein (CDP), the homologue of the Drosophila melanogaster Cut protein, contains four DNA binding domains that function in pairs. Cooperation between Cut repeat 3 and the Cut homeodomain allows stable DNA binding to the ATCGAT motif, an activity previously shown to be upregulated in S phase. Here we showed that the full-length CDP/Cut protein is incapable of stable DNA binding and that the ATCGAT binding activity present in cells involves a 110-kDa carboxy-terminal peptide of CDP/Cut. A vector expressing CDP/Cut with Myc and hemagglutinin epitope tags at either end generated N- and C-terminal products of 90 and 110 kDa, suggesting that proteolytic cleavage was involved. In vivo pulse/chase labeling experiments confirmed that the 110-kDa protein was derived from the full-length CDP/Cut protein. Proteolytic processing was weak or not detectable in G(0) and G(1) but increased in populations of cells enriched in S phase, and the appearance of the 110-kDa protein coincided with the increase in ATCGAT DNA binding. Interestingly, the amino-truncated and the full-length CDP/Cut isoforms exhibited different transcriptional properties in a reporter assay. We conclude that proteolytic processing of CDP/Cut at the G(1)/S transition generates a CDP/Cut isoform with distinct DNA binding and transcriptional activities. These findings, together with the cleavage of the Scc1 protein at mitosis, suggest that site-specific proteolysis may play an important role in the regulation of cell cycle progression.

p62dok negatively regulates CD2 signaling in Jurkat cells.

p62(dok) belongs to a newly identified family of adaptor proteins. In T cells, the two members that are predominantly expressed, p56(dok) and p62(dok), are tyrosine phosphorylated upon CD2 or CD28 stimulation, but not upon CD3 ligation. Little is known about the biological role of Dok proteins in T cells. In this study, to evaluate the importance of p62(dok) in T cell function, we generated Jurkat clones overexpressing p62(dok). Our results demonstrate that overexpression of p62(dok) in Jurkat cells has a dramatic negative effect on CD2-mediated signaling. The p62(dok)-mediated inhibition affects several biochemical events initiated by CD2 ligation, such as the increase of intracellular Ca(2+), phospholipase C gamma 1 activation, and extracellular signal-regulated kinase 1/2 activation. Importantly, these cellular events are not affected in the signaling cascade induced by engagement of the CD3/TCR complex. However, both CD3- and CD2-induced NF-AT activation and IL-2 secretion are impaired in p62(dok)-overexpressing cells. In addition, we show that CD2 but not CD3 stimulation induces p62(dok) and Ras GTPase-activating protein recruitment to the plasma membrane. These results suggest that p62(dok) plays a negative role at multiple steps in the CD2 signaling pathway. We propose that p62(dok) may represent an important negative regulator in the modulation of the response mediated by the TCR.

CCAAT displacement activity involves CUT repeats 1 and 2, not the CUT homeodomain.

The CCAAT displacement protein, the homolog of the Drosophila melanogaster CUT protein, contains four DNA-binding domains: three CUT repeats (CR1, CR2, and CR3) and the CUT homeodomain (HD). Using a panel of fusion proteins, we found that a CUT repeat cannot bind to DNA as a monomer, but that certain combinations of domains exhibit high DNA-binding affinity: CR1+2, CR3HD, CR1HD, and CR2HD. One combination (CR1+2) exhibited strikingly different DNA-binding kinetics and specificities. CR1+2 displayed rapid on and off rates and bound preferably to two C(A/G)AT sites, organized as direct or inverted repeats. Accordingly, only CR1+2 was able to bind to the CCAAT sequence, and its affinity was increased by the presence of a C(A/G)AT site at close proximity. A purified CCAAT displacement protein/CUT protein exhibited DNA-binding properties similar to those of CR1+2; and in nuclear extracts, the CCAAT displacement activity also required the simultaneous presence of a C(A/G)AT site. Moreover, CR1+2, but not CR3HD, was able to displace nuclear factor Y. Thus, the CCAAT displacement activity requires the presence of an additional sequence (CAAT or CGAT) and involves CR1 and CR2, but not the CUT homeodomain.

Exon/intron structure and alternative transcripts of the CUTL1 gene.

The human CUTL1 gene (Cut-like 1) is a candidate tumor suppressor gene located on chromosome 7 at band 22, a region that is frequently deleted in several human cancers. The gene spans at least 340kb and contains 33 exons. Synthesis of five different transcripts involves two promoter regions, two polyadenylation sites and seven alternative splicing events. The two polyadenylation sites are located at the ends of exons 24 and 33 and are separated by approximately 40kb. Transcription is initiated in two genomic regions, giving rise to alternate first exons which are spliced to a common exon 2. All transcripts contain exons 2 to 14, but differ in their 3' regions. Exon 14 can be spliced alternatively to the beginning or the middle of exon 15, or to exon 25, generating transcripts with exons 15 to 24 or exons 25 to 33. Moreover, exon 16 can be spliced out from the mature transcripts that contain exons 15 to 24. Overall, five distinct transcripts are generated as a result of alternative transcription initiation, splicing and polyadenylation. We discuss potential mechanisms by which alternate polyadenylation site usage may affect alternative splicing events and vice versa.

Effects of new triphenylethylene platinum(II) complexes on the interaction with phosphatidylcholine liposomes.

In a previous work we synthesized a class of new antineoplastic drugs by coupling a cisplatin derivative to a triphenylethylene moiety similar to the antiestrogen, tamoxifen. These drugs differ in the number of hydroxy functions on the triphenylethylene rings and in the length of the linking arm. To gain more insight into the cellular mechanism by which these new drugs act on cells, we studied, using differential scanning calorimetry, the effects of these compounds on the phase transition of membrane phospholipid (distearoyl phosphatidyl choline (DSPC)), and correlated these effects to drug cytotoxicity. The drugs without hydroxy function showed the highest cytotoxicity and induced little change on the thermogram of DSPC. Contrarily, the drugs bearing two or three hydroxy groups were less toxic, but induced important modifications of the thermogram. We suggest that the drugs with no hydroxy group enter the membrane, with the triphenylethylene moiety localized deep within the hydrophobic core of the bilayer and do not affect the cooperativity region (C2-C8). In contrast, drugs which bear hydroxy groups on the triphenylethylene rings system perturb the phospholipid molecular arrangement; this may be due either to the additional steric hindrance of the hydroxy functions in the core of the bilayer, or to their hydrophilic effect on the polar head of the lipid. In vitro, the cytotoxic effect of these drugs seems not to be related to their affinity for the estrogen receptor. We suggest that the addition of a triphenylethylene moiety to the platinum(II) complexes increases the hydrophobicity, and consequently the resulting drugs become more permeable to the membrane, particularly the non-hydroxylated triphenylethylene derivatives.