Antitumour potential of BPT: a dual inhibitor of cdk4 and tubulin polymerization.

The marine natural product fascaplysin (1) is a potent Cdk4 (cyclin-dependent kinase 4)-specific inhibitor, but is toxic to all cell types possibly because of its DNA-intercalating properties. Through the design and synthesis of numerous fascaplysin analogues, we intended to identify inhibitors of cancer cell growth with good therapeutic window with respect to normal cells. Among various non-planar tryptoline analogues prepared, N-(biphenyl-2-yl) tryptoline (BPT, 6) was identified as a potent inhibitor of cancer cell growth and free from DNA-binding properties owing to its non-planar structure. This compound was tested in over 60 protein kinase assays. It displayed inhibition of Cdk4-cyclin D1 enzyme in vitro far more potently than many other kinases including Cdk family members. Although it blocks growth of cancer cells deficient in the mitotic-spindle checkpoint at the G0/G1 phase of the cell cycle, the block occurs primarily at the G2/M phase. BPT inhibits tubulin polymerization in vitro and acts as an enhancer of tubulin depolymerization of paclitaxel-stabilized tubulin in live cells. Western blot analyses indicated that, in p53-positive cells, BPT upregulates the expression of p53, p21 and p27 proteins, whereas it downregulates the expression of cyclin B1 and Cdk1. BPT selectively kills SV40-transformed mouse embryonic hepatic cells and human fibroblasts rather than untransformed cells. BPT inhibited the growth of several human cancer cells with an IC50<1 µM. The pharmacokinetic study in BALB/c mice indicated good plasma exposure after intravenous administration. It was found to be efficacious at 1/10th the maximum-tolerated dose (1000 mg/kg) against human tumours derived from HCT-116 (colon) and NCI-H460 (lung) cells in SCID (severe-combined immunodeficient) mice models. BPT is a relatively better anticancer agent than fascaplysin with an unusual ability to block two overlapping yet crucial phases of the cell cycle, mitosis and G0/G1. Its ability to effectively halt tumour growth in human tumour-bearing mice would suggest that BPT has the potential to be a candidate for further clinical development.

Oncogenic and drug-sensitive NTRK1 rearrangements in lung cancer.

We identified new gene fusions in patients with lung cancer harboring the kinase domain of the NTRK1 gene that encodes the high-affinity nerve growth factor receptor (TRKA protein). Both the MPRIP-NTRK1 and CD74-NTRK1 fusions lead to constitutive TRKA kinase activity and are oncogenic. Treatment of cells expressing NTRK1 fusions with inhibitors of TRKA kinase activity inhibited autophosphorylation of TRKA and cell growth. Tumor samples from 3 of 91 patients with lung cancer (3.3%) without known oncogenic alterations assayed by next-generation sequencing or fluorescence in situ hybridization demonstrated evidence of NTRK1 gene fusions.

Polymeric nanoparticle formulation of octapeptide (NP-OP): in vitro release and in vivo effect in common marmosets, Callithrix jacchus Linn.

Octapeptide (OP)/FSH-Receptor Binding Inhibitor-8 (FRBI-8), is a synthetic peptide corresponding to N-terminal sequence of purified fraction of Follicle Stimulating Hormone Binding-Inhibitor (FSHBI), isolated earlier from human ovarian follicular-fluid. In order to avoid the repeated drug-administration, OP-loaded, polymeric polylactide (PLA) nanoparticle formulation (NP-OP), was developed using multiple-emulsion technique. This yielded an average particle size of 120 nm with 70% encapsulation-efficiency. In vitro release profile of NP-OP showed sustained release of OP for 21 days. In vivo anti-fertility studies were conducted in marmosets. Results indicated that control animals conceived in the same cycle while two of three treated animals failed to conceive in treatment cycle. The in vivo studies thus corroborate with in vitro release of OP, demonstrating its anti-fertility activity in 66% of animals.

Pre-ischemic exercise reduces matrix metalloproteinase-9 expression and ameliorates blood-brain barrier dysfunction in stroke.

Exercise reduces ischemia and reperfusion (I/R) injury in the rat stroke model. We investigated whether pre-ischemic exercise ameliorates blood-brain barrier (BBB) dysfunction in stroke by reducing matrix metalloproteinase (MMP)-9 expression and strengthening basal lamina. Adult male Sprague-Dawley rats were subjected to a 30 min exercise program on a treadmill 5 days a week for 3 weeks. Stroke was induced by a 2-h middle cerebral artery (MCA) occlusion using an intraluminal filament in the exercised and non-exercised groups. Brain infarction was measured and neurological deficits were scored. BBB dysfunction was determined by examining brain edema and Evans Blue extravasation. Expression of collagen IV, the major component of basal lamina essential for maintenance of the endothelial permeability barrier, was quantitatively detected by Western blot and immunocytochemistry. Ex vivo techniques were used to compare collagen IV-labeled vessels in response to ischemic insult. Temporal relationship of expression of MMP-9 and its endogenous inhibitor, the tissue inhibitors of metalloproteinase-1 (TIMP-1), was determined by real-time PCR for mRNA and Western blot for protein during reperfusion. Brain edema and Evans Blue leakage were both significantly (P<0.01) reduced after stroke in the exercised group, in association with reduced brain infarct volume and neurological deficits. Western blot analysis indicated that exercise enhanced collagen IV expression and reduced the collagen loss after stroke. Immunocytochemistry demonstrated that collagen IV-labeled vessels were significantly (P<0.01) increased in exercised rats. In the ex vivo study, after exercised brains were incubated with ischemic brain tissue, a significantly (P<0.01) higher level of collagen IV-labeled vessels was observed as compared with non-exercised brains following the same treatment. The ex vivo study also revealed a key role of MMP-9 in exercise-strengthened collagen IV expression against I/R injury. TIMP-1 protein levels were significantly (P<0.01) increased by exercise. Our results indicate that pre-ischemic exercise reduces brain injury by improving BBB function and enhancing basal lamina integrity in stroke. This study suggests that the neuroprotective effect of physical exercise is associated with an imbalance of MMP-9 and TIMP-1 expression.

Identification of the region 285-309 of follicle stimulating hormone receptor as a bioneutralizing epitope.

Structure-function relationship studies of the follicle stimulating hormone and its receptor assume importance as this hormone is essential for folliculogenesis and spermatogenesis in females and males, respectively. The interaction between the hormone and the receptor is complex and not well understood. In vitro studies using synthetic peptides from the extracellular domain of the receptor and corresponding antipeptide antibodies have suggested that the 285-309 region is surface-oriented. Antipeptide antibodies to this region also inhibit hormone-receptor interaction in a dose-dependent manner and the mechanism of inhibition appears to be competitive in nature. To test this hypothesis in an animal model, antibodies to peptide 285-309 from rat follicle stimulating hormone receptor (FSHR) were developed and characterized. These antibodies were able to detect FSHR in rat ovaries by immunohistochemistry. Further, these antibodies were administered into adult female rats and their effect on fertility status was monitored. These antibodies were found to neutralize the biological activity of endogenous receptor, which resulted in the induction of infertility in the treated animals. Thus, bioneutralization of FSHR has been achieved by targeting its region 285-309 in an in vivo system.

Secondary structure analysis of synthetic peptides of the extracellular domain of the human follicle-stimulating hormone receptor.

Follicle-stimulating hormone receptor (FSHR) is a glycoprotein hormone receptor and possesses a large extracellular domain (ECD) instrumental in hormone binding. The ECD is characterized by the presence of leucine-rich repeat (LRR) structures made up of alpha-helices flanked by beta-strands. Our previous studies with the synthetic peptides corresponding to the potentially surface-oriented regions of the ECD had led to the identification of some of these regions in either FSH-binding or FSH-induced cAMP production or both. This study was undertaken with an aim to correlate the findings made in vitro with the secondary structures of the respective peptides. Accordingly, all peptides were screened for their secondary structures in different biochemical environments. This study correlates the observed alpha-helical signature with the previously demonstrated activity in signal generation for peptides 15-31 and 216-235 hFSHR, while FSH binding is correlated with the maintenance of beta-sheet structure in peptides 285-300 and 297-310 hFSHR as observed in vitro.

Identification of the structural and functional determinants of the extracellular domain of the human follicle stimulating hormone receptor.

The extracellular domain (ECD) of the human follicle-stimulating hormone receptor (hFSHR) is believed to be the major determinant of hormone selectivity. Different discrete, discontinuous regions on the ECD of the hFSHR have been suggested to be crucial for hormone binding. However, the role of the ECD in signal transduction is not well understood. This study provides some insight into these aspects of the structure-function relationship of the ECD of hFSHR. Ten peptides were selected from the ECD on the basis of their ability to be surface oriented, synthesized by the solid-phase method using fluorenylmethyloxycarbonyl chemistry, purified and characterized. They were further studied for their ability to modulate both human follicle-stimulating hormone (hFSH)-FSHR binding and cAMP generation. Competitive inhibition studies showed that, of all the peptides studied, peptides 285-300 and 297-310 hFSHR were able to inhibit hFSH binding to FSHR. Both peptides function as weak competitive inhibitors of hFSH-FSHR binding. Peptides 285-300 hFSHR, 216-235 hFSHR, 184-195 hFSHR, 79-89 hFSHR and 15-31 hFSHR were observed to inhibit FSH-induced cAMP production. In summary, this study suggests that discrete, functional domains of the ECD have a role in hormone binding and signal transduction. Region 285-300 has been identified as a novel region crucial for both FSH binding and cAMP generation.

Attempt to map the receptor binding sites of human follicle-stimulating hormone using disulfide peptides of its beta-subunit indicates major involvement of the regions around disulfide bonds Cys28-Cys82 and Cys32-Cys84 in receptor binding of the hormone.

Follicle-stimulating hormone (FSH) is a heterodimeric glycoprotein hormone secreted by the anterior pituitary. It plays a very important role in folliculogenesis in females and is responsible for spermatogenesis in males. The alpha-subunit which is common within a species and the beta-subunit which is hormone-specific are held together by noncovalent association. This association is very essential for the biological activity of the hormone. Each of these subunits are highly cross-linked by disulfide bonds which appear to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. This study was initiated to delineate the role of the disulfide bonds of hFSH beta in receptor binding of the hormone. Five intermolecular and one intramolecular disulfide peptides corresponding to the disulfide bonds found in hFSH beta were synthesized and screened along with their linear counterparts, for their ability to competitively inhibit the radiolabelled [125I]hFSH from binding to the FSH receptor containing membranes from the testis of immature rats. The disulfide peptides Cys28-Cys82 and Cys32-Cys84 were found to be the most potent in inhibiting radiolabelled hFSH from binding to its receptor. The results suggest the involvement of the regions around disulfide bonds Cys28-Cys82 and Cys32-Cys84 in receptor binding of the hormone. The studies also suggest the involvement of beta L2 and beta L3 loop regions in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hFSH.

Disulfide bonds Cys(9)-Cys(57), Cys(34)-Cys(88) and Cys(38)-Cys(90) of the beta-subunit of human chorionic gonadotropin are crucial for heterodimer formation with the alpha-subunit: experimental evidence for the conclusions from the crystal structure of hCG.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The alpha- and beta-subunits of hCG are highly cross-linked internally by disulfide bonds that seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. This paper describes the results of our studies on the role of the disulfide bonds of hCG-beta in heterodimer formation with the alpha-subunit. Six disulfide peptides incorporating each of the six disulfide bonds of hCG-beta were screened, along with their linear counterparts, for their ability to competitively inhibit the recombination of alpha- and beta-subunits. The disulfide peptides Cys (9-57), Cys (34-88) and Cys (38-90) were found to inhibit the alpha/beta recombination whereas the remaining three disulfide peptides viz. Cys (23-72), Cys (26-110) and Cys (93-100) did not exhibit any inhibition activity. Interestingly, none of the linear peptides could inhibit the alpha/beta recombination. Results clearly demonstrate that the disulfide bonds Cys(9)-Cys(57), Cys(34)-Cys(88) and Cys(38)-Cys(90) of the beta-subunit of hCG are crucial for heterodimer formation with the alpha-subunit thus providing experimental confirmation of the conclusions from the crystal structure of the hormone.

A detailed study of the L2beta long-loop region of human chorionic gonadotrophin suggests it to be spatially close to, but not part of, the receptor-binding site.

An in-depth study of the L2beta long-loop region of human chorionic gonadotrophin (hCG), earlier identified to be a conformational bioneutralization epitope and receptor-binding site of the hormone, was carried out. The linear 38-57 hCGbeta peptide and the corresponding cyclic disulphide peptide were synthesized and antipeptide antibodies developed. Binding studies with antibodies to the linear peptide, and with hCGbeta, hCG and human LH suggest that part of the region is buried at the alpha/beta interface and part exposed in hCG. Observation of the surface exposure of residues 47-53 from the crystal structure of hCG was confirmed by epitope mapping studies of the region. The region is not unique to hCG as a majority of the antibodies to both the linear and cyclic peptides did not exhibit the required specificity. Competitive inhibition studies with the linear and cyclic peptides failed to show inhibition of radiolabelled hCG binding to its receptors. However, both the antipeptide antibodies were able to bioneutralize the hormone in an in vivo assay. Taken together, these results seem to indicate that the L2beta long-loop region is not a receptor-binding site of hCG but spatially close to it.

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its beta-subunit: possible involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) in receptor binding of the hormone.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The alpha- and beta-subunits of hCG are highly cross-linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGbeta in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGbeta were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [125I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9-57) was found to be approximately 4-fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23-72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) of the beta-subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.

Autologous biological response modification of the gonadotropin receptor.

It is generally held with respect to heterotrimeric guanine nucleotide binding protein-coupled receptors that binding of ligand stabilizes a conformation of receptor that activates adenylyl cyclase. It is not formally appreciated if, in the case of G-protein-coupled receptors with large extracellular domains (ECDs), ECDs directly participate in the activation process. The large ECD of the glycoprotein hormone receptors (GPHRs) is 350 amino acids in length, composed of seven leucine-rich repeat domains, and necessary and sufficient for high affinity binding of the glycoprotein hormones. Peptide challenge experiments to identify regions in the follicle-stimulating hormone (FSH) receptor (FSHR) ECD that could bind its cognate ligand identified only a single synthetic peptide corresponding to residues 221-252, which replicated a leucine-rich repeat domain of the FSHR ECD and which had intrinsic activity. This peptide inhibited human FSH binding to the human FSHR (hFSHR) and also inhibited human FSH-induced signal transduction in Y-1 cells expressing recombinant hFSHR. The hFSHR-(221-252) domain was not accessible to anti-peptide antibody probes, suggesting that this domain resides at an interface between the hFSHR ECD and transmembrane domains. CD spectroscopy of the peptide in dodecyl phosphocholine micelles showed an increase in the ordered structure of the peptide. CD and NMR spectroscopies of the peptide in trifluoroethanol confirmed that hFSHR-(221-252) has the propensity to form ordered secondary structure. Importantly and consistent with the foregoing results, dodecyl phosphocholine induced a significant increase in the ordered secondary structure of the purified hFSHR ECD as well. These data provide biophysical evidence of the influence of environment on GPHR ECD subdomain secondary structure and identify a specific activation domain that can autologously modify GPHR activity.

Identification of bioneutralization epitopes of human follicle stimulating hormone in the regions 31-52 and 66-75 of its beta-subunit.

The crucial role played by follicle stimulating hormone (FSH) in regulating both male and female reproduction and the possibilities of developing contraceptive methods for males by blocking the function of the hormone, makes it important to delineate the hormone-specific bioneutralization epitopes of human follicle stimulating hormone (hFSH) on its beta-subunit. Predictive methods were used to identify the potential surface-oriented regions of hFSH-beta. Peptides corresponding to these regions, i.e. 31-52, 66-75 and 86-95 hFSH-beta, were synthesized, anti-peptide antibodies were elicited in rabbits and the properties of these antisera to bind hFSH and neutralize its biological activity were assessed. Anti-31-52 hFSH-beta antisera bound hFSH specifically, whereas anti-66-75 and anti-86-95 hFSH-beta antisera did not show any detectable binding, proving the region 31-52 hFSH-beta to be a specific antigenic determinant of hFSH. The bioneutralizing abilities of the anti-peptide antibodies were assessed by measuring the hFSH-induced progesterone secretion by rat granulosa cells in vitro. Antibodies to 31-52 and 66-75 hFSH-beta neutralized the bioactivity of hFSH, but anti-86-95 hFSH-beta antibodies did not. Furthermore, the three linear peptides and two disulphide looped peptides of 31-52 hFSH-beta and 86-95 hFSH-beta were also subjected to the in-vitro granulosa cell assay. The linear peptides 31-52 hFSH-beta and 66-75 hFSH-beta and the cyclic 31-52 hFSH-beta disulphide loop peptide significantly inhibited the hFSH-induced progesterone secretion by rat granulosa cells, but the linear 86-95 hFSH-beta peptide and the corresponding cyclic disulphide loop peptide did not. The results clearly show that the regions 31-52 and 66-75 of hFSH-beta harbor bioneutralization epitopes of the hormone. The studies also indicate that cyclization of the linear 31-52 hFSH-beta peptide greatly enhances receptor recognition and that the region 66-75 hFSH-beta may also be involved in hormone-receptor interaction.

Studies on the delineation of the hormone-specific antigenic determinants of human follicle-stimulating hormone.

Human follicle-stimulating hormone (hFSH) is a key hormone regulating both male and female reproduction. The present study attempts to delineate the hFSH-specific antigenic determinants on its beta-subunit. Predictive methods were used to identify the potential surface-oriented regions of hFSH-beta. Peptides corresponding to these regions, namely, 31-52, 66-75 and 86-95 hFSH-beta, were synthesized and conjugated to diphtheria toxoid. Antipeptide antibodies, elicited in rabbits by immunization with the conjugates, were screened for their ability to bind to hFSH-beta and hFSH. Anti-31-52 hFSH-beta antisera bound to both hFSH-beta and hFSH, whereas anti-66-75 and anti-86-95 hFSH-beta antisera did not show any detectable binding. Furthermore, screening of anti-hFSH antisera showed significant binding only to 31-52 hFSH-beta. These results identify the region 31-52 hFSH-beta as a hormone-specific antigenic determinant of hFSH.

Comparison of antibodies raised against the peptide 10-24 of chicken riboflavin carrier protein (cRCP) by classical and multiple antigen peptide (MAP) approaches.

Riboflavin carrier protein is an essential protein required for the growth and development of the embryo and hence for the maintenance of pregnancy. Our efforts to delineate the antigenic determinants of chicken riboflavin carrier protein (cRCP) resulted in the identification of a bioneutralization epitope in the region 10-24 of cRCP. The present work compares the properties of the antibodies raised against the peptide epitope by classical and multiple antigen peptide (MAP) system approaches. The extent of cross-reaction of the antibodies to the MAP construct with the parent protein was found to be significantly less as compared to the antibodies raised against the peptide-diphtheria toxoid conjugate. Furthermore, the bioneutralizing ability of the antisera to the MAP construct was also found to be very poor. The results suggest that there are serious limitations in the ability of antibodies raised against MAP constructs to cross-react with the native proteins.

Regulation of follicular maturation by human ovarian follicular fluid peptide.

Follicular development, ovulation and luteal function are controlled by gonadotrophins. However, recent evidence indicates that local factors are also responsible for the regulation of folliculogenesis. In addition to their endocrine action on pituitary gonadotrophins, inhibin, activin and follistatin also have a paracrine role in follicular maturation. An ovarian follicular fluid peptide (OFFP) has been identified from sheep and humans. Purification of OFFP has been achieved by ultrafiltration and gel chromatography with further purification by fast performance liquid chromatography and reversed phase-high pressure liquid chromatography. OFFP is a small (< 5 kDa) peptide that competes with FSH in binding to granulosa cells in vitro and inhibits progesterone secretion from granulosa cells in culture. Immunohistochemical localization revealed the presence of OFFP mainly in granulosa cells of ovarian follicles. Furthermore, the peptide caused apoptosis in granulosa cells and induced follicular atresia. OFFP may act indirectly on oocytes via its effect on granulosa cells. The peptide from ovarian follicular fluid appears to have an important autocrine or paracrine role in the regulation of folliculogenesis.

Termination of pregnancy in mice following administration of antibodies to the pentadecapeptide 10-24 of chicken riboflavin carrier protein: identification of a bioneutralizing epitope of chicken riboflavin carrier protein.

Our studies have shown that the administration of antibodies to chicken riboflavin carrier protein (cRCP) or disulfide-reduced carboxymethylated cRCP (RCM-RCP) leads to termination of pregnancy in mice. In an attempt to delineate the bioneutralizing epitopes, a combination of chemical modification and predictive methods was used. The results led to the identification of the region 10-24 of cRCP as a potential candidate. A single injection of antipeptide antibodies to pregnant mice on day 11 resulted in 100% pregnancy termination. This was accompanied by a drastic reduction in circulatory progesterone as early as 24 h after the antibody administration. These results thus demonstrate that the amino acid sequence 10-24 of cRCP harbours a bioneutralizing epitope of cRCP.

Studies on the carboxyl terminal peptides of human seminal plasma inhibin (HSPI). Chemical synthesis and in vivo biological activity of the disulfide loop peptide 67-94 of HSPI.

Observation of contradictory results with the in vitro assays for inhibin-like activity of the carboxyl terminal 28 amino acid peptide 67-94 with a disulfide loop, of human seminal plasma inhibin (HSPI), prompted us to synthesize both the linear and the cyclic peptides and test their ability to suppress the circulating levels of follicle stimulating hormone (FSH) in vivo in adult male rats. The linear peptide [Cys(Acm)73,87] 67-94 of HSPI was synthesized by solid-phase peptide synthesis using fluorenylmethyloxycarbonyl (Fmoc) chemistry and a continuous-flow technology. The peptide was cyclized by direct iodine oxidation of the S-diacetamidomethyl peptide in dilute solution. In the in vivo assay the linear peptide did not affect the levels of FSH, whereas the cyclic peptide suppressed the levels of FSH significantly. Thus, the carboxyl terminal region of HSPI does have inhibin-like activity and perhaps has the active core of the protein.

Studies on the delineation of the antigenic determinants of chicken riboflavin carrier protein (cRCP). Identification of a determinant in the region 10-24 of the protein.

As riboflavin carrier proteins play a critical role in the maintenance of pregnancy, studies on the immunology of these proteins have become very relevant. This paper describes our attempts to identify the sequential antigenic determinants of chicken riboflavin carrier protein (cRCP). Potential surface oriented regions of cRCP were identified by constructing acrophilicity and hydrophilicity profiles of the protein. Of the three regions identified, the pentadecapeptide 10-24 of cRCP forms the subject of the present study. The pentadecapeptide amide was synthesized by solid phase synthesis using Fmoc chemistry. Immunization of rabbits with the peptide-diphtheria toxoid conjugate elicited high titres of the antipeptide antibodies, revealing the high immunogenicity of the peptide. The antipeptide antibodies bound to both cRCP and reduced carboxymethylated RCP (RCM-RCP). Antisera to RCM-RCP showed significant binding to the peptide showing thereby that the region 10-24 of cRCP is perhaps one of the major epitopes of RCM-RCP. Thus, the studies have resulted in the identification of the region 10-24 as an antigenic determinant of cRCP.