Structural plasticity of a transmembrane peptide allows self-assembly into biologically active nanoparticles.

Significant efforts have been devoted to the development of nanoparticular delivering systems targeting tumors. However, clinical application of nanoparticles is hampered by insufficient size homogeneity, difficulties in reproducible synthesis and manufacturing, frequent high uptake in the liver, systemic toxicity of the carriers (particularly for inorganic nanoparticles), and insufficient selectivity for tumor cells. We have found that properly modified synthetic analogs of transmembrane domains of membrane proteins can self-assemble into remarkably uniform spherical nanoparticles with innate biological activity.

Normalization of proliferation and tight junction formation in bladder epithelial cells from patients with interstitial cystitis/painful bladder syndrome by d-proline and d-pipecolic acid derivatives of antiproliferative factor.

Interstitial cystitis/painful bladder syndrome is a chronic bladder disorder with epithelial thinning or ulceration, pain, urinary frequency and urgency, for which there is no reliably effective therapy. We previously reported that interstitial cystitis/painful bladder syndrome bladder epithelial cells make a glycopeptide antiproliferative factor or 'APF' (Neu5Acα2-3Galβ1-3GalNAcα-O-TVPAAVVVA) that induces abnormalities in normal cells similar to those in interstitial cystitis/painful bladder syndrome cells in vitro, including decreased proliferation, decreased tight junction formation, and increased paracellular permeability. We screened inactive APF derivatives for their ability to block antiproliferative activity of asialylated-APF ('as-APF') in normal bladder cells and determined the ability of as-APF-blocking derivatives to normalize tight junction protein expression, paracellular permeability, and/or proliferation of interstitial cystitis/painful bladder syndrome cells.

Structure-Activity Studies on Antiproliferative Factor (APF) Glycooctapeptide Derivatives.

Antiproliferative factor (APF), a sialylated glycopeptide secreted by explanted bladder epithelial cells from interstitial cystitis/painful bladder syndrome (IC/PBS) patients, and its unsialylated analogue (as-APF) significantly decrease proliferation of bladder epithelial cells and/or certain carcinoma cell lines in vitro. We recently reported a structure-activity relationship profile for the peptide portion of as-APF and revealed that truncation of the C-terminal alanine did not significantly affect antiproliferative activity. To better understand the structural basis for the maintenance of activity of this truncated eight amino acid as-APF (as-APF8), we synthesized several amino acid-substituted derivatives and studied their ability to inhibit bladder epithelial cell proliferation in vitro as well as their solution conformations by CD and NMR spectroscopy.

Development of antiproliferative phenylmaleimides that activate the unfolded protein response.

The current paper presents the synthesis and evaluation of a series of maleimides that were designed to inhibit the Cdc25 phosphatase by alkylation of catalytically essential cysteine residues. Although in HepB3 cell culture assays the analogues did exhibit antiproliferative IC(50) values ranging from sub-micromolar to greater than 100 microM, inhibition of Cdc25 through cysteine alkylation could not be demonstrated. It was also found that analysis using fluorescence activated cell sorting (FACS) following treatment with the most potent analogue (1t) did not provide data consistent with inhibition at one specific point in the cell cycle, as would be expected if Cdc25A were inhibited.

Structure-activity relationship studies for the peptide portion of the bladder epithelial cell antiproliferative factor from interstitial cystitis patients.

We performed comprehensive structure-activity relationship (SAR) studies on the peptide portion of antiproliferative factor (APF), a sialylated frizzled-8 related glycopeptide that inhibits normal bladder epithelial and urothelial carcinoma cell proliferation. Glycopeptide derivatives were synthesized by solid-phase methods using standard Fmoc chemistry and purified by RP-HPLC; all intermediate and final products were verified by HPLC-MS and NMR analyses. Antiproliferative activity of each derivative was determined by inhibition of (3)H-thymidine incorporation in primary normal human bladder epithelial cells.

Growth inhibition of hepatocellular carcinoma cells in vitro and in vivo by the 8-methoxy analog of WMC79.

HKH40A (RTA 502), an optimized 8-methoxy analog of the unsymmetrical bifunctional antitumor agent WMC79, was found to be potently active against liver cancer cell growth in vitro and in vivo. Studies on selected human hepatocellular carcinoma (HCC) cell lines with differing p53 status (HepG2, Hep3B, and PLC/PRF/5), revealed that drug-mediated growth inhibition was independent of p53 status. FACS analysis showed an accumulation of cells in S-phase within 24 h of treatment with 100 nM HKH40A.

Bombesin marine toxin conjugates inhibit the growth of lung cancer cells.

Hemiasterlin (Hem) and dolastatin (Dol) are marine natural products which are cytotoxic for cancer cells. Hem, a tripeptide, and Dol, a hexapeptide, were conjugated with linkers (L) to the universal BB agonist DPhe-Gln-Trp-Ala-Val-betaAla-His-Phe-Nle-NH2(BA1) and the effects of the Hem-BB and Dol-BB conjugates investigated on NCI-H1299 lung cancer cells. Hem-LA-BA1 and Hem-LB-BA1 inhibited specific (125I-Tyr4)BB binding to NCI-H1299 cells, which have BB2 receptors (R), with IC50 values of 15 and 25 nM, respectively.

Rationally designed inhibitors identify STAT3 N-domain as a promising anticancer drug target.

Activation of the signal transducer and activator of transcription 3 (STAT3) is frequently detected in many cancer types. Activated STAT3 may participate in oncogenesis by stimulating cell proliferation and resisting apoptosis, as well as promoting tumor angiogenesis, invasion, and migration. Many STAT3-dependent cellular responses are mediated through interactions with other proteins, and the amino-terminal domain (N-domain) of STAT3 was proposed to be responsible for this.

Design and synthesis of novel and potent inhibitors of the type II transmembrane serine protease, matriptase, based upon the sunflower trypsin inhibitor-1.

Matriptase, initially isolated from human breast cancer cells in culture, is a member of the emerging class of type II transmembrane serine proteases. Matriptase blockade could potentially modulate tumorigenesis and metastasis in vivo. Sunflower trypsin inhibitor-1 (1, SFTI-1), isolated from sunflower seeds, exhibits very potent matriptase inhibitory activity.

Optimization of naphthalimide-imidazoacridone with potent antitumor activity leading to clinical candidate (HKH40A, RTA 502).

Unsymmetrical bifunctional antitumor agent WMC79 was further optimized to generate compound 7b that not only inhibited the growth of many tumor cell lines, but caused rapid apoptosis. Unlike the parent compound, 7b is toxic to both p53 positive and negative cancer cells. It has potent in vivo activity against xenografts of human colon and pancreatic tumors in athymic mice.

Design and synthesis of paclitaxel conjugated with an ErbB2-recognizing peptide, EC-1.

The selective delivery of therapeutic agents to receptors overexpressed in cancer cells without harming the rest of the body is a major challenge in clinical oncology today. In this study, we report the design and synthesis of paclitaxel (PTX) conjugated with an erbB2-recognizing peptide (EC-1). The cyclic peptide EC-1 specifically binds to the extracellular domain of ErbB2 and selectively inhibits proliferation of breast cancer cells overexpressing ErbB2.

Synthesis, biological activity, and crystal structure of potent nonnucleoside inhibitors of HIV-1 reverse transcriptase that retain activity against mutant forms of the enzyme.

In an ongoing effort to develop novel and potent nonnucleoside HIV-1 reverse transcriptase (RT) inhibitors that are effective against the wild type (WT) virus and clinically observed mutants, 1,2-bis-substituted benzimidazoles were synthesized and tested. Optimization of the N1 and C2 positions of benzimidazole led to the development of 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimidazole (1) (IC50 = 0.2 microM, EC50 = 0.

Synthesis and biological activity of 5-aza-ellipticine derivatives.

Novel 5-aza-ellipticine derivatives were synthesized and tested as antitumor agents. The new compounds were prepared more readily than the analogous ellipticine derivatives, which are known to be potent anti-tumor agents Although the novel 5-aza-ellipticine derivatives are not as biologically active as their corresponding ellipticine analogues, the new compounds represent a new, readily accessible class of heteroaromatic catalytic inhibitors of topoisomerase II and possible anti-tumor agents.

CKAP4/p63 is a receptor for the frizzled-8 protein-related antiproliferative factor from interstitial cystitis patients.

Antiproliferative factor (APF) is a low molecular weight sialoglycopeptide that is secreted by bladder cells from interstitial cystitis patients and is a potent inhibitor of both normal bladder epithelial and bladder carcinoma cell proliferation. We hypothesized that APF may produce its antiproliferative effects by binding to a transmembrane receptor. This study demonstrates that cytoskeleton-associated protein 4/p63 (CKAP4/p63), a type II transmembrane receptor, binds with high affinity to APF.

PM-20, a novel inhibitor of Cdc25A, induces extracellular signal-regulated kinase 1/2 phosphorylation and inhibits hepatocellular carcinoma growth in vitro and in vivo.

We have synthesized several new phenyl maleimide compounds, which are potent growth inhibitors of several human tumor cell lines. Among these, PM-20 was the most potent with an IC50 of 700 nmol/L for Hep3B human hepatoma cell growth. Two other derivatives, PM-26 and PM-38, did not inhibit Hep3B cell growth even at 100 micromol/L.

WMC-79, a potent agent against colon cancers, induces apoptosis through a p53-dependent pathway.

WMC-79 is a synthetic agent with potent activity against colon and hematopoietic tumors. In vitro, the agent is most potent against colon cancer cells that carry the wild-type p53 tumor suppressor gene (HCT-116 and RKO cells: GI50<1 nmol/L, LC50 approximately 40 nmol/L). Growth arrest of HCT-116 and RKO cells occurs at the G1 and G2-M check points at sublethal concentrations (10 nmol/L) but the entire cell population was killed at 100 nmol/L.

A new synthetic agent with potent but selective cytotoxic activity against cancer.

The synthesis of novel unsymmetrical bifunctional antitumor agents was accomplished by linking an imidazoacridone moiety to another polycyclic heteroaromatic moiety via linkers of various length and rigidity. These compounds bind to cellular DNA, but it is hypothesized that biological effects become manifested when the drug-DNA complexes interact with critical DNA binding proteins that are involved in repair and transcription. The most promising compound of the series, 4ad (WMC79), consists of an imidazoacridone linked to a 3-nitronaphthalimide moiety via a 1,4-dipropanopiperazine linker.

Transmembrane inhibitors of P-glycoprotein, an ABC transporter.

Drug resistance mediated by ABC transporters such as P-glycoprotein (P-gp) continues to be a major impediment to effective cancer chemotherapy. We have developed a panel of highly specific peptide inhibitors of P-gp based on the structure of the transmembrane domains of the transporter. These peptides are thought to exert their inhibitory action by disrupting the proper assembly of P-gp.

HIV-1 reverse transcriptase variants: molecular modeling of Y181C, V106A, L100I, and K103N mutations with nonnucleoside inhibitors using Monte Carlo simulations in combination with a linear response method.

The energies and physical descriptors for the binding of 21 novel 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-benzimidazole (BPBI) analogs to HIV-1 reverse transcriptase (RT) variants Y181C, L100I, V106A, and K103N have been determined using Monte Carlo (MC) simulations. The crystallographic structure of the lead compound, 4-methyl BPBI, was used as a starting point to model the inhibitors in both the mutant bound and the unbound states. The energy terms and physical descriptors obtained from the calculations were reasonably correlated with the respective experimental EC50 values for the inhibitors against the various mutant RTs.

Caspase-mediated apoptosis and caspase-independent cell death induced by irofulven in prostate cancer cells.

Irofulven (hydroxymethylacylfulvene) is a novel antitumor drug, which acts by alkylating cellular macromolecular targets. The drug is a potent inducer of apoptosis in various types of tumor cells, whereas it is nonapoptotic in normal cells. This study defined molecular responses to irofulven involving mitochondrial dysfunction and leading to death of prostate tumor LNCaP-Pro5 cells.

The development of VIP-ellipticine conjugates.

The mechanism by which vasoactive intestinal peptide (VIP)-ellipticine (E) conjugates are cytotoxic for human lung cancer cells was investigated. VIP-alanyl-leucyl-alanyl-leucyl-alanine (ALALA)-E and VIP-leucyl-alanyl-leucyl-alanine (LALA)-E inhibited (125)I-VIP binding to NCI-H1299 cells with an IC50 values of 0.5 and 0.

An antiproliferative factor from interstitial cystitis patients is a frizzled 8 protein-related sialoglycopeptide.

Approximately 1 million people in the United States suffer from interstitial cystitis, a chronic painful urinary bladder disorder characterized by thinning or ulceration of the bladder epithelial lining; its etiology is unknown. We have identified a glycosylated frizzled-related peptide inhibitor of cell proliferation that is secreted specifically by bladder epithelial cells from patients with this disorder. This antiproliferative factor (APF) profoundly inhibits bladder cell proliferation by means of regulation of cell adhesion protein and growth factor production.

Small molecule toxins targeting tumor receptors.

Targeting toxic therapeutics to tumors through receptors over expressed on the surface of cancer cells can reduce systemic toxicity and increase the effectiveness of the targeted compounds. Small molecule targeted therapeutics have a number of advantages over toxic immunoconjugates including better tumor penetration, lack of neutralizing host immune response and superior flexibility in selection of drug components with optimal specificity, potency and stability in circulation. Three major components of the targeted drug, the toxic warhead, tumor-specific ligand and the linker can influence the properties of each other and thus have to be optimized for each system.

Mutational analysis of ABCG2: role of the GXXXG motif.

ABCG2 (BCRP/MXR/ABCP) is a half-transporter associated with multidrug resistance that presumably homodimerizes for function. It has a conserved GXXXG motif in its first transmembrane segment, a motif that has been linked with dimerization in other proteins, e.g.

Bisimidazoacridones: 2. Steady-state and time-resolved fluorescence studies of their diverse interactions with DNA.

Several bisimidazoacridones (BIA) are potent, selective antineoplastic agents, whereas others have potent anti-human immunodeficiency virus activity. BIA are bifunctional agents that consist of two imidazoacridone (IA) chromophores held together by various linkers. Interaction of BIA with DNA has been postulated to be required for their biological activity.