Variation of Donor and Acceptor Strength in Analogues of Brooker's Merocyanine and Generalization to Various Classes of Charge Transfer Compounds.

Derivatives of Brooker's merocyanine (BM) have been investigated, which possess different donors and acceptors and therefore vary their donor-acceptor strength SDA. The 00 energies have been extracted from the spectra and compared. Under basic conditions, where the neutral (merocyanine) form is present, the absorption energies for all compounds are similar, whereas there is a large difference for acidic conditions where the cationic (cyanine) form is present. This behavior could be explained by a simple theoretical model involving the dependence of the excitation energy Δ E01 on SDA. This model can be generalized to describe in a consistent way two different well-known classes of neutral chromophores with a certain degree of charge separation, namely merocyanine I (TICT) and merocyanine II (often betainic) compounds. Merocyanines I are characterized by a medium polar aromatic ground state and a zwitterionic quinoid excited state and hence positive solvatochromism, whereas merocyanines II are formally characterized by a zwitterionic aromatic ground state and a less polar quinoid excited state and, accordingly, by negative solvatochromism. On increasing the donor-acceptor strength SDA sufficiently, merocyanines II can, however, move to the so-called overcritical region with the excited state dominated by the zwitterionic valence bond structure. For many of the merocyanine II molecules investigated here, a weakly positive solvatochromic behavior is observed indicating that the ground state contains less of the zwitterionic valence bond wave function than the excited state and that these compounds belong to the overcritical region. The fluorescence spectra have been analyzed in terms of the Franck-Condon model and confirm these conclusions.

Experimental and theoretical study of excited-state structure and relaxation processes of betaine-30 and of pyridinium model compounds.

The long-wavelength absorption band of 2,6-diphenyl-4-(2,4,6-triphenylpyridinium-1-yl) phenolate (betaine-30, B30) in ethanol and 1-chlorobutane shifts to the blue by cooling in the temperature range of 294-128 K. In addition, B30 shows fluorescence in both solvents at 77 K, which is absent at room temperature. The study of the ground and excited state of B30 and its model compound 4-(pyridinium-1-yl)-phenolate PyPo by DFT/TDDFT calculations indicates that for the perpendicular conformation, the ground state possesses a charge-separated closed-shell hole pair (hp) electronic configuration, and the S(1) state corresponds to a biradicaloid electronic structure (dd) with a small dipole moment caused by an unpaired electron on each of the orthogonal fragments. Following the absorption process, there is a driving force for geometrical relaxation within the S(1) state toward an orthogonal arrangement of the phenolate and the pyridinium ring. In this final S(1) equilibrium geometry, the energy gap between the excited and the ground state is strongly reduced and causes very efficient radiationless deactivation of the S(1) state at room temperature. At 77 K, the viscous barrier hindering large-amplitude motion enables the appearance of the fluorescence from the partially or nonrelaxed excited Franck-Condon structure. By variation of the donor and acceptor strength of the two moieties, the energy gap for perpendicular systems can be tuned, allowing, in principle, the switching between the two cases hp < dd and hp > dd. This enables a new access to the efficient construction of mnemonic systems and NLO dyes.

Photophysical and complexation properties of benzenesulfonamide derivatives with different donor and acceptor moieties.

Pyrrolobenzosulfonamide, indolobenzosulfonamide and carbazolobenzosulfonamide derivatives with different acceptor groups were synthesized and their photophysical properties were compared. The electron donor linking sites are found to influence the emission characteristics of these compounds while acceptor linking sites have no noticeable effects on the spectral properties. P2-A5 which is a C-C linked pyrrole derivative exhibited different spectral properties from the C-N linked pyrrole derivatives. The complexation properties of the molecules were also investigated employing Na (I), Ca (II), Li (I), Mg (II), Zn (II) and Cu (II) ions.

BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models.

Genetic alterations in the kinase domain of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) patients are associated with sensitivity to treatment with small molecule tyrosine kinase inhibitors. Although first-generation reversible, ATP-competitive inhibitors showed encouraging clinical responses in lung adenocarcinoma tumors harboring such EGFR mutations, almost all patients developed resistance to these inhibitors over time. Such resistance to first-generation EGFR inhibitors was frequently linked to an acquired T790M point mutation in the kinase domain of EGFR, or upregulation of signaling pathways downstream of HER3. Overcoming these mechanisms of resistance, as well as primary resistance to reversible EGFR inhibitors driven by a subset of EGFR mutations, will be necessary for development of an effective targeted therapy regimen. Here, we show that BIBW2992, an anilino-quinazoline designed to irreversibly bind EGFR and HER2, potently suppresses the kinase activity of wild-type and activated EGFR and HER2 mutants, including erlotinib-resistant isoforms. Consistent with this activity, BIBW2992 suppresses transformation in isogenic cell-based assays, inhibits survival of cancer cell lines and induces tumor regression in xenograft and transgenic lung cancer models, with superior activity over erlotinib. These findings encourage further testing of BIBW2992 in lung cancer patients harboring EGFR or HER2 oncogenes.

Charge-transfer transitions in twisted stilbenoids: interchangeable features and generic distinctions of single- and double-bond twists.

The charge-transfer transitions relevant to single- and double-bond photochemical twisting have been studied in the framework of the biradicaloid state theory using the AM1 method for a family of donor-acceptor-substituted stilbenoids and a series of sparkle-simulated model stilbenes. Features in common and mutually interchangeable properties for the two transition types as well as their peculiarities are revealed; they are considered in relation to the varied donor-acceptor strength of the substituents, with particular attention given to the occurrence of S0-S1 state conical intersections. The difference in critical points at which the conical intersections occur for double-bond and single-bond twisted stilbenoids is shown to be related to the splitting of the cyanine limit of their planar counterparts.

Bifunctional charge transfer operated fluorescent probes with acceptor and donor receptors. 1. Biphenyl-type sensor molecules with protonation-induced anti-energy gap rule behavior.

On the basis of biphenyl (b) type molecules bpb-R substituted with a 2,2':6',2' '-terpyridine acceptor (bp) and either amino-type donor receptors (R = dimethylamino (DMA), A15C5 = monoaza-15-crown-5) or nonbinding substituents (R = CF(3), H, OMe) of various donor strengths, we developed a family of charge transfer (CT) operated monofunctional and bifunctional fluorescent sensors for protons and metal ions. These molecules are designed to communicate the interaction of an analyte with the acceptor and the donor receptor differing in basicity and cation selectivity by clearly distinguishable spectral shifts and intensity modulations in absorption and in emission as well as in fluorescence lifetime. From the dependence of the fluorescence spectra, fluorescence quantum yields, and fluorescence lifetimes of bpb-R on solvent polarity and proton concentration, the photophysics of bpb-R and their protonated analogues can be shown to be governed by the relaxation to a CT state of forbidden nature and by the switching between anti-energy and energy gap law type behaviors. This provides the basis for analytically favorable red shifted emission spectra in combination with comparatively high fluorescence quantum yields. Accordingly, bpb-H and bpb-OMe are capable of ratiometric emission signaling of protons. bpb-DMA reveals a protonation-induced ON-OFF-ON switching of its emission.

Bifunctional charge transfer operated fluorescent probes with acceptor and donor receptors. 2. Bifunctional cation coordination behavior of biphenyl-type sensor molecules incorporating 2,2':6',2' '-terpyridine acceptors.

Based on donor (D)-acceptor (A) biphenyl (b) type molecules, a family of fluorescent reporters with integrated acceptor receptors and noncoordinating and coordinating donor substituents of varying strength has been designed for ratiometric emission sensing and multimodal signaling of metal ions and protons. In part 2 of this series on such charge transfer (CT) operated mono- and bifunctional fluorescent devices, the cation coordination behavior of the sensor molecules bpb-R equipped with a proton- and cation-responsive 2,2':6',2' '-terpyridine (bp) acceptor and either amino-type donor receptors (R = DMA, A15C5 = monoaza-15-crown-5) or nonbinding substituents (R = CF(3), H, OMe) is investigated employing the representative metal ions Na(I), Ca(II), Zn(II), Hg(II), and Cu(II) and steady-state and time-resolved fluorometry. The bpb-R molecules, the spectroscopic behavior and protonation behavior of which have been detailed in part 1 of this series, present rare examples for CT-operated bifunctional fluorescent probes that can undergo consecutive and/or simultaneous analyte recognition. The analyte-mediated change of the probes' intramolecular CT processes yields complexation site- and analyte-specific outputs, i.e., absorption and fluorescence modulations in energy, intensity, and lifetime. As revealed by the photophysical studies of the cation complexes of these fluoroionophores and the comparison to other neutral and charged D-A biphenyls, the spectroscopic properties of the acceptor chelates of bpb-R and A- and D-coordinated bpb-R are governed by CT control of an excited-state barrier toward formation of a forbidden charge transfer state, by the switching between analytically favorable anti-energy and common energy gap law type behavior, and by the electronic nature of the ligated metal ion. This accounts for the astonishingly high fluorescence quantum yields of the acceptor chelates of bpb-R equipped with weak or medium-sized donors and the red emission of D- and A-coordinated bpb-R observed for nonquenching metal ions.

CT-operated bifunctional fluorescent probe based on a pretwisted donor-donor-biphenyl.

Taking into account the structural requirements for TICT-type sensor molecules, a general synthetic route to derive pH and cation-responsive pretwisted donor (D)-donor (D) biphenyls (b) equipped with donor receptors is developed and a first model compound containing a mono aza-15-crown-5 and a DMA receptor is synthesized, see Scheme 1. The spectroscopic properties of this new bifunctional D-D biphenyl are studied in the non-polar and polar solvents cyclohexane, acetonitrile, and methanol. Protonation as well as complexation studies are performed with the representative metal ions Na(I), K(I), Ca(II), Ag(I), Zn(II), Cd(II), Hg(II), and Pb(II) to reveal the potential of this molecule for communication of whether none, only one, or both binding sites are engaged in analyte coordination by spectroscopically distinguishable outputs. The results are compared to those obtained with closely related donor (D)-acceptor (A) substituted biphenyl-type sensor molecules and are discussed within the framework of neutral and ionic D-A biphenyls.

A highly selective telomerase inhibitor limiting human cancer cell proliferation.

Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues. Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation. We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo. Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression. Telomerase inhibition and telomere shortening also result in a marked reduction of the tumorigenic potential of drug-treated tumour cells in a mouse xenograft model. This model was also used to demonstrate in vivo efficacy with no adverse side effects and uncomplicated oral administration of the inhibitor. These findings indicate that potent and selective, non-nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities.

Phase transitions and hydrogen bonding in a bipolar phosphocholine evidenced by calorimetry and vibrational spectroscopy.

As a model for natural archaebacterial bolalipids, we have synthesized omega-hydroxybehenylphosphocholine (HBPC, HO-(CH(2))(22)-OP(O(-)(2))O-(CH(2))(2)-N+(CH(3))(3)) and investigated it, by Fourier-transform infrared and Raman spectroscopy and differential scanning calorimetry, both as fully hydrated dispersions (varying temperature) and as aligned films (varying hydration) in terms of particular structural features predestining such bipolar lipids for their occurrence in extremophilic organisms. The phase behavior of HBPC in dispersions depends on sample pretreatment as it comprises metastabilities in annealed samples. However, main transition proceeds consistently near 81 degrees C. Some (extra) deal of headgroup (phosphate) hydration accompanying a gel-gel phase transition near 66 degrees C appears to precede chain melting. Studies with HBPC films revealed lamellar interdigitated-like solid phases with an extraordinarily strong omega-OH--OPO(-) omega-OH--OPO(-) omega-OH hydrogen-bond pattern formed along both sides of the resulting monolayers. The "clamping" effect inherent to such structures provides a clue to explain the relatively high main-transition temperature of HBPC assemblies.

Species-crossreactive scFv against the tumor stroma marker "fibroblast activation protein" selected by phage display from an immunized FAP-/- knock-out mouse.

BACKGROUND: Fibroblast activation protein (FAP) is a type II membrane protein expressed on tumor stroma fibroblasts in more than 90% of all carcinomas. FAP serves as a diagnostic marker and is potential therapeutic target for treatment of a wide variety of FAP+ carcinomas. Murine tumor stroma models and FAP-specific antibodies are required to investigate the functional role of FAP in tumor biology and its usefulness for drug targeting. We here describe the development of antibodies with crossreactivity for human (hFAP) and murine FAP (mFAP), which share 89% amino acid identity. MATERIAL AND METHODS: An FAP-/- mouse was sequentially immunized with recombinant murine and human FAP-CD8 fusion proteins. Immunoglobulin cDNA derived from hyperimmune spleen cells was used for the construction of a combinatorial single chain Fv (scFv) library. Phage display selection of FAP-specific scFv was performed on immobilized hFAP followed by selection on cells expressing murine FAP. RESULTS: High-affinity, species-crossreactive, FAP-specific scFv were isolated upon sequential phage display selection. A bivalent derivative (minibody M036) constructed thereof was applied for immunohistochemical analyses and allowed detection of FAP expression on stroma cells of different human carcinomas as well as on murine host stroma in a tumor xenograft model. CONCLUSIONS: MB M036, derived from phage display selected species crossreactive scFv, is suitable for tumor stroma targeting and will be a valuable tool in the analyses of the functional role of FAP in tumor biology as well as in the evaluation of the suitability of FAP for drug targeting.

Molecular cloning and characterization of EndoGlyx-1, an EMILIN-like multisubunit glycoprotein of vascular endothelium.

EndoGlyx-1, the antigen identified with the monoclonal antibody H572, is a pan-endothelial human cell surface glycoprotein complex composed of four different disulfide-bonded protein species with an apparent molecular mass of approximately 500 kDa. Here, we report the purification and peptide analysis of two EndoGlyx-1 subunits, p125 and p140, and the identification of a common, full-length cDNA with an open reading frame of 2847 base pairs. The EndoGlyx-1 cDNA encodes a protein of 949 amino acids with a predicted molecular mass of 105 kDa, found as an entry for an unnamed protein with unknown function in public data bases. A short sequence tag matching the cDNA of this gene was independently discovered by serial analysis of gene expression profiling as a pan-endothelial marker, PEM87. Bioinformatic evaluation classifies EndoGlyx-1 as an EMILIN-like protein composed of a signal sequence, an N-terminal EMI domain, and a C-terminal C1q-like domain, separated from each other by a central coiled-coil-rich region. Biochemical and carbohydrate analysis revealed that p125, p140, and the two additional EndoGlyx-1 subunits, p110 and p200, are exposed on the cell surface. The three smaller subunits show a similar pattern of N-linked and O-linked carbohydrates, as shown by enzyme digestion. Because the two globular domains of EndoGlyx-1 p125/p140 show structural features shared by EMILIN-1 and Multimerin, two oligomerizing glycoproteins implicated in cell-matrix adhesion and hemostasis, it will be of interest to explore similar functions for EndoGlyx-1 in human vascular endothelium.

Expression of the fibroblast activation protein during mouse embryo development.

Human Fibroblast Activation Protein (FAP), a member of the serine prolyl oligopeptidase family, is a type II cell surface glycoprotein that acts as a dual-specificity dipeptidyl-peptidase (DPP) and collagenase in vitro. Its restricted expression pattern in embryonic mesenchyme, in wound healing and in reactive stromal fibroblasts of epithelial cancers, has suggested a role for the FAP protease in extracellular matrix degradation or growth factor activation in sites of tissue remodeling. The FAP homologue in Xenopus laevis has been reported to be induced in the thyroid hormone-induced tail resorption program during tadpole metamorphosis supporting a role for FAP in tissue remodeling processes during embryonic development. However, Fap-deficient mice show no overt developmental defects and are viable. To study the expression of FAP during mouse embryogenesis, a second Fap-deficient mouse strain expressing beta-Galactosidase under the control of the Fap promoter was generated by homologous recombination (Fap-/- lacZ mice). FAP deficiency was confirmed by the absence of FAP-specific dipeptidyl-peptidase activity in detergent-soluble extracts isolated from 17.5 d.p.c. Fap-/- lacZ embryos. We report that Fap-/- lacZ mice express beta-Galactosidase at regions of active tissue remodeling during embryogenesis including somites and perichondrial mesenchyme from cartilage primordia.

Human antibody derivatives against the fibroblast activation protein for tumor stroma targeting of carcinomas.

The fibroblast activation protein (FAP) is selectively expressed on activated fibroblasts of the tumor stroma on more than 90% of lung, breast and colon carcinomas. The high prevalence and abundance of FAP(+) stroma make it a promising target for in vivo diagnosis and therapy of a variety of carcinomas. We describe the humanization of the murine FAP-specific MAb, F19, which has already been clinically used for in vivo diagnostic purposes. Using phage display technology and human V-repertoires, VL and VH regions of F19 were replaced by analogous human V-regions while retaining the original HCDR3 sequence in order to maintain F19 epitope specificity. The resulting human single-chain fragments of immunoglobulin variable regions (scFv 34, scFv 18) showed affinities of 6 nM on cell membrane-bound FAP. scFv 34 was expressed as a bivalent minibody (Mb 34). The antigen-binding characteristics of Mb 34 were comparable to the parental and a complementarity-determining region (CDR)-grafted version of F19. This was revealed by binding competition studies, FACS analyses and immunohistochemistry on various tumor samples including breast, colon and lung carcinomas. Importantly, compared with the CDR-grafted humanized scFv version of F19, the V-regions of the selected human scFv 34 showed sequence identity with the parental antibody (Ab) only over the short, 15-amino acid long HCDR3. Thus, a largely reduced xenoantigenic potential is expected. These human Ab derivatives are suitable to develop novel therapeutic concepts with broad applicability for a wide variety of histological carcinomas based on tumor stroma targeting.

Population pharmacokinetics of antifibroblast activation protein monoclonal antibody F19 in cancer patients.

AIMS: The population pharmacokinetics of 131I-mAbF19, a radiolabelled murine monoclonal antibody against fibroblast activation protein and a potential antitumour stroma agent, were investigated during two phase I studies in cancer patients. METHODS: 131I-mAbF19 serum concentration-time data were obtained in 16 patients from two studies involving imaging and dosimetry in colorectal carcinoma and soft tissue sarcoma. Doses of 0.2, 1 and 2 mg antibody were administered as 60 min intravenous infusions. The data were analysed by nonlinear mixed effect modelling. RESULTS: The data were described by a two-compartment model. Population mean values were 109 ml h(-1) for total serum clearance, 3.1 l for the volume of distribution of the central compartment, and 4.9 l for the volume of distribution at steady state. Mean terminal half-life was 38 h. Intersubject variability was high, but no patient covariates could be identified that further explained this variability. In particular, there was no influence of tumour type or mAbF19 dose. CONCLUSIONS: The pharmacokinetics of antistromal mAbF19 were well defined in these two studies with different solid tumour types, and were comparable with those of other murine monoclonal antibodies that do not bind to normal tissue antigens or blood cells.

Generation of human high-affinity antibodies specific for the fibroblast activation protein by guided selection.

Four completely human antibody derivatives [single-chain-antibody fragments (scFvs)] with specificity for the general tumor stroma marker fibroblast activation protein (FAP) were isolated by guided selection. Highly diverse IgG, IgM and IgD isotypes comprising heavy-chain variable domain libraries were generated using cDNAs derived from diverse lymphoid organs of a multitude of donors. Three of the human scFvs were converted into bivalent minibodies and expressed in eukaryotic cells for further functional characterization. Binding-competition studies and analysis by fluorescence-activated cell sorting showed high-affinity binding (10--20 nM) for two clones and recognition of the same epitope as the murine guiding antibody. The minibodies were successfully used for immunohistology of a variety of human carcinoma biopsies, revealing specific staining of stromal fibroblasts. Therefore, they should be suitable for in vivo diagnostic and tumor-targeting studies and, because of their completely human origin, be superior to murine or humanized antibody derivatives.

X-ray diffraction and spectroscopic studies of oleic acid-sodium oleate.

The sodium oleate-oleic acid (1:1) complex (NaHOl(2)) is characterized using X-ray diffraction, FT-IR photoacoustic spectroscopy, FT-Raman spectroscopy, and DSC. The special arrangement of hydrogen-bonded pairs of carboxylic acid and carboxylate groups into unique "head-group" is supported by frequency shifts and partial or total disappearance of characteristic vibrations of carboxylic acid dimer and of carboxylate groups. The well-ordered state of hydrocarbon chains is demonstrated by the existence of sharp Raman bands in the C-C stretching region (1000-1150 cm-1) and other conformationally sensitive modes. The FT-Raman results suggest that the transition at about 32 degrees C involves the cooperative melting of methyl- and carboxyl/carboxylate-sided hydrocarbon chains. From the X-ray diffraction data it is clear that this transition is associated with the disintegration of the hydrogen-bonded carboxylate-carboxylic acid complex, followed by the separate formation of oleic acid and sodium oleate. The packing of hydrocarbon chain in the acid-soap complex is different from the parent oleic acid or sodium oleate. The hydrocarbon chains in the NaHOl(2) form more stable packing (O subcell) in comparison to that of oleic acid. A temperature composition phase diagram is presented.

Molecular cloning and characterization of endosialin, a C-type lectin-like cell surface receptor of tumor endothelium.

Endosialin, the antigen identified with monoclonal antibody FB5, is a highly restricted 165-kDa cell surface glycoprotein expressed by tumor blood vessel endothelium in a broad range of human cancers but not detected in blood vessels or other cell types in many normal tissues. Functional analysis of endosialin has been hampered by a lack of information about its molecular structure. In this study, we describe the purification and partial amino acid sequencing of endosialin, leading to the cloning of a full-length cDNA with an open reading frame of 2274 base pairs. The endosialin cDNA encodes a type I membrane protein of 757 amino acids with a predicted molecular mass of 80.9 kDa. The sequence matches with an expressed sequence tag of unknown function in public data bases, named TEM1, which was independently linked to tumor endothelium by serial analysis of gene expression profiling. Bioinformatic evaluation classifies endosialin as a C-type lectin-like protein, composed of a signal leader peptide, five globular extracellular domains (including a C-type lectin domain, one domain with similarity to the Sushi/ccp/scr pattern, and three EGF repeats), followed by a mucin-like region, a transmembrane segment, and a short cytoplasmic tail. Carbohydrate analysis shows that the endosialin core protein carries abundantly sialylated, O-linked oligosaccharides and is sensitive to O-sialoglycoprotein endopeptidase, placing it in the group of sialomucin-like molecules. The N-terminal 360 amino acids of endosialin show homology to thrombomodulin, a receptor involved in regulating blood coagulation, and to complement receptor C1qRp. This structural kinship may indicate a function for endosialin as a tumor endothelial receptor for as yet unknown ligands, a notion now amenable to molecular investigation.

Blue-fluorescent antibodies.

The forte of catalytic antibodies has resided in the control of the ground-state reaction coordinate. A principle and method are now described in which antibodies can direct the outcome of photophysical and photochemical events that take place on excited-state potential energy surfaces. The key component is a chemically reactive optical sensor that provides a direct report of the dynamic interplay between protein and ligand at the active site. To illustrate the concept, we used a trans-stilbene hapten to elicit a panel of monoclonal antibodies that displayed a range of fluorescent spectral behavior when bound to a trans-stilbene substrate. Several antibodies yielded a blue fluorescence indicative of an excited-state complex or "exciplex" between trans-stilbene and the antibody. The antibodies controlled the isomerization coordinate of trans-stilbene and dynamically coupled this manifold with an active-site residue. A step was taken toward the use of antibody-based photochemical sensors for diagnostic and clinical applications.

Phosphorylation-dependent proline isomerization catalyzed by Pin1 is essential for tumor cell survival and entry into mitosis.

Pin1, a member of the parvulin family of peptidyl-prolyl cis-trans isomerases (PPIases) has been implicated in the G2-M transition of the mammalian cell cycle. Pin1 interacts with a series of mitotic phosphoproteins, including Polo-like kinase-1, Cdc25C, and Cdc27, and is thought to act as a phosphorylation-dependent PPIase for these target molecules. Pin1 recognizes phosphorylated serine-proline or threonine-proline peptide-bonds in test substrates up to 1300-fold better than in the respective unphosphorylated peptides. To test directly whether Pin1 regulates the G2-M transition and/or progression through mitosis by catalyzing phosphorylation-dependent prolyl isomerization of essential mitotic targets, we examined the consequences of Pin1 depletion, achieved by (a) overexpression of Pin1 antisense RNA, (b) overexpression of dominant-negative Pin1, and (c) by a known small-molecule Pin1-PPIase inhibitor, juglone. The results of all of the three lines of investigation show that the catalytic activity of Pin1 is essential for tumor cell survival and entry into mitosis.