Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multidomain protein that includes a Rho GTPase Activating Protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5-to 20-fold enhanced activity for RhoA, RhoB, RhoC and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-δ1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC.

The potency of nucleotide antagonists at P2Y1 receptors was enhanced by replacing the ribose moiety with a constrained carbocyclic ring (Nandanan, et al. J. Med. Chem. 2000, 43, 829-842). We have now synthesized ring-constrained methanocarba analogues (in which a fused cyclopropane moiety constrains the pseudosugar ring) of adenine and uracil nucleotides, the endogenous activators of P2Y receptors. Methanocarba-adenosine 5'-triphosphate (ATP) was fixed in either a Northern (N) or a Southern (S) conformation, as defined in the pseudorotational cycle. (N)-Methanocarba-uridine was prepared from the 1-amino-pseudosugar ring by treatment with beta-ethoxyacryloyl cyanate and cyclization to form the uracil ring. Phosphorylation was carried out at the 5'-hydroxyl group through a multistep process: Reaction with phosphoramidite followed by oxidation provided the 5'-monophosphates, which then were treated with 1,1'-carbonyldiimidazole for condensation with additional phosphate groups. The ability of the analogues to stimulate phospholipase C through activation of turkey P2Y1 or human P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors stably expressed in astrocytoma cells was measured. At recombinant human P2Y1 and P2Y2 receptors, (N)-methanocarba-ATP was 138- and 41-fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)-methanocarba-ATP activated P2Y11 receptors with a potency similar to ATP. (N)-Methanocarba-uridine 5'-triphosphate (UTP) was equipotent to UTP as an agonist at human P2Y2 receptors and also activated P2Y4 receptors with an EC(50) of 85 nM. (N)-Methanocarba-uridine 5'-diphosphate (UDP) was inactive at the hP2Y6 receptor. The vascular effects of (N)-methanocarba-UTP and (N)-methanocarba-UDP were studied in a model of the rat mesenteric artery. The triphosphate was more potent than UTP in inducing a dilatory P2Y4 response (pEC(50) = 6.1 +/- 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y6 receptor-mediated contractile response. Our results suggest that new nucleotide agonists may be designed on the basis of the (N) conformation that favors selectivity for P2Y1, P2Y2, P2Y4, and P2Y11 receptors.

Combining molecular dynamics (MD) in a hydrated phospholipids (DOPC) bilayer, Monte Carlo search, and synthesis of locked nucleotide analogues we discovered that the Southern conformation of the ribose is preferred for ligand recognition by the P2Y 6 receptor. 2′-Deoxy-(S)-methanocarbaUDP was found to be a full agonist of the receptor and displayed a 10-fold higher potency than the corresponding flexible 2′-deoxyUDP. MD results also suggested a conformational change of the second extracellular loop consequent to agonist binding.P2Y receptors are a family of class A G protein-coupled receptors (GPCRs), variously activated by extracellular purine and pyrimidine nucleotides. 1,2 The family is composed of two distinct subgroups, which differ in overall sequence similarity, mechanism of ligand recognition, and coupling to second messengers. The P2Y 1 -like subgroup encompasses the P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 receptors, which couple mainly to the stimulation of phospholipase C (PLC) via G q . On the other hand, the P2Y 12 -like subgroup encompasses the P2Y 12 , P2Y 13 , and P2Y 14 receptors, which couple mainly to the inhibition of adenylyl cyclase (AC) via G i . The P2Y 6 receptor is the only P2Y receptor whose natural agonist is uridine-5′-diphosphate (UDP, 1). It is the least studied subtype of the P2Y 1 -like subgroup, due to the lack of potent and selective ligands. It has recently been shown that the activation of the P2Y 6 receptor induces Ca 2+ and Cl − secretion in mouse trachea. Hence, P2Y 6 agonists may be useful to stimulate electrolyte transport in the airways of cystic fibrosis patients. 3It is known that the five-membered ring of the ribose moiety of nucleosides and nucleotides can adopt a range of conformations, which can be exhaustively described by two parameters indicative of puckering: P (phase angle of pseudorotation, schematically represented as a pseudorotational cycle) and θ (puckering amplitude). 4,5 Using conformationally-locked nucleotide analogues, we deduced a preference for the Northern (N) hemisphere of the pseudorotational cycle (2′-exo, 3′-endo) for the P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 11 receptors. 4,6, 7,8 However, the UDP analogue 2 locked in the (N) conformation by a (N)-methanocarba Figure 1). 7 Hence, prior to this work, the P2Y 6 receptor has been the most elusive subtype of the P2Y 1 -like receptors and the conformation of the ribose moiety of UDP required for the recognition of the ligand was not established.Here we report the discovery of the biologically-active conformation of the sugar moiety of UDP, which we accomplished by means of an interdisciplinary approach combining advanced molecular modeling techniques with the synthesis of conformationally locked nucleotide analogs. This finding represents a breakthrough toward the understanding of the biology of the P2Y 6 receptor, as it may be the key to the design of the long needed pharmacological tools.To obtain a refined model of the P2Y 6 receptor that could be exploited to identify the conformation ...

With the long-term goal of developing receptor subtype-selective high affinity agonists for the uracil nucleotide-activated P2Y receptors we have carried out a series of structure activity and molecular modeling studies of the human P2Y2 and P2Y4 receptors. UTP analogues with substitutions in the 2'-position of the ribose moiety retained capacity to activate both P2Y2 and P2Y4 receptors. Certain of these analogues were equieffective for activation of both receptors whereas 2'-amino-2'-deoxy-UTP exhibited higher potency for the P2Y2 receptor and 2'-azido-UTP exhibited higher potency for the P2Y4 receptor. 4-Thio substitution of the uracil base resulted in a UTP analogue with increased potency relative to UTP for activation of both the P2Y2 and P2Y4 receptors. In contrast, 2-thio substitution and halo- or alkyl substitution in the 5-position of the uracil base resulted in molecules that were 3-30-fold more potent at the P2Y2 receptor than P2Y4 receptor. 6-Aza-UTP was a P2Y2 receptor agonist that exhibited no activity at the P2Y4 receptor. Stereoisomers of UTPalphaS and 2'-deoxy-UTPalphaS were more potent at the P2Y2 than P2Y4 receptor, and the R-configuration was favored at both receptors. Molecular docking studies revealed that the binding mode of UTP is similar for both the P2Y2 and P2Y4 receptor binding pockets with the most prominent dissimilarities of the two receptors located in the second transmembrane domain (V90 in the P2Y2 receptor and I92 in the P2Y4 receptor) and the second extracellular loop (T182 in the P2Y2 receptor and L184 in the P2Y4 receptor). In summary, this work reveals substitutions in UTP that differentially affect agonist activity at P2Y2 versus P2Y4 receptors and in combination with molecular modeling studies should lead to chemical synthesis of new receptor subtype-selective drugs.

Preference for the northern (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of P2Y(1) receptors was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute (Nandanan et al. J. Med. Chem. 2000, 43, 829-842). We have now combined the ring-constrained (N)-methanocarba modification with other functionalities at the 2-position of the adenine moiety. A new synthetic route to this series of bisphosphate derivatives was introduced, consisting of phosphorylation of the pseudoribose moiety prior to coupling with the adenine base. The activity of the newly synthesized analogues was determined by measuring antagonism of 2-methylthio-ADP-stimulated phospholipase C (PLC) activity in 1321N1 human astrocytoma cells expressing the recombinant human P2Y(1) receptor and by using the radiolabeled antagonist [(3)H]2-chloro-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate 5 in a newly developed binding assay in Sf9 cell membranes. Within the series of 2-halo analogues, the most potent molecule at the hP2Y(1) receptor was an (N)-methanocarba N(6)-methyl-2-iodo analogue 12, which displayed a K(i) value in competition for binding of [(3)H]5 of 0.79 nM and a K(B) value of 1.74 nM for inhibition of PLC. Thus, 12 is the most potent antagonist selective for the P2Y(1) receptor yet reported. The 2-iodo group was substituted with trimethyltin, thus providing a parallel synthetic route for the introduction of an iodo group in this high-affinity antagonist. The (N)-methanocarba-2-methylthio, 2-methylseleno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues bound less well, exhibiting micromolar affinity at P2Y(1) receptors. An enzymatic method of synthesis of the 3',5'-bisphosphate from the corresponding 3'-monophosphate, suitable for the preparation of a radiophosphorylated analogue, was explored.

The structure-activity relationships and molecular modeling of the uracil nucleotide activated P2Y6 receptor have been studied. Uridine 5'-diphosphate (UDP) analogues bearing substitutions of the ribose moiety, the uracil ring, and the diphosphate group were synthesized and assayed for activity at the human P2Y6 receptor. The uracil ring was modified at the 4 position, with the synthesis of 4-substituted-thiouridine 5'-diphosphate analogues, as well as at positions 2, 3, and 5. The effect of modifications at the level of the phosphate chain was studied by preparing a cyclic 3',5'-diphosphate analogue, a 3'-diphosphate analogue, and several dinucleotide diphosphates. 5-Iodo-UDP 32 (EC50 = 0.15 microM) was equipotent to UDP, while substitutions of the 2'-hydroxyl (amino, azido) greatly reduce potency. The 2- and 4-thio analogues, 20 and 21, respectively, were also relatively potent in comparison to UDP. However, most other modifications greatly reduced potency. Molecular modeling indicates that the beta-phosphate of 5'-UDP and analogues is essential for the establishment of electrostatic interactions with two of the three conserved cationic residues of the receptor. Among 4-thioether derivatives, a 4-ethylthio analogue 23 displayed an EC50 of 0.28 microM, indicative of favorable interactions predicted for a small 4-alkylthio moiety with the aromatic ring of Y33 in TM1. The activity of analogue 19 in which the ribose was substituted with a 2-oxabicyclohexane ring in a rigid (S)-conformation (P = 126 degrees , 1'-exo) was consistent with molecular modeling. These results provide a better understanding of molecular recognition at the P2Y6 receptor and will be helpful in designing selective and potent P2Y6 receptor ligands.

Importance: Although several reports are available on the use of conventional and cultured limbal epithelium using various substrates in the treatment of limbal stem cell deficiency (LSCD), the patient populations studied have been largely adults. Thus, to our knowledge, the outcomes of this procedure exclusively in a pediatric population have not been reported previously. Objective: To report the outcomes of autologous ex vivo cultivated limbal epithelial transplantation (CLET) in pediatric patients with LSCD after ocular burns.

Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5′-triphosphate agonists at P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 11 receptors, but not P2Y 6 receptors, was established using a ring-constrained methanocarba (a 3.1.0-bicyclohexane) ring as a ribose substitute (Kim et al. J. Med. Chem. 2002, 45, 208-218.). We have now combined the ringconstrained (N)-methanocarba modification of adenine nucleotides with other functionalities known to enhance potency at P2 receptors. The potency of the newly synthesized analogues was determined in the stimulation of phospholipase C through activation of turkey erythrocyte P2Y 1 or human P2Y 1 and P2Y 2 receptors stably expressed in astrocytoma cells. An (N)-methanocarba-2-methylthio-ADP analogue displayed an EC 50 at the hP2Y 1 receptor of 0.40 nM and was 55-fold more potent than the corresponding triphosphate and 16-fold more potent than the riboside 5′-diphosphate. 2-Cl-(N)-methanocarba-ATP and its N 6 -Me analogue were also highly selective, full agonists at P2Y 1 receptors. The (N)-methanocarba-2-methylthio and 2-chloromonophosphate analogues were full agonists exhibiting micromolar potency at P2Y 1 receptors, while the corresponding ribosides were inactive. Although β,γ-methylene-ATP was inactive at P2Y receptors, β,γ-methylene-(N)-methanocarba-ATP was a potent hP2Y 1 receptor agonist with an EC 50 of 160 nM and was selective versus hP2Y 2 and hP2Y 4 receptors. The rates of hydrolysis of Northern (N) and Southern (S) methanocarba analogues of AMP by rat 5′-ectonucleotidase were negligible. The rates of hydrolysis of the corresponding triphosphates by recombinant rat NTPDase1 and 2 were studied. Both isomers were hydrolyzed by NTPDase 1 at about half the rate of ATP hydrolysis. The (N) isomer was hardly hydrolyzed by NTPDase 2, while the (S) isomer was hydrolyzed at one-third of the rate of ATP hydrolysis. This suggests that new, more stable and