The synthesis of the nuclear lamina protein lamin A requires the prenylation-dependent processing of its precursor protein, prelamin A. Unlike p2lr", which undergoes similar initial posttranslational modifications, maturation of lamin A results in the proteolytic removal of the prenylated portion of the molecule. We have used an in vitro prenylation system to demonstrate the nature of the prenyl substituent on prelamin A to be a farnesyl group. Further, the in vitro farnesylation of prelamin A requires an intact cysteinealiphatic-aliphatic-other (CAAX) amino acid sequence motif at its carboxyl terminus. The effect of blocking the prenylation of prelamin A on its localization and assembly into the nuclear lamina was investigated by indirect immunofluorescence. Expression of wild-type prelamin A in lovastatin-treated cells showed that nonprenylated prelamin A accumulated as nucleoplasmic particles. Upon addition of mevalonate to lovastatintreated cells, the wild-type lamin A was incorporated into the lamina within 3 hr. Expression of a mutant lamin A in which the carboxyl-terminal 21 amino acids were deleted resulted in a lamin molecule that was directly assembled into the lamina. These results indicate that the carboxyl-terminal peptide of prelamin A blocks its proper assembly into the nuclear lamina and that the prenylation-initiated removal of this peptide can occur in the nucleus.The nuclear lamina is a polymeric protein structure that lines the inner nuclear membrane. In most mammalian cells, it consists of three major proteins, lamins A-C (for review, see ref. 1). Mature lamin A is synthesized from a larger precursor protein (2, 3) and lacks the carboxyl-terminal 18 amino acids predicted by the cDNA sequence (4). Conversion ofthe lamin A precursor, prelamin A, to lamin A is dependent upon the isoprenylation of prelamin A (5). Prelamin A is an example of a class of proteins terminating in the sequence cysteinealiphatic-aliphatic-other (CAAX), which is prenylated at the consensus cysteine (for reviews, see refs. 6 and 7). This motif, shared by such proteins as p215 and the a-type mating factor of Saccharomyces, entrains a series of posttranslational processing steps. These include prenylation at the CAAX cysteine with either a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) isoprenoid followed by proteolytic removal of the A-A-X amino acids and carboxyl methylation of the now terminal cysteine. Evidence has been reported that the B-type lamins also undergo these prenylationdependent processing reactions (8-10). However, specific steps for the processing of prelamin A have not been directly demonstrated. Activities capable of catalyzing the carboxylterminal processing of a-factor and p2l's in vitro have been described (11,12). For p21'S, these activities were localized to the cytosolic and microsomal compartments (12, 13).Prelamin A and the yeast a-factor share an additional reaction subsequent to these carboxyl-terminal processing events. Both are subject to an endoproteolytic cleavage of their resp...

In the present study we have investigated whether pharmacophore models may account for the activity and selectivity of the known cyclooxygenase-2 (COX-2) selective inhibitors of the phenylsulfonyl tricyclic series, i.e., Celecoxib (1) and Rofecoxib (3), and whether transferring this structural information onto the frame of a nonsteroidal antiinflammatory drug (NSAID), known to tightly bind the enzyme active site, may be useful for designing novel COX-2 selective inhibitors. With this aim we have developed a pharmacophore based on the geometric disposition of chemical features in the most favorable conformation of the COX-2 selective inhibitors SC-558 (2; analogue of Celecoxib (1)) and Rofecoxib (3) and the more restrained compounds 4 (DFU) and 5. The pharmacophore model contains a sulfonyl S atom, an aromatic ring (ring plane A) with a fixed position of the normal to the plane, and an additional aromatic ring (ring plane B), both rings forming a dihedral angle of 290 degrees +/- 10 degrees. The final disposition of the pharmacophoric groups parallels the geometry of the ligand SC-558 (2) in the known crystal structure of the COX-2 complex. Moreover, the nonconserved residue 523 is known to be important for COX-2 selective inhibition; thus, the crystallographic information was used to position an excluded volume in the pharmacophore, accounting for the space limits imposed by this nonconserved residue. The geometry of the final five-feature pharmacophore was found to be consistent with the crystal structure of the nonselective NSAID indomethacin (6) in the COX-2 complex. This result was used to design indomethacin analogues 8 and 9 that exhibited consistent structure-activity relationships leading to the potent and selective COX-2 inhibitor 8a. Compound 8a (LM-1685) was selected as a promising candidate for further pharmacological evaluation.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by extensive local invasion and systemic spread. In this study, we employed a three-dimensional organoid model of human pancreatic cancer to characterize the molecular alterations critical for invasion. Time-lapse microscopy was used to observe invasion in organoids from 25 surgically resected human PDAC samples in collagen I. Subsequent lentiviral modification and small-molecule inhibitors were used to investigate the molecular programs underlying invasion in PDAC organoids. When cultured in collagen I, PDAC organoids exhibited two distinct, morphologically defined invasive phenotypes, mesenchymal and collective. Each individual PDAC gave rise to organoids with a predominant phenotype, and PDAC that generated organoids with predominantly mesenchymal invasion showed a worse prognosis. Collective invasion predominated in organoids from cancers with somatic mutations in the driver gene SMAD4 (or its signaling partner TGFBR2). Reexpression of SMAD4 abrogated the col-lective invasion phenotype in SMAD4-mutant PDAC organoids, indicating that SMAD4 loss is required for collective invasion in PDAC organoids. Surprisingly, invasion in passaged SMAD4mutant PDAC organoids required exogenous TGFb, suggesting that invasion in SMAD4-mutant organoids is mediated through noncanonical TGFb signaling. The Rho-like GTPases RAC1 and CDC42 acted as potential mediators of TGFb-stimulated invasion in SMAD4-mutant PDAC organoids, as inhibition of these GTPases suppressed collective invasion in our model. These data suggest that PDAC utilizes different invasion programs depending on SMAD4 status, with collective invasion uniquely present in PDAC with SMAD4 loss.Significance: Organoid models of PDAC highlight the importance of SMAD4 loss in invasion, demonstrating that invasion programs in SMAD4-mutant and SMAD4 wild-type tumors are different in both morphology and molecular mechanism.