Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization.

Synthesis and bioactivity of 2,4-diacyl analogues of paclitaxel.

The 2,4-diacyl paclitaxel analogues 8a-8r were prepared from paclitaxel by acylation of 4-deacetyl-2-debenzoylpaclitaxel 1,2-carbonate (3) followed either by hydrolysis of the carbonate and acylation or by direct treatment of the carbonate with an aryllithium. Some of the resulting derivatives showed significantly improved tubulin assembly activity and cytotoxicity as compared with paclitaxel; in some cases this improvement was especially significant for paclitaxel-resistant cell lines.

Synthesis and biological evaluation of 2-acyl analogues of paclitaxel (Taxol).

The anticancer drug paclitaxel (Taxol) has been converted to a large number of 2-debenzoyl-2-aroyl derivatives by three different methods. The bioactivities of the resulting analogues were determined in both tubulin polymerization and cytotoxicity assays, and several analogues with enhanced activity as compared with paclitaxel were discovered. Correlation of cytotoxicity in three cell lines with tubulin polymerization activity showed reasonable agreement. Among the cell lines examined, the closest correlation with antitubulin activity was observed with a human ovarian carcinoma cell line.

Distances between the paclitaxel, colchicine, and exchangeable GTP binding sites on tubulin.

Distances between the paclitaxel, colchicine, and exchangeable GTP binding sites on tubulin polymers have been probed using fluorescence spectroscopy. Techniques for measuring fluorescence resonance energy transfer (FRET) between fluorescent or chromophoric ligands for each binding site were employed. 2-Debenzoyl-2-(m-aminobenzoyl)paclitaxel (2-AB-PT) was the fluorophore ligand for the paclitaxel binding site; thiocolchicine, allocolchicine, and MDL 27048 were probes for the colchicine site, and 2'(or 3')-O-(trinitrophenyl)guanosine 5'-triphosphate (TNP-GTP) was the fluorophore ligand for the exchangeable GTP site. The distance between the colchicine and paclitaxel binding sites was determined with two different acceptor ligands in the colchicine site. An average distance distribution of 17 A was found in both cases. Energy transfer between 2-AB-PT bound to the paclitaxel site and TNP-GTP (acceptor) bound to the exchangeable GTP site was observed in the polymer. The average distance distribution between the fluorophores was 16.0 A, but the half-width of the distribution was large (17.9 A), which indicates that energy transfer between more than one donor-acceptor pair occurred in the system. One interpretation of this result is that 2-AB-PT serves as an energy transfer donor for two GTP sites, one contained on the same subunit and one on an adjacent protofilament. No FRET was observed between ligands bound to the colchicine and exchangeable GTP sites, indicating that the result of colchicine binding on the GTP region of beta-tubulin is a long range, allosteric effect. The results from these experiments are interpreted in terms of known structural features of microtubules.

Identification of the structural region of taxol that may be responsible for cytokine gene induction and cytotoxicity in human ovarian cancer cells.

PURPOSE: Interleukin-8 (IL-8) is a pleiotropic chemokine with both chemoattractant and angiogenic properties. In addition to its cytotoxic effects on ovarian cancer cells, taxol can transcriptionally activate genes such as IL-8 that may play a role in tumorigenesis. Utilizing IL-8 as a prototypic marker of tumor-derived modulators of growth, we undertook a systematic study of taxol and 11 structurally modified taxol analogs to identify the region of the taxane skeleton responsible for IL-8 gene induction. METHODS: The human ovarian cancer cell line OVCA-420 was exposed to taxol or taxol analogs. IL-8 gene induction was assessed by Northern blot analysis after 6 h and cytotoxicity after 72 h. RESULTS: Changes in the southern hemisphere (C-1 to C-4) of the taxane skeleton had greater effects on IL-8 induction than changes in the northern hemisphere (C-7 to C-11). Some of the taxol analogs modified at positions C-1 and/or C-2 with increased hydrophobicity induced IL-8 expression more than threefold over that induced by taxol or taxotere and more than 20-fold over control cells. Cells that failed to induce IL-8 gene expression in response to taxol were only marginally responsive to the analogs unless first primed with IL-1beta. Modifications to the northern hemisphere did not alter taxol's effect on IL-8 expression in human cells, but did influence TNFalpha expression in murine macrophage cells, suggesting species and/or gene specificity. We found a direct correlation between IL-8 induction and cytotoxicity, in that analogs that dramatically upregulated IL-8 expression proved to be the most cytotoxic, inhibiting cell survival by > 90%. CONCLUSION: Taken together our results demonstrate that changes in the southern hemisphere of the taxane skeleton influence both the gene induction and cytotoxic potential of taxol in human ovarian cancer cells.

Synthesis and biological activity of A-nor-paclitaxel analogues.

A number of paclitaxel analogues with a 5-membered A-ring (A-nor-paclitaxels, or (15-->1)-abeo-paclitaxels) have been prepared in order to determine whether analogues of this class might have improved bioactivity as compared with paclitaxel. Most of the compounds synthesized were less active than paclitaxel, but one analogue was equivalent to paclitaxel in a tubulin-assembly assay, and another analogue was more cytotoxic than paclitaxel in two different cell lines of the NCI screen.

Interaction of a fluorescent derivative of paclitaxel (Taxol) with microtubules and tubulin-colchicine.

A fluorescent derivative of paclitaxel, 2-debenzoyl-2-(m-aminobenzoyl)paclitaxel (2-AB-PT), has been prepared. 2-AB-PT induces microtubule assembly in vitro, but is about 3-fold less potent than paclitaxel itself. The absorption and emission characteristics of 2-AB-PT were analyzed as a function of solvent. It was found that both spectra were perturbed by specific solvent effects when the solvent contained a hydrogen bond donor. The absorption and fluorescence spectra of 2-AB-PT bound to microtubules could not be mimicked by a single solvent, but the absorption and emission maxima of the tubulin-bound species could be duplicated by a solvent mixture of DMSO and water. These results indicate that the fluorophore binding site on the microtubule is in an environment of intermediate polarity that is accessible to a hydrogen bond donor in the vicinity of the m-amino group. In addition, tubulin fluorescence is quenched in the 2-AB-PT/microtubule complex, and energy transfer from tubulin to 2-AB-PT is apparent. These results indicate that substituents on the C-2 position of paclitaxel associate with tubulin when bound to the microtubule. 2-AB-PT binding to microtubules was quantitatively analyzed by fluorescence titrations. Two classes of binding sites for 2-AB-PT on microtubules were found. The high affinity site has an apparent association constant (K1app) of 2.0 (+/- 0.9) x 10(7) M-1 and an apparent binding stoichiometry (n1app) of 0.8 (+/- 0.1) sites/tubulin dimer in the microtubule. The apparent association constant for the lower affinity site is about 100-fold less than that of the higher affinity site (K2app = 2.1 (+/- 0.7) x 10(5) M-1), and the stoichiometry of the lower affinity site or class of sites (n2app) was found to be 1.3 +/- 0.1. Paclitaxel blocked 2-AB-PT binding to the high affinity site. No binding of 2-AB-PT to unassembled tubulin was observed, but the emission spectrum of 2-AB-PT in the presence of the tubulin-colchicine complex resembled the emission spectrum of the ligand bound to microtubules. It was previously shown that paclitaxel can induce GTPase activity in the tubulin-colchicine complex, indicating that paclitaxel can bind to unassembled tubulin in its complex with colchicine [Carlier, M.-F., & Pantaloni, D. (1983) Biochemistry 22, 4814-4822]. Rigorous characterization of the aggregation state of the protein under these conditions demonstrates that 2-AB-PT is also capable of binding to the tubulin-colchicine complex.