Structure-based design, synthesis and in vitro characterization of potent 17beta-hydroxysteroid dehydrogenase type 1 inhibitors based on 2-substitutions of estrone and D-homo-estrone.

In search for specific drugs against steroid-dependent cancers we have developed a novel set of potent inhibitors of steroidogenic human 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD 1). The X-ray structure of 17beta-HSD 1 in complex with estradiol served as basis for the design of the inhibitors. 2-Substituted estrone and D-homo-estrone derivatives were synthesized and tested for 17beta-HSD 1 inhibition. The best 17beta-HSD 1 inhibitor, 2-phenethyl-D-homo-estrone, revealed an IC(50) of 15 nM in vitro. The inhibitory potency of compounds is comparable or better to that of previously described inhibitors. An interaction within the cofactor binding site is not necessary to obtain this high binding affinity for substances developed.

Inhibitory effects of fluorine-substituted estrogens on the activity of 17beta-hydroxysteroid dehydrogenases.

In search for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (h17beta-HSD1) a specific group of steroids with interesting properties including novel compounds was investigated. Several estratriene derivatives with fluorine-substitution in position 17 of the steroidal scaffold were synthesised and tested in vitro towards recombinant h17beta-HSD1, 2, 4, 5 and 7. Moderate, mostly unselective inhibitors of h17beta-HSD1 and h17beta-HSD2 and a selective inhibitor of h17beta-HSD5 were identified. The structure-activity relationship with respect to inhibitory strengths and selectivity of these compounds on five h17beta-HSDs is discussed.

Dimerization and enzymatic activity of fungal 17beta-hydroxysteroid dehydrogenase from the short-chain dehydrogenase/reductase superfamily.

BACKGROUND: 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. SDR proteins usually function as dimers or tetramers and 17beta-HSDcl is also a homodimer under native conditions. RESULTS: We have investigated here which secondary structure elements are involved in the dimerization of 17beta-HSDcl and examined the importance of dimerization for the enzyme activity. Sequence similarity with trihydroxynaphthalene reductase from Magnaporthe grisea indicated that Arg129 and His111 from the alphaE-helices interact with the Asp121, Glu117 and Asp187 residues from the alphaE and alphaF-helices of the neighbouring subunit. The Arg129Asp and His111Leu mutations both rendered 17beta-HSDcl monomeric, while the mutant 17beta-HSDcl-His111Ala was dimeric. Circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. The three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of NADP+, but both monomers showed no enzymatic activity. CONCLUSION: We have shown by site-directed mutagenesis and structure/function analysis that 17beta-HSDcl dimerization involves the alphaE and alphaF helices of both subunits. Neighbouring subunits are connected through hydrophobic interactions, H-bonds and salt bridges involving amino acid residues His111 and Arg129. Since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. A detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor.

A new method to determine the structure of the metal environment in metalloproteins: investigation of the prion protein octapeptide repeat Cu(2+) complex.

Since high-intensity synchrotron radiation is available, "extended X-ray absorption fine structure" spectroscopy (EXAFS) is used for detailed structural analysis of metal ion environments in proteins. However, the information acquired is often insufficient to obtain an unambiguous picture. ENDOR spectroscopy allows the determination of hydrogen positions around a metal ion. However, again the structural information is limited. In the present study, a method is proposed which combines computations with spectroscopic data from EXAFS, EPR, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM). From EXAFS a first picture of the nearest coordination shell is derived which has to be compatible with EPR data. Computations are used to select sterically possible structures, from which in turn structures with correct H and N positions are selected by ENDOR and ESEEM measurements. Finally, EXAFS spectra are re-calculated and compared with the experimental data. This procedure was successfully applied for structure determination of the Cu(2+) complex of the octapeptide repeat of the human prion protein. The structure of this octarepeat complex is rather similar to a pentapeptide complex which was determined by X-ray structure analysis. However, the tryptophan residue has a different orientation: the axial water is on the other side of the Cu.

Micellar environments induce structuring of the N-terminal tail of the prion protein.

In the physiological form, the prion protein is a glycoprotein tethered to the cell surface via a C-terminal glycosylphosphatidylinositol anchor, consisting of a largely alpha-helical globular C-terminal domain and an unstructured N-terminal portion. This unstructured part of the protein contains four successive octapeptide repeats, which were shown to bind up to four Cu(2+) ions in a cooperative manner. To mimic the location of the protein on the cell membrane and to analyze possible structuring effects of the lipid/water interface, the conformational preferences of a single octapeptide repeat and its tetrameric form, as well of the fragment 92-113, proposed as an additional copper binding site, were comparatively analyzed in aqueous and dodecylphosphocholine micellar solution as a membrane mimetic. While for the downstream fragment 92-113 no conformational effects were detectable in the presence of DPC micelles by CD and NMR, both the single octapeptide repeat and, in an even more pronounced manner, its tetrameric form are restricted into well-defined conformations. Because of the repetitive character of the rigid structural subdomain in the tetrarepeat molecule, the spatial arrangement of these identical motifs could not be resolved by NMR analysis. However, the polyvalent nature of the repetitive subunits leads to a remarkably enhanced interaction with the micelles, which is not detectably affected by copper complexation. These results strongly suggest interactions of the cellular form of PrP (PrP(c)) N-terminal tail with the cell membrane surface at least in the octapeptide repeat region with preorganization of these sequence portions for copper complexation. There are sufficient experimental facts known that support a physiological role of copper complexation by the octapeptide repeat region of PrP(c) such as a copper-buffering role of the PrP(c) protein on the extracellular surface.

Synthesis and conformational characterization of peptides related to the neck domain of a fungal kinesin.

The Y362K mutation in the neck domain of conventional kinesin from Neurospora crassa provokes a significant reduction of the rate of movement along microtubules. Since the alpha-helical coiled-coil structure of the neck region is implicated in the mechanism of the processive movement of kinesins, a series of peptides related to the heptad region 338-379 of the wild-type and the variant fungal kinesinswere synthesized as monomers and as N-terminal disulfide dimers, crosslinked to favour self-association into coiled-coil structures entropically. A comparison of the dichroic properties of the peptides and the effects of trifluoroethanol and peptide concentration clearly confirmed the strong implication of the single point mutation in destabilizing the intrinsic propensity of the peptides to fold into the supercoiled conformation. That there is a correlation between the stability of the coiled-coil and rate of movement of the kinesin is confirmed.

Calpastatin exon 1B-derived peptide, a selective inhibitor of calpain: enhancing cell permeability by conjugation with penetratin.

The ubiquitous calpains, mu- and m-calpain, have been implicated in essential physiological processes and various pathologies. Cell-permeable specific inhibitors are important tools to elucidate the roles of calpains in cultivated cells and animal models. The synthetic N-acetylated 27-mer peptide derived from exon B of the inhibitory domain 1 of human calpastatin (CP1B) is unique as a potent and highly selective reversible calpain inhibitor, but is poorly cell-permeant. By addition of N-terminal cysteine residues we have generated a disulfide-conjugated CP1B with the cell-penetrating 16-mer peptide penetratin derived from the third helix of the Antennapedia homeodomain protein. The inhibitory potency and selectivity of CP1B for calpain versus cathepsin B and L, caspase 3 and the proteasome was not affected by the conjugation with penetratin. The conjugate was shown to efficiently penetrate into living LCLC 103H cells, since it prevents ionomycin-induced calpain activation at 200-fold lower concentration than the non-conjugated inhibitor and is able to reduce calpain-triggered apoptosis of these cells. Penetratin-conjugated CP1B seems to be a promising alternative to the widely used cell-permeable peptide aldehydes (e.g. calpain inhibitor 1) which inhibit the lysosomal cathepsins and partially the proteasome as well or even better than the calpains.

A conserved tyrosine in the neck of a fungal kinesin regulates the catalytic motor core.

The neck domain of fungal conventional kinesins displays characteristic properties which are reflected in a specific sequence pattern. The exchange of the strictly conserved Tyr 362, not present in animals, into Lys, Cys or Phe leads to a failure to dimerize. The destabilizing effect is confirmed by a lower coiled-coil propensity of mutant peptides. Whereas the Phe substitution has only a structural effect, the Lys and Cys replacements lead to dramatic kinetic changes. The steady state ATPase is 4- to 7-fold accelerated, which may be due to a faster microtubule-stimulated ADP release rate. These data suggest that an inhibitory effect of the fungal neck domain on the motor core is mediated by direct interaction of the aromatic ring of Tyr 362 with the head, whereas the OH group is essential for dimerization. This is the first demonstration of a direct influence of the kinesin neck region in regulation of the catalytic activity.

Epoxysuccinyl peptide-derived cathepsin B inhibitors: modulating membrane permeability by conjugation with the C-terminal heptapeptide segment of penetratin.

Besides its physiological role in lysosomal protein breakdown, extralysosomal cathepsin B has recently been implicated in apoptotic cell death. Highly specific irreversible cathepsin B inhibitors that are readily cell-permeant should be useful tools to elucidate the effects of cathepsin B in the cytosol. We have covalently functionalised the poorly cell-permeant epoxysuccinyl-based cathepsin B inhibitor [R-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH; R=OMe] with the C-terminal heptapeptide segment of penetratin (R=epsilonAhx-Arg-Arg-Nle-Lys-Trp-Lys-Lys-NH2). The high inhibitory potency and selectivity for cathepsin B versus cathepsin L of the parent compound was not affected by the conjugation with the penetratin heptapeptide. The conjugate was shown to efficiently penetrate into MCF-7 cells as an active inhibitor, thereby circumventing an intracellular activation step that is required by other inhibitors, such as the prodrug-like epoxysuccinyl peptides E64d and CA074Me.