The 2.9 Å resolution crystal structure of apo wild-type GroEL was determined for the first time and represents the reference structure, facilitating the study of structural and functional differences observed in GroEL variants. Until now the crystal structure of the mutant Arg13Gly, Ala126Val GroEL was used for this purpose. We show that, due to the mutations as well as to the presence of a crystallographic symmetry, the ring–ring interface was inaccurately described. Analysis of the present structure allowed the definition of structural elements at this interface, essential for understanding the inter-ring allosteric signal transmission. We also show unambiguously that there is no ATP-induced 102° rotation of the apical domain helix I around its helical axis, as previously assumed in the crystal structure of the (GroEL-KMgATP)14 complex, and analyze the apical domain movements. These results enabled us to compare our structure with other GroEL crystal structures already published, allowing us to suggest a new route through which the allosteric signal for negative cooperativity propagates within the molecule. The proposed mechanism, supported by known mutagenesis data, underlines the importance of the switching of salt bridges.

The 3D structure of a complex of the anti-Alzheimer drug galanthamine with Torpedo californica acetylcholinesterase is reported. Galanthamine, a tertiary alkaloid extracted from several species of Amarylidacae, is so far the only drug that shows a dual activity, being both an acetylcholinesterase inhibitor and an allosteric potentiator of the nicotinic response induced by acetylcholine and competitive agonists, The X-ray structure, at 2.5 Angstrom resolution, shows an unexpected orientation of the ligand within the active site, as well as unusual protein-ligand interactions. The inhibitor binds at the base of the active site gorge, interacting with both the acyl-binding pocket and the principal quaternary ammonium-binding site. However, the tertiary amine group of galanthamine does not directly interact with Trp84, A docking study using the program AUTODOCK correctly predicts the orientation of galanthamine in the active site. The docked lowest-energy structure has a root mean square deviation of 0.5 Angstrom with respect to the corresponding crystal structure of the complex, The observed binding mode explains the affinities of a series of structural analogs of galanthamine and provides a rational basis for structure-based drug design of synthetic derivatives with improved pharmacological properties. Proteins 2001;42:182-191. (C) 2000 Wiley-Liss, Inc.

The crystal structure of Torpedo californica (Tc) acetylcholinesterase (AChE) carbamoylated by the physostigmine analogue 8-(cis-2,6-dimethylmorpholino)octylcarbamoyleseroline (MF268) is reported at 2.7 A resolution. In the X-ray structure, the dimethylmorpholinooctylcarbamic moiety of MF268 is covalently bound to the catalytic serine, which is located at the bottom of a long and narrow gorge. The alkyl chain of the inhibitor fills the upper part of the gorge, blocking the entrance of the active site. This prevents eseroline, the leaving group of the carbamoylation process, from exiting through this path. Surprisingly, the relatively bulky eseroline is not found in the crystal structure, thus implying the existence of an alternative route for its clearance. This represents indirect evidence that a "back door" opening may occur and shows that the release of products via a "back door" is a likely alternative for this enzyme. However, its relevance as far as the mechanism of substrate hydrolysis is concerned needs to be established. This study suggests that the use of properly designed acylating inhibitors, which can block the entrance of catalytic sites, may be exploited as a general approach for investigating the existence of "back doors" for the clearance of products.

In the search for potential nucleoside/non-nucleoside mixed type inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, we synthesized a new set of rifamycin S derivatives, containing AZT connected via its hydroxyl at 5′ C, through a spacer, to the third C of rifamycin S. The length of the spacer was eight, nine or 14 atoms. Rifamycin S was also used in its 21, 23-O, O-isopropylidene derivative form, and in one case thymidine replaced AZT. These nucleosidyl rifamycins were weak inhibitors of isolated HIV-1 reverse transcriptase. The inhibitory power was weak most probably because their large molecular volume hindered the inhibition process. With the exception of the thymidine derivative, the AZT derivatives, at concentrations in the range 0.04–0.07 μM, proved non-toxic and inhibited the replication of HIV-1 in C8166 T lymphocytes. This activity appears to be owing to AZT released by the derivatives upon hydrolysis in solution. The present compounds require further development as mixed type reverse transcriptase inhibitors and can be considered non-toxic lipophilic prodrugs of AZT.

Glycosylrifamycins, a new type of semisynthetic rifamycin derivatives, can be easily obtained by reaction of 3-(2-aminoethylthio)rifamycin SV (2) with a glycosyl compound carrying a coupling group, such as isothiocyanate or carboxy. We prepared O-acetylated and free glucopyranosyl and arabinopyranosyl derivatives of rifamycin S and SV (see 3-10). Additionally, derivatives with D-saccharo-1,4-lactone and with shikimic acid were obtained (see 11-15). Glycosylrifamycins show an interesting inhibitory power on Gram-positive bacteria (Table).

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Several samples of oversulfated chondroitin and dermatan were obtained by chemical sulfation and by SAX-HPLC enrichment. The starting products and oversulfated products were tested as potential inhibitors of human leukocyte elastase, an enzyme hypothesized to be involved in the etiology of diseases such as emphysema, atherosclerosis, and rheumatoid arthritis. Chemical oversulfation (SO3H/COOH 1.6-3.2), preferentially occurring at C-6 of galactosamine residues, was found generally to increase the inhibitory power on elastase. Chemically oversulfated galactosaminoglycans thus have potential as therapeutic agents, considering that they produce non-significant effects on the hemocoagulative system. Two naturally oversulfated dermatans sulfate (SO3H/COOH ca. 1.2), mainly oversulfated at C-2 of iduronic acid residues, showed comparatively higher anticoagulant activity (in the HC-II mediated thrombin inhibition test).

Three types of open ansa-chain rifamycin S derivatives have been prepared: derivatives with the ansa-chain open at C(29) and the original dihydrofuranone ring; derivatives with the ansa-chain open at C(29) and a furane ring; derivatives with the ansa-chain at open NH-C(15). Only derivatives of the first type are weak inhibitors of HIV-1 reverse transcriptase (IC50 ca.300 microM) while derivatives of the two other types are inactive. It has been hypothesized that the active derivatives inhibit the viral enzyme interacting through the groups C(14)H3, C(13)H3, and C(1)O at the same site as the well-known inhibitors TIBO and Nevirapine. In particular C(13)H3 must be unhindered and in an appropriate position out of the plane containing the chromophore-rings. The open ansa-chain seems to play the role of a lipophylic substituent.

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The catalytic hydrogenation of rifamycin S(2) over Pd/C, followed by oxidation with K3[Fe(CN)6], generates a pair of 16,17,18,19-tetrahydrorifamycins S(3/4), epimeric at C(16). The use of PtO2 as catalyst leads to the hydrogenation also of the C(28)=C(29) bond giving, after oxidation by K3[Fe(CN)6], a mixture of the epimers (16,R)- and (16S)-16,17,18,19,28,29-hexahydrorifamycins S(5/6). Furthermore, we synthesized the (16R)- and (16S)-3-bromo derivatives 7/8 and (16,R)- and (16S)-3-(piperidin-1-yl) derivatives 9/10. The determination of the X-ray crystal structure of the most abundant epimer 4 of the tetrahydrorifamycins allowed the assignment of the absolute configuration at C(16)of all derivatives. A structure-activity relationship study showed that in general the (16R)-epimers are more potent inhibitors of bacterial RNA polymerase than the (16S)-epimers.

In search of new types of semisynthetic derivatives of the natural antibiotic rifamycin S (1), we synthesized 25-O-deacetyl-27,28-didehydro-27-demethoxy-11-deoxo-11,29-epoxy-28,29-dihydro-21,23-O-isopropylidenerifamycin S(3). Its X-ray crystal structure shows a new type of ansa-chain with an 11,29-epoxy moiety, the loss of the MeO group on C(27), and the shift of the C = C bond from C(28), C(29) to C(27), C(28). These modifications result in a conformational rearrangement of the whole ansa-chain, nonetheless the overall spatial shape of the molecule is still close to that of most rifamycins. As found in other 11-deoxo-11-hydroxyrifamycin-S derivatives, the chromophore rings of 3 give rise to pi-pi-association in the crystal.

29 Rifamycins were tested for inhibition of Reverse Transcriptase (RT) as potential anti HIV drugs. Two purified commercial enzymes from M-MuLV and RAV-2 were used. Anti-RT activity was also measured on a crude lysate of HIV-1. The results show that some derivatives have interesting levels of activity on isolated M-MuL V and RA V-2 RTs, while they are less active on the RT in the crude HIV-1 lysate. The active derivatives include oximes and hydrazones, alkylaminoderivatives, open ansa-chain derivatives and derivatives carrying a modified nucleoside.

The inhibitory activity of a series of 2- and 4-quinolinehydrazones on retroviral reverse transcriptase has been studied on enzymes from M-MuLV, RAV-2, and on a crude lysate of HIV-1, assuming the first two enzymes as potential models of the third. The highest activity is mainly found in lipophilic, water soluble 4-quinolinehydrazones. The inhibitory activity of these compounds decreases in changing from the M-MuLV to the RAV-2, and HIV-1 enzymes, in this order.

C14H20O5, M(r) = 268.31, monoclinic, P2(1), a = 5.0680 (5), b = 19.519 (2), c = 7.3968 (8) angstrom, beta = 106.03 (1)-degrees, V = 703.3 (1) angstrom 3, Z = 2, D(x) = 1.27 g cm-3, lambda(Cu K-alpha) = 1.54184 angstrom, mu = 7.55 cm-1, F(000) = 288, T = 298 K, R = 0.029 for 1286 reflections with F(o) greater-than-or-equal-to 4-sigma(F(o)). The absolute configurations of C4, C5, C6, C7 and C13 are R, S, R, S and S respectively. The C1-C2 and C8-C9 double bonds have E and Z configurations respectively. The determination of the absolute configuration of the title compound also allows that of its trans-bromohydrin derivative to be established.

Lysosomal hyaluronidase is responsible for the degradation of hyaluronan, a component of the extracellular matrix, in degenerative disorders of the joints. It has been hypothesized that the administration of chondroitin sulfate (both a component of the extracellular matrix and a substrate for hyaluronidase) could compete for this enzyme and reduce the degradation process. The present study shows that a mixture of chondroitin 4-sulfate and chondroitin 6-sulfate is a good competitor of hyaluronan for hyaluronidase. The digestion of hyaluronan is reduced in proportion to the amount of competing chondroitin. The competitive ability is dependent on the 4-sulfate, 6-sulfate composition of the chondroitin mixture. Mixtures richer in the 4-sulfate isomer are more effective. The enzymatic reactions have been monitored by HPLC and PAGE.