Acetylcholinesterase complexed with bivalent ligands related to huperzine a: experimental evidence for species-dependent protein-ligand complementarity

Acetylcholinesterase (AChE) inhibitors improve the cognitive abilities of Alzheimer patients. (-)-Huperzine A [(-)-HupA], an alkaloid isolated from the club moss, Huperzia serrata, is one such inhibitor, but the search for more potent and selective drugs continues. Recently, alkylene-linked dimers of 5-amino-5,6,7,8-tetrahydroquinolinone (hupyridone, 1a), a fragment of HupA, were shown to serve as more potent inhibitors of AChE than (-)-HupA and monomeric 1a. We soaked two such dimers, (S,S)-(-)-bis(10)-hupyridone [(S,S)-(-)-2a] and (S,S)-(-)-bis(12)-hupyridone [(S,S)-(-)-2b] containing, respectively, 10 and 12 methylenes in the spacer, into trigonal TcAChE crystals, and solved the X-ray structures of the resulting complexes using the difference Fourier technique, both to 2.15 A resolution. The structures revealed one HupA-like 1a unit bound to the "anionic" subsite of the active-site, near the bottom of the active-site gorge, adjacent to Trp84, as seen for the TcAChE/(-)-HupA complex, and the second 1a unit near Trp279 in the "peripheral" anionic site at the top of the gorge, both bivalent molecules thus spanning the active-site gorge. The results confirm that the increased affinity of the dimeric HupA analogues for AChE is conferred by binding to the two "anionic" sites of the enzyme. Inhibition data show that (-)-2a binds to TcAChE approximately 6-7- and > 170-fold more tightly than (-)-2b and (-)-HupA, respectively. In contrast, previous data for rat AChE show that (-)-2b binds approximately 3- and approximately 2-fold more tightly than (-)-2a and (-)-HupA, respectively. Structural comparison of TcAChE with rat AChE, as represented by the closely related mouse AChE structure (1maa.pdb), reveals a narrower gorge for rat AChE, a perpendicular alignment of the Tyr337 ring to the gorge axis, and its conformational rigidity, as a result of hydrogen bonding between its hydroxyl group and that of Tyr341, relative to TcAChE Phe330. These structural differences in the active-site gorge explain the switch in inhibitory potency of (-)-2a and 2b and the larger dimer/(-)-HupA potency ratios observed for TcAChE relative to rat AChE. The results offer new insights into factors affecting protein-ligand complementarity within the gorge and should assist the further development of improved AChE inhibitors.     

Hydroxyl radical probe of the calmodulin-melittin complex interface by electrospray ionization mass spectrometry

The calcium-dependent interaction of calmodulin and melittin is studied through the application of a radical probe approach in which solutions of the protein and peptide and protein alone are subjected to high fluxes of hydroxyl and other oxygen radicals on millisecond timescales. These radicals are generated by an electrical discharge within an electrospray ion source of a mass spectrometer. Condensation of the electrosprayed droplets followed by proteolytic digestion of both calmodulin and melittin has identified residues in both which participate in the interaction and/or are shielded from solvent within the protein complex. Consistent with other theoretical models and available experimental data, the tryptophan residue of melittin at position 19 is shown to be critical to the formation of the complex with the C-terminal domain of peptide enveloped by and protected from oxidation upon binding to the protein. Furthermore, the N-terminal domain (to residue 36) and tyrosine at position 99 in calmodulin are significantly protected from limited oxidation upon the binding of melittin while exposing the phenylalanine residue at position 92 of the flexible loop domain. The N-terminus (through residue 36) of calmodulin is shown to lie in closer proximity to the melittin helix than its C-terminal counterpart (residues 127-148) based upon the protection levels measured at reactive residues within these segments of the protein.     

New polycyclic diamine scaffolds from dimerization of 3-alkyl-1,4-dihydropyridines in acidic medium

[reaction: see text] 3-Alkyl-1,4-dihydropyridines dimerize in acidic medium, at low temperature, to give polycyclic imminium salts derivatives that were reduced to afford new polycyclic diamine scaffolds. The reaction can be extended to enantiopure series starting from R-(+)- or S-(-)-1-phenylethylamine. Long exposure of the polycyclic imminium salt intermediates to air moisture at 20 degrees C resulted in formation of new amide derivatives. This is probably due to the addition of water followed by an intramolecular oxido-reduction process.     

Improved synthesis of functionalized 5,6-dihydro-pyrimido[4,5-b][1,4]oxazepines

Novel synthetic approaches toward 5,6-dihydro-pyrimido[4,5-b][1,4]oxazepines were reported that led to successful introduction of poorly reactive anilines and various 2-hydroxybenzaldehydes to this therapeutically relevant scaffold. More extensive SAR studies on this scaffold hence became possible.     

On radial weighting effects in gaussian expansions of self-consistent field atomic orbitals

Gaussian expansions of the SCF functions for the first row atoms, boron through fluorine, in ground and low-lying electronic states have been generated under a wide range of radial weighting conditions by a full least-squares procedure. Typical results are presented and the quality of the wavefunctions obtained are analyzed in terms of regional electron densities and a variety of expectation values including energies. A novel method for recursive evaluation of repeated integrals of the error function, F _l (,), is adopted and analyzed. These integrals are central quantities in the least-squares procedure employed.

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Zusammenfassung Entwicklungen nach Gaußfunktionen für SCF-Funktionen von Elementen der ersten Reihe (Bor bis Fluor) wurden für den Grundzustand und niedrige angeregte Zustände einer Methode der kleinsten Quadrate berechnet, wobei eine Reihe von radialen Gewichtsfaktoren benutzt wurden. Einige typische Ergebnisse werden mitgeteilt und die Qualität der erhaltenen Wellenfunktion wird mit Hilfe von regionalen Elektronendichten und einer Reihe von Erwartungswerten sowie der Energie geprüft. Eine neue Methode für die rekursive Auswertung der mehrfachen Integrale der Fehlerfunktion F _l (, ) wird angewendet und analysiert. Diese Integrale sind von zentraler Bedeutung in der benutzten Methode der kleinsten Quadrate.

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Résumé Engendrement de représentations gaussiennes des fonctions SCF pour les atomes de la première ligne, du bore au fluor, dans les états électroniques fondamentaux et faiblement excités, avec un large éventail de conditions de pondération radiale obtenues par une procédure de moindres carrés. Des résultats typiques sont présentés et les qualités des fonctions d'onde obtenues sont analysées en fonction des densités électroniques par régions et de différentes valeurs moyennes dont les énergies. Adoption et analyse d'une nouvelle méthode pour l'évaluation récursive d'intégrales répétées de la fonction d'erreur F _l (,). Ces intégrales sont des quantités centrales dans les procédures de moindres carrés utilisées.

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Research performed under the auspices of the U.S. Atomic Energy Commission.

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Summer student program participant, 1971, from Swarthmore College, Swarthmore, Pa.     

Molecular structures of two unexpected metal complexes obtained from additions of coordinated phosphines to acetylenes

The syntheses and structures of two new compounds are reported. The first compound, [Cr(CO)₄]₂[C₄F₂(PPh₂)₄], obtained from Cr(CO)₄(PPh₂H)₂ and CF₃CCF₃ in the presence of one equivalent of BuLi has a structure with the ligand 1,2,3,4-tetrakis(diphenylphosphino)-1,4-difluorobutadiene chelating to two Cr(CO)₄ groups via the 1,4 and 2,3 phosphine groups. A mechanism for the formation of this compound is suggested which involves sequential deprotonation of a phosphine, nucleophilic attack on the fluorocarbon, and fluoride ion elimination. The second compound, CrC₃₆H₂₈P₂O₇, arises from a similar base promoted reaction of Cr(CO)₄-(PPhH₂)₂ and PhCCCOOEt. Here the expected initial product from cyclization of these reactants acts as a nucleophile to attack a second equivalent of the acetylene. The intermediate carbanion from this reaction can undergo a ring closure by displacement of OEt^-, giving the observed product.