Miguel Feliz

Molecular recycling within amyloid fibrils

Amyloid fibrils are thread-like protein aggregates with a core region formed from repetitive arrays of β-sheets oriented parallel to the fibril axis. Such structures were first recognized in clinical disorders, but more recently have also been linked to a variety of non-pathogenic phenomena ranging from the transfer of genetic information to synaptic changes associated with memory. The observation that many proteins can convert into similar structures in vitro has suggested that this ability is a generic feature of polypeptide chains. Here we have probed the nature of the amyloid structure by monitoring hydrogen/deuterium exchange in fibrils formed from an SH3 domain using a combination of nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. The results reveal that under the conditions used in this study, exchange is dominated by a mechanism of dissociation and re-association that results in the recycling of molecules within the fibril population. This insight into the dynamic nature of amyloid fibrils, and the ability to determine the parameters that define this behaviour, have important implications for the design of therapeutic strategies directed against amyloid disease.

Isonicotinic Acid Hydrazide Conversion to Isonicotinyl-NAD by Catalase-peroxidases

Activation of the pro-drug isoniazid (INH) as an anti-tubercular drug in Mycobacterium tuberculosis involves its conversion to isonicotinyl-NAD, a reaction that requires the catalase-peroxidase KatG. This report shows that the reaction proceeds in the absence of KatG at a slow rate in a mixture of INH, NAD+, Mn2+, and O2, and that the inclusion of KatG increases the rate by >7 times. Superoxide, generated by either Mn2+- or KatG-catalyzed reduction of O2, is an essential intermediate in the reaction. Elimination of the peroxidatic process by mutation slows the rate of reaction by 60% revealing that the peroxidatic process enhances, but is not essential for isonicotinyl-NAD formation. The isonicotinyl-NAD•+ radical is identified as a reaction intermediate, and its reduction by superoxide is proposed. Binding sites for INH and its co-substrate, NAD+, are identified for the first time in crystal complexes of Burkholderia pseudomallei catalase-peroxidase with INH and NAD+ grown by co-crystallization. The best defined INH binding sites were identified, one in each subunit, on the opposite side of the protein from the entrance to the heme cavity in a funnel-shaped channel. The NAD+ binding site is ∼20 Å from the entrance to the heme cavity and involves interactions primarily with the AMP portion of the molecule in agreement with the NMR saturation transfer difference results.

Alkaloids from Narcissus confusus

Abstract We report the isolation of haemanthamine, galanthamine, pretazettine and the new alkaloid N-formylgalanthamine from Narcissus confusus plants. Structural determination was carried out by spectroscopic analyses and application of two-dimensional NMR spectroscopy.

Two alkaloids from Narcissus requienii

The aerial part of Narcissus requienii (Amaryllidaceae) was found to contain pseudolycorine as the major alkaloid, and two new phenolic bases: 2-O-acetylpseudolycorine and 1-O-acetylpseudolycorine. The present communication reports the complete assignments of the NMR spectra of pseudolycorine, and the structure elucidation of the other comnounds by means of 2D 1H-1H and 1H-13C NMR chemical shift correlation experiments.

Synthetic ligands able to interact with the P53 tetramerization domain. Towards understanding a protein surface recognition event

The applied interaction of synthetic molecules with defined regions of protein surfaces is an emerging strategy for the modulation of protein activity and/or stability. In spite of recent advances, the design of these molecules is not trivial. Among the most challenging aspects in designing these compounds is that they must compete with water molecules for interaction with polar patches of protein surfaces. Herein is reported the preparation of an arginine‐rich peptide that interacts in aqueous solution with a very hydrophilic patch at the surface of the tetramerization domain of the tumor suppressor protein p53. The interaction has been studied by several complementary techniques. By using this peptide as a template, a library of peptides has been prepared and evaluated in order to examine the different factors that contribute to the recognition event. The conclusions extracted from this work could be useful for the design of ligands directed at highly hydrophilic protein surface patches.

Synthetic applications of 2-cyanopiperidines. Model studies in the synthesis of bridged indole alkaloids

Abstract Acid cyclization of 4-(indol-1- and -3-ylmethyl)-2-cyanopiperidines 5 led to the bridged tetracyclic indole- systems 1 and 2. The required 2-cyanopiperidines 5 were prepared by catalytic hydrogenation of 2-cyano-1,2,3,6-tetrahydropyridines 4 and by DIBAL reduction of lactams 7 followed by cyanide ion addition to the intermediate enamine.

Total synthesis and stereochemical reassignment of the indole alkaloid vinoxine

Abstract The first total synthesis of the indole alkaloid vinoxine and the reassignment of the relative configuration at carbon-16 in this alkaloid is reported.

13C dynamic NMR studies on restricted rotation about C-N bond in 2-aryl-1-formyl-4-piperidones

The synthesis of 1-formyl-2-(3-indolyl)-4-piperidone, trans-1-formyl-2-(3-indolyl)-3-methyl-4-piperidone, and their 4,4-ethyleneacetals is described. The introduction of a formyl group on the piperidone nitrogen induces a change in the piperidine ring conformation such that the 2-indolyl group is axial. However, in the 1-formyl-2-indolyl-4-piperidone 4,4-ethyleneacetals the indolyl group adopts an equatorial disposition due to a severe steric syn-diaxial interaction with the C-4 substituent. 13 C-Dnmr is used to investigate the amide rotational barriers in a series of 2-aryl-1-formyl-4-piperidones

Computer-Aided Design of Fragment Mixtures for NMR-Based Screening

Fragment-based drug discovery is widely applied both in industrial and in academic screening programs. Several screening techniques rely on NMR to detect binding of a fragment to a target. NMR-based methods are among the most sensitive techniques and have the further advantage of yielding a low rate of false positives and negatives. However, NMR is intrinsically slower than other screening techniques; thus, to increase throughput in NMR-based screening, researchers often assay mixtures of fragments, rather than single fragments. Herein we present a fast and straightforward computer-aided method to design mixtures of fragments taken from a library that have minimized NMR signal overlap. This approach enables direct identification of one or several active fragments without the need for deconvolution. Our approach entails encoding of NMR spectra into a computer-readable format that we call a fingerprint, and minimizing the global signal overlap through a Monte Carlo algorithm. The scoring function used favors a homogenous distribution of the global signal overlap. The method does not require additional experimental work: the only data required are NMR spectra, which are generally recorded for each compound as a quality control measure before its insertion into the library.

Malaysiatin, the first cyclic heptapeptide from a marine sponge

Abstract Malaysiatin ( 1 ), the first homodetic cycloheptapeptide from a marine source, has been isolated from the cytotoxic and antimicrobial extracts of the marine sponge Pseudaxinyssa sp. , collected in Borneo. The unusual structure of malaysiatin, which contains homodipeptide and homotripeptide fragments, was elucidated by extensive spectroscopic analysis that suggests an all-L stereochemistry.