Ildikó Nagy

Structure–activity relationship of antiparasitic and cytotoxic indoloquinoline alkaloids, and their tricyclic and bicyclic analogues

Based on the indoloquinoline alkaloids cryptolepine (1), neocryptolepine (2), isocryptolepine (3) and isoneocryptolepine (4), used as lead compounds for new antimalarial agents, a series of tricyclic and bicyclic analogues, including carbolines, azaindoles, pyrroloquinolines and pyrroloisoquinolines was synthesized and biologically evaluated. None of the bicyclic compounds was significantly active against the chloroquine-resistant strain Plasmodium falciparum K1, in contrast to the tricyclic derivatives. The tricyclic compound 2-methyl-2H-pyrido[3,4-b]indole (9), or 2-methyl-beta-carboline, showed the best in vitro activity, with an IC(50) value of 0.45 microM against P. falciparum K1, without apparent cytotoxicity against L6 cells (SI>1000). However, this compound was not active in the Plasmodium berghei mouse model. Structure-activity relationships are discussed and compared with related naturally occurring compounds.

A novel COCH mutation, V104del, impairs folding of the LCCL domain of cochlin and causes progressive hearing loss

Recently the causative gene of autosomal dominant sensorineural nonsyndromic late onset hearing loss (DFNA9) has been identified as the COCH gene, which lies in the DFNA9 region of human chromosome 14 (gene map locus 14q12–q13). Molecular analysis of cases of DFNA9 have identified several families with five different mutations in this gene.1–5 The cochlin protein encoded by COCH is an extracellular matrix protein that contains an LCCL domain and two von Willebrand type A domains.1 Interestingly, all mutations causing DFNA9 type deafness disorder affect the LCCL domain of cochlin. In view of the central role of this domain in the DFNA9 disorder, we initiated studies on it to explore the molecular basis of the disease.6 We found that most DFNA9 mutations affected conserved structural elements of the LCCL fold and disrupted the proper folding of this domain; recombinant mutant LCCL domains expressed in Escherichia coli failed to adopt the wild type fold and instead formed insoluble aggregates.7 It is noteworthy that insoluble deposits are detected in temporal bone sections of individuals affected by DFNA9.8 These results support the notion that the DFNA9 mutations may act through a gain of function mechanism; it is the presence of the abnormal protein that causes the disease. According to this view, accumulation of deposits in vestibular and cochlear nerve channels leads to strangulation and progressive degeneration of the dendrites, and loss of cochlear and vestibular neurones. Our present investigation was aimed at identifying novel mutations affecting the LCCL domain of cochlin in order to gain more insight into the pathomechanism of DFNA9. Hungarian patients selected according to the diagnostic criteria of DFNA9 were screened for the presence of mutations affecting the LCCL domain. These studies identified one person heterozygous for a novel mutation in the coding region of …

Expression and characterization of the olfactomedin domain of human myocilin.

The olfactomedin-domain has been first identified in olfactomedin, an extracellular matrix protein of the olfactory neuroepithelium. Members of this extracellular domain-family have since been shown to be present in several metazoan proteins, such as latrophilins, myocilins, and noelins, but their biological function is unknown. The olfactomedin-domain of myocilin is of considerable interest, since mutations affecting this domain are associated with primary open angle glaucoma. In order to define structural features of this domain-type we have expressed the olfactomedin-domain of human myocilin in Pichia pastoris. The olfactomedin-domain contains a single disulphide-bond connecting Cys-245 and Cys-433 residues; secondary structure predictions and circular dichroism studies indicate that it consists primarily of beta-strands. It is noteworthy that the majority of mutations associated with severe forms of glaucoma affect residues that reside in conserved secondary structural elements of the olfactomedin-domain or are otherwise critical for the integrity of this protein-fold.

Carrier design: Synthesis and conformational studies of poly (L-lysine) based branched polypeptides with hydroxyl groups in the side chains

In the present study the development of a new series of branched polypeptides that contain hydroxyl groups in side chains is reported. Serine or threonine were attached by 1-hydroxy-benzotriazole catalyzed active ester method to the N-terminals of oligo (DL-alanine) chains grafted to a polylysine backbone resulted in poly[Lys-(Ser1-DL-Alam)] (SAK) and poly-[Lys-(Thri-DL-Alam)] (TAK). Ser was coupled also directly to the η-amino groups of polylysine followed by polymerization of N-carboxy-DL-alanine anhydride resulting oligo (DL-Ala) chain terminals. In this way a reverse sequence was built up in the side chain corresponding to the poly[Lys-(DL-Alam-Seri)] (ASK). The number of hydroxyl groups in the polymer was increased by the synthesis of a branched polypeptide with oligo (DL-serine) branches instead of oligo (DL-alanine) ones—poly[Lys-(DL-Serm)] (SK). Classification of solution conformations of branched polypeptides was carried out by CD spectroscopy performed in water solution of various pH values and ionic strengths. Incorporation of single Ser residues in poly[Lys-(Xi)]-type polypeptides markedly promotes the formation of ordered structure without resulting precipitation even in high salt concentration. The presence of branches with multiple DL-Ser residues resulted in a slightly decreased ability of the polypeptide backbone to adopt an ordered conformation. Comparison of the CD properties of the SAK-ASK pair demonstrates that these compounds are similar, showing an increased tendency to form an ordered spatial arrangement in solution at elevated pH or ionic strength; however, differences in their CD spectra suggest that SAK has higher capability to form regular conformation under comparable conditions. The replacement of Ser by the Thr residue in poly[Lys-(Xi-DL-Alam)] induced a conformational transition and TAK exhibited a more helical structure. These results might indicate that not only hydrophobic or ionic attraction, but also H-bond interaction, can play a role in the formation and/or stabilization of ordered conformation of branched polypeptides. Findings with the hydroxyl group containing polymers reported in this paper can also explain their prolonged shelf stability and high water solubility.

INTERACTION OF BRANCHED CHAIN POLYMERIC POLYPEPTIDES WITH PHOSPHOLIPID MODEL MEMBRANES

The surface properties at the air/water interface and the interaction of branched chain polymeric polypeptides with a general formula poly[Lys-(DL-Alam-X1)], where X = Π (AK), Ser (SAK), or Glu (EAK), with phospholipids were investigated. Polylysine derivatives with polycationic (SAK, AK) or amphoteric (EAK) were capable to spread and form stable monomolecular layers. The stability of monolayers at the air/water interface was dependent on the side-chain terminal amino acid residue of polymers and can be described by SAK < AK < EAK order. The area per amino acid residue values calculated from compression isotherms were in the same range as compared to those of linear poly-α-amino acids and proteins. Moreover, these polymers interact with phospholipid monomolecular layers composed of dipalmitoyl phosphatidyl choline (DPPC) or DPPC/PG (PG: phosphatidyl glycerol; 95/5, mol/mol). Data obtained from compression isotherms of phospholipids spread on aqueous polymer solutions at different initial surface pressure indicated that insertion into lipid monolayers for SAK or AK is more pronounced than for EAK. The interaction between branched polypeptides and phospholipid membranes was further investigated using lipid bilayers with DPPC/PG and fluorescent probes located either at the polar surface [1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) sodium anilino naphthalene sulfonate (ANS)] or within the hydrophobic core (DPH) of the liposome. Changes in fluorescence intensity and in polarization were observed when TMA-DPH or ANS, but not DPH were used. Comparative data also indicate that all three polymers interact only with the outer surface of the bilayer, but even the most marked penetration of polycationic polypeptide (SAK) did not result in alteration of the ordered state of the alkyl chains in the bilayer. Taken together, data obtained from mono- or bilayer experiments suggest that the interaction between branched polymers and phospholipids are highly dependent on the charge properties (Ser vs Glu) and on the identity (Ser vs Ala) of side-chain terminating amino acids. The binding of polymers to the model membranes could be mainly driven by electrostatic forces, but the significant role of hydrophilic properties in case of SAK cannot be excluded.

The second von Willebrand type A domain of cochlin has high affinity for type I, type II and type IV collagens

MINT‐6796048: type I collagen (uniprotkb:P02452) binds (MI:0407) to cochlin‐vWA2 uniprotkb:O43405) by surface plasmon resonance (MI:0107) MINT‐6796166: type III collagen (uniprotkb:P02462) binds (MI:0407) to cochlin‐vWA2 (uniprotkb:O43405) by surface plasmon resonance (MI:0107) MINT‐6796062: type II collagen (uniprotkb:P02458) binds (MI:0407) to cochlin‐vWA2 (uniprotkb:O43405) by surface plasmon resonance (MI:0107)

New Preparative Route to Hetaryldienes and Azadienes

Tetrazolylacroleins easily available via a four step pathway from 2-aminopyridines proved to be suitable starting compounds for preparation of new tetrazolyldiene systems. Reaction with reagents containing active methylene group gave a series of new dienes, whereas hydroxylamines yielded nitrones. The new dienes underwent cyclizations with electron withdrawn dienophiles to yield tetrazolyl substituted new polycycles, and the nitrones gave fused isoxazolines. The electron demand of the tetrazolyldienes in cycloadditions was interpreted by calculation of frontier orbital energy levels.

Synthesis and transformations of tetrazolylacroleins

1-Methoxy-4-aryltetrazolyldienes have been subjected to oxidative degradation to yield tetrazolyl acroleins. These compounds when reacted with 1,1-dimethylhydrazine gave 1-dimethylamino-4-aryltetrazolyl-1-azadienes. Both the acroleins and the new 1-azadienes underwent ring transformation in reaction with fumaronitrile to afford pyrazolyl derivatives.

Conjugation of HS-Oligopeptides with Polymeric Branched Chain Polypeptides Containing Multiple Amino Groups:

For the preparation of bioconjugates containing polymeric polypeptides with well-defined structure and composition, we systematically studied 3-(2-pyridyldithio)propionic acid N-hydroxy-succinimide ester (SPDP). SPDP as amino- and thiol-reactive heterobifunctional coupling agent is mainly used for protein-based conjugates, and very little data are available on its application for the modification of polymers. In this communication, we describe the effect of polymer/oligopeptide structure and of the reaction condition on the incorporation of oligopeptides with free thiol group (CAVKDEL, CTGRGDSP) into polymeric polypeptides possessing multiple amino groups. For these studies, linear poly[L-lysine] with free e-amino groups and its XAK-type branched polypeptide derivatives {poly[Lys(X i -DL-Ala m )] (i < 1, m ∼ 3, XAK)} either with polycationic character {X =O (AK), X = Ser (SAK)} or with amphoteric nature {X = Glu (EAK)} were utilized. First, the polymers were modified with SPDP under various conditions, and the degree of substitution was determined. We found that the efficacy of the nucleophilic substitution of NH 2 groups with SPDP depended not only on the pH and the concentration of the coupling reagent but also on the polymer composition, mainly on the pK a of the branch-terminal amino group of the polymers. SPDP-modified polymeric polypeptides were reacted with the HS-oligopeptides, and the effects of polymer/oligopeptide structure as well as the reaction conditions (pH, peptide/(SSP)polymer molar ratio) on the composition of the product were evaluated. The results suggest that acidic pH is more favorable for the thiol-disulphide exchange, and the side chain composition of the polymers had a pronounced effect while the chemical structure of oligopeptides had only moderate influence on the average degree of substitution.

Selective hydroboration of dieneamines. Formation of hydroxyalkylphenothiazines as MDR modulators

N-dienylphenothiazines synthesized from tetrazolo[1,5-a]pyridinium salts by treatment with phenothiazine were subjected to catalytic hydrogenation to yield N-butylphenothiazines, whereas transformation of these dienes with borane dimethyl sulfide (BH(3) × Me(2)S) resulted in selective hydroboration of one double bond and full reduction of the other double bond to give 2-hydroxybutylphenothiazines. Position of the hydroxyl group was supported by NMR spectroscopy and verified by X-ray analysis. Comparison of MDR modulatory activity of the new derivatives revealed that the hydroxybutyl compounds are promising candidates for development of novel MDR inhibitors.