Ivica Malnar

Valosin containing protein (VCP) interacts with macrolide antibiotics without mediating their anti-inflammatory activities

In addition to antibacterial activity, some macrolide antibiotics, such as azithromycin and clarithromycin, also exhibit anti-inflammatory properties in vitro and in vivo, although the targets and mechanism(s) of action remain unknown. The aim of the present study was to identify protein targets of azithromycin and clarithromycin which could potentially explain their anti-inflammatory effects. Using chemical proteomics approach, based on compound-immobilized affinity chromatography, valosin containing protein (VCP) was identified as a potential target of the macrolides. Validation studies confirmed the interaction of macrolides and VCP and gave some structural characteristics of this interaction. Cell based assays however, including the use of gene silencing and the study of VCP specific cellular functions in J774.A1 (murine macrophage) and IB3-1 (human cystic fibrotic epithelial) cell lines, failed to confirm an association between the binding of the macrolides to VCP and anti-inflammatory effects. These findings suggest the absence of an abundant high affinity protein target and the potential involvement of other biological molecules in the anti-inflammatory activity of macrolides.

Synthesis, NMR and X-ray structure analysis of macrolide aglycons

Macrolide aglycons (E)-9-hydroxyimino-6-O-methylerythronolide A (4), 9a-aza-9-deoxo-9,9-dihydro-9a,11-O-dimethyl-9a-homoerythronolide A (5) and 9a-aza-9-deoxo-9,9-dihydro-9a-homoerythronolide A (6) were prepared by multistep syntheses. A conformational study of these new macrolide aglycons was performed using single crystal X-ray crystallography to gain information about the solid state, while a combination of NMR spectroscopy and molecular modelling was employed to study the solution structures. The crystal structures were found to be stabilised by a complex network of hydrogen bonds and van der Waals interactions. To some extent, the same building motif of infinite molecular chains held together by O–H···O hydrogen bonds was present in the crystal structure of all three compounds. Thorough analysis and comparison of the obtained solid state structures with their solution counterparts showed no significant differences between them, confirming the constrained flexibility of the macrocyclic ring. Moreover, in all three compounds, in both solution and solid state, the macrolactone ring adopts energetically more favoured folded-out conformations.

A comparison of in vitro ADME properties and pharmacokinetics of azithromycin and selected 15-membered ring macrolides in rodents

The purpose of this study was to evaluate the impact of structural modifications on the 15-membered macrolactone ring and/or substituents on the in vitro ADME properties and in vivo pharmacokinetic (PK) profile for selected derivatives in rodents in comparison to azithromycin. Azithromycin and seven selected 15-membered macrolide derivatives, modified either by removal of the sugar moieties, replacement of the amine with a lactam, or addition of lipophilic substituents, were screened in several in vitro ADME assays and in vivo PK studies in rodents. In vitro ADME profiling included assessment of passive permeability and P-gp substrate, metabolic stability in liver microsomes and hepatocytes, as well as CYP direct inhibition measurements. In vivo PK studies were performed in rats (Sprague–Dawley), mice (Balb/c), and P-gp wild-type and deficient mice (CF-1™). Different structural modifications on the azithromycin scaffold resulted in substantial changes in disposition kinetics and oral bioavailability in both rodent species. However, these differences in vivo cannot be predicted based on in vitro results since most of these molecules are classified in the same category. Therefore, in the case of 15-membered ring macrolides, the in vitro ADME screens presented here seem to have low predictive value for in vivo prediction, making their use as routine in vitro screens prior to PK assessments questionable.

Determination of aqueous stability and degradation products of series of coumarin dimers

The stability in aqueous solution of five classes of coumarin dimers (I-V, compounds 1-29) was studied by HPLC-MS/MS at various pH values. The relationship between chemical structure and stability is discussed. It was found that dimeric compounds with strong electron withdrawing groups (EWGs) on the α-carbon to the bridging C-atom are stable at all pH values, whereas other derivatives undergo retro-Michael addition at rates which are also affected by the substituents on the aromatic rings. In some cases formation of stable isomers or oxidation products was observed. In order to evaluate their developability and potential for progression to in vivo studies, representative compounds were tested in an in vitro microsomal stability assay.

Study of lipophilicity and membrane partition of 4-hydroxycoumarins by HPLC and PCA

Physicochemical properties provide reliable guidance in optimization of pharmacological efficiency and ADME profile of small chemical compounds. Their high-throughput determination is regularly based on application of HPLC techniques. In this study CHI and CHI IAM of 32 4-hydroxycoumarin analogs were measured by HPLC with methanol gradient at pHs 2.8 and 7.0. Results were analyzed by PCA in terms of computed descriptors in order to identify space for optimization of their phospholipids affinity and lipophilicity for which predictive software failed to produce reliable estimations. The chromatographic behavior of studied 4-hydroxycoumarins was typical of acidic compounds. The CHI(2.8), CHI(7.0), CHI IAM(2.8) and CHI IAM(7.0) values were all considerably cross-correlated in accordance with their prevailing lipophilic character. Structure-retention relationship (SRR) analysis furthermore revealed that H-bond accepting capacity and dipolar interactions with methanol generally shorten their retention times. However, deviations from the linear trends were noticed for R3/R5-substituted derivatives able to form intramolecular contacts with the 4-O(H) group and characterized by more uniform electron density at 2-O and 4-O atoms and quite different acidity/H-bond donating capacity than the rest of derivatives. Thus, CHI and CHI IAM determinations and SRR analysis are fast and efficiently pointed to ways of modifying biological activities of 4-hydroxycoumarins.

Around the macrolide – Impact of 3D structure of macrocycles on lipophilicity and cellular accumulation

The aim of this study was to investigate lipophilicity and cellular accumulation of rationally designed azithromycin and clarithromycin derivatives at the molecular level. The effect of substitution site and substituent properties on a global physico-chemical profile and cellular accumulation of investigated compounds was studied using calculated structural parameters as well as experimentally determined lipophilicity. In silico models based on the 3D structure of molecules were generated to investigate conformational effect on studied properties and to enable prediction of lipophilicity and cellular accumulation for this class of molecules based on non-empirical parameters. The applicability of developed models was explored on a validation and test sets and compared with previously developed empirical models.

Structure and conformational analysis of spiroketals from 6-O-methyl-9(E)-hydroxyiminoerythronolide A

Summary Three novel spiroketals were prepared by a one-pot transformation of 6-O-methyl-9(E)-hydroxyiminoerythronolide A. We present the formation of a [4.5]spiroketal moiety within the macrolide lactone ring, but also the unexpected formation of a 10-C=11-C double bond and spontaneous change of stereochemistry at position 8-C. As a result, a thermodynamically stable structure was obtained. The structures of two new diastereomeric, unsaturated spiroketals, their configurations and conformations, were determined by means of NMR spectroscopy and molecular modelling. The reaction kinetics and mechanistic aspects of this transformation are discussed. These rearrangements provide a facile synthesis of novel macrolide scaffolds.