Insulin polymorphism induced by two polyphenols: new crystal forms and advances in macromolecular powder diffraction

Abstract

This study focuses on the polymorphism of human insulin (HI) upon the binding of the phenolic derivatives p-coumaric acid or trans-resveratrol over a wide pH range. The determination of the structural behaviour of HI via X-ray powder diffraction (XRPD) and single-crystal X-ray diffraction (SCXRD) is reported. Four distinct polymorphs were identified, two of which have not been reported previously. The intermediate phase transitions are discussed. One of the novel monoclinic polymorphs displays the highest molecular packing among insulin polymorphs of the same space group to date; its structure was elucidated by SCXRD. XRPD data collection was performed using a variety of instrumental setups and a systematic comparison of the acquired data is presented. A laboratory diffractometer was used for screening prior to high-resolution XRPD data collection on the ID22 beamline at the European Synchrotron Radiation Facility. Additional measurements for the most representative samples were performed on the X04SA beamline at the Swiss Light Source (SLS) using the MYTHEN II detector, which allowed the detection of minor previously untraceable impurities and dramatically improved the d-spacing resolution even for poorly diffracting samples.

New perspectives in macromolecular powder diffraction using single-photon-counting strip detectors: high-resolution structure of the pharmaceutical peptide octreotide

“…MYTHEN II is a general-purpose detector, with intrinsic angular resolution of 3.7 mdeg (angle 2), very high counting efficiency and dynamic range as well as fast (ms) acquisition times. Due to its simultaneous covering of 120 2, it requires minimum sample exposure time, up to 60 times shorter than that required with crystal analyzer detectors, thus enabling the collection of data with enhanced counting statistics before radiation damage effects arise (Gozzo et al, 2010;Triandafillidis et al, 2020). MYTHEN II is a singlephoton-counting detector, which means that a discriminator threshold can be set, allowing one to overcome the problem of a background affected by fluorescence.…”

High-throughput macromolecular polymorph screening via an NMR and X-ray powder diffraction synergistic approach: the case of human insulin co-crystallized with resorcinol derivatives

“…High angular and d-spacing resolutions do not necessarily need to be combined in the same data sets, and we demonstrate with the present study that a consistent combination of different sets of XRPD data recorded on the same protein powders can indeed be the key to success for their structure analysis. Synchrotron XRPD with single-photon-counting detectors and laboratory XRPD (Spiliopoulou et al, 2021;Triandafillidis et al, 2020;Margiolaki, 2019;Margiolaki & Wright, 2008; provide in fact precious complementary information to the ultra-high-resolution synchrotron XRPD data for a successful exhaustive structure analysis of proteins.…”

“…The excellent germicidal action exhibited by the resorcinol derivatives is due to a combination of their physical ability to lower surface tension, which can make these derivatives penetrate more easily through the membranes, and chemical reactivity associated with the two hydroxyl groups (Durairaj, 2005;Klarmann, 1926;Magaard & Ekeströ m, 1975). Phenol, resorcinol and their products have been used in cocrystallization experiments with HI, resulting in a diverse assortment of polymorphs (Fili et al, 2015;Karavassili et al, 2017Karavassili et al, , 2012Triandafillidis et al, 2020;Valmas et al, 2015). 4-Bromoresorcinol and 4-chlororesorcinol could prove to be efficient alternatives, owing to their similar structure, further expanding the HI ligand library available today.…”

Συνδυαστική Χρήση Τεχνικών Ακτίνων-Χ Για Το Δομικό Χαρακτηρισμό Βιολογικών Μακρομορίων Φαρμακευτικού Ενδιαφέροντος

“…Τα τριχοειδή μετρήθηκαν σε πολλές διαδοχικές θέσεις για να περιοριστούν οι συνέπειες του radiation damage, λόγω της υψηλής λαμπρότητας της δέσμης του σύγχροτρον, με τη μέτρηση σε κάθε θέση να διαρκεί ~2 min. Περαιτέρω μετρήσεις από τα πιο αντιπροσωπευτικά δείγματα πραγματοποιήθηκαν στο σταθμό MS-X04SA του SLS (Willmott et al, 2013), με μήκος κύματος λ = 1.3004392( 8 Από τον υπολογισμό του συντελεστής Matthews (Matthews, 1968;Kantardjieff and Rupp, 2003), o οποίος με δεδομένο το μοριακό βάρος της πρωτεΐνης (5.8 kDa), λαμβάνει την τιμή 2.01 Å αντίστοιχα (Triandafillidis, Parthenios et al, 2020). Για την αρχική επεξεργασία των διαγραμμάτων περίθλασης, την ταυτοποίηση του αριθμού των ανακλάσεων καθώς και για την πρωταρχική ανάλυσή τους χρησιμοποιήθηκε το λογισμικό XDSAPP (Sparta et al, 2016) ενώ για τη σύμπτυξη (merging) των 2 σετ δεδομένων χρησιμοποιήθηκε το πρόγραμμα XSCALE (Kabsch, 2010).…”

“…προσδετών που χρησιμοποιούνται ήδη, χωρίς να είναι τοξικός, βοηθώντας παράλληλα στην ανακούφιση και κάποιων παθήσεων που επιβαρύνουν επιπρόσθετα την υγεία των διαβητικών.Οι κρυσταλλώσεις πραγματοποιήθηκαν σύμφωνα με το πρωτόκολλο που αναφέρεται εκτενώς στη σχετική δημοσίευση(Triandafillidis, Parthenios et al, 2020) και περιγράφεται συνοπτικά στον παρακάτω πίνακα. Mix of 0.5 ml protein solution (1 to 5) and 250 μl pH buffer mixture (Na2HPO4/KH2PO4), 2 Μ (of different pH values) in an Eppendorf tube 7.…”