Effects of Ca2+ ions on the horseshoe crab coagulation cascade triggered by lipopolysaccharide.

The lipopolysaccharide (LPS)-triggered horseshoe crab coagulation cascade is composed of three protease zymogens, prochelicerase C (proC), prochelicerase B (proB) and the proclotting enzyme (proCE). In this study, we found that Ca 2+ ions increase the production of the clotting enzyme as a result of a cascade reaction reconstituted by recombinant proteins of wild-type (WT) proC, WT proB and WT proCE. We divided the cascade into three stages: autocatalytic activation of WT proC on the surface of LPS into WT α-chelicerase C (Stage 1); activation of WT proB on the surface of LPS into WT chelicerase B by WT α-chelicerase C (Stage 2) and activation of WT proce into WT CE by chelicerase B (Stage 3). Ca2+ ions enhanced the proteolytic activation in Stage 2, but not those in Stages 1 and 3. Moreover, we performed isothermal titration calorimetry to clarify the interaction of LPS or the recombinant zymogens with Ca2+ ions. LPS interacted with Ca2+ ions at an association constant of Ka = 4.7 × 104 M-1, but not with any of the recombinant zymogens. We concluded that LPS bound with Ca2+ ions facilitates the chain reaction of the cascade as a more efficient scaffold than LPS itself.

From structure to function - Ligand recognition by myeloid C-type lectin receptors.

The relevance of protein-glycan interactions in immunity has long been underestimated. Yet, the immune system possesses numerous classes of glycan-binding proteins, so-called lectins. Of specific interest is the group of myeloid C-type lectin receptors (CLRs) as they are mainly expressed by myeloid cells and play an important role in the initiation of an immune response. Myeloid CLRs represent a major group amongst pattern recognition receptors (PRRs), placing them at the center of the rapidly growing field of glycoimmunology. CLRs have evolved to encompass a wide range of structures and functions and to recognize a large number of glycans and many other ligands from different classes of biopolymers. This review aims at providing the reader with an overview of myeloid CLRs and selected ligands, while highlighting recent insights into CLR-ligand interactions. Subsequently, methodological approaches in CLR-ligand research will be presented. Finally, this review will discuss how CLR-ligand interactions culminate in immunological functions, how glycan mimicry favors immune escape by pathogens, and in which way immune responses can be affected by CLR-ligand interactions in the long term.

Valorphins alter physicochemical characteristics of phosphatidylcholine membranes: Datasets on lipid packing, bending rigidity, specific electrical capacitance, dipole potential, vesicle size.

Endogenous hemorphins are being intensively investigated as therapeutic agents in neuropharmacology, and also as biomarkers in mood regulation, inflammation and oncology. The datasets collected herein report physicochemical parameters of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes in the presence of VV-hemorphin-5 (Val-Val-Tyr-Pro-Trp-Thr-Gln) and analogues, modified at position 1 and 7 by the natural amino acid isoleucine or the non-proteinogenic 2-aminoisobutyric, 2,3-diaminopropanoic or 2,4-diaminobutanoic amino acids. These peptides have been previously screened for nociceptive activity and were chosen accordingly. The present article contains fluorescence spectroscopy data of Laurdan- and di-8-ANEPPS- labelled large unilamellar vesicles (LUV) providing the degree of hydration and dipole potential of lipid bilayers in the presence of VV-hemorphin-5 analogues. Lipid packing is accessible from Laurdan intensity profiles and generalized polarization datasets reported herein. The data presented on fluorescence intensity ratios of di-8-ANEPPS dye provide dipole potential values of phosphatidylcholine-valorphin membranes. Vesicle size and electrophoretic mobility datasets included refer to the effect of valorphins on the size distribution and ζ -potential of POPC LUVs. Investigation of physicochemical properties of peptides such as diffusion coefficients and heterogeneous rate constant relates to elucidation of transport mechanisms in living cells. Voltammetric data of valorphins are presented together with square-wave voltammograms of investigated peptides for calculation of their heterogeneous electron transfer rate constants. Datasets from the thermal shape fluctuation analysis of quasispherical 'giant' unilamellar vesicles (GUV) are provided to quantify the influence of hemorphin incorporation on the membrane bending elasticity. Isothermal titration calorimetric data on the thermodynamics of peptide-lipid interactions and the binding affinity of valorphin analogues to phosphatidylcholine membranes are reported. Data of frequency-dependent deformation of GUVs in alternating electric field are included together with the values of the specific electrical capacitance of POPC-valorphin membranes. The datasets reported in this article can underlie the formulation and implementation of peptide-based strategies in pharmacology and biomedicine.

Interaction and binding mechanism of lipid oxidation products to sturgeon myofibrillar protein in low temperature vacuum heating conditions: Multispectroscopic and molecular docking approaches.

In this work, the binding mechanism of myofibrillar protein (MP) with malondialdehyde and 4-hydroxy-2-nonenal under low temperature vacuum heating was investigated via multispectroscopic and molecular docking. The results showed that binding interaction and increasing temperature caused significant changes in the conformations as well as a decrease in the value of protein intrinsic fluorescence, surface hydrophobicity, and fluorescence excitation-emission matrix spectra. Furthermore, the decrease in α-helix and ß-turn, increase in ß-sheet and a random coil of MP, imply the MP molecules to be more unfolded. Isothermal titration calorimetry and molecular docking results showed that main driving force for binding with MP was hydrogen bond, and the binding ability of malondialdehyde was superior to that of 4-hydroxy-2-nonenal. Moreover, increasing the heating temperature was beneficial to the binding reaction and intensified the conformational transition of MP. These results will provide a reference for further studies on the lipid and protein interaction of sturgeon.

PP2A is activated by cytochrome c upon formation of a diffuse encounter complex with SET/TAF-Iß.

Intrinsic protein flexibility is of overwhelming relevance for intermolecular recognition and adaptability of highly dynamic ensemble of complexes, and the phenomenon is essential for the understanding of numerous biological processes. These conformational ensembles-encounter complexes-lack a unique organization, which prevents the determination of well-defined high resolution structures. This is the case for complexes involving the oncoprotein SET/template-activating factor-Iß (SET/TAF-Iß), a histone chaperone whose functions and interactions are significantly affected by its intrinsic structural plasticity. Besides its role in chromatin remodeling, SET/TAF-Iß is an inhibitor of protein phosphatase 2A (PP2A), which is a key phosphatase counteracting transcription and signaling events controlling the activity of DNA damage response (DDR) mediators. During DDR, SET/TAF-Iß is sequestered by cytochrome c (Cc) upon migration of the hemeprotein from mitochondria to the cell nucleus. Here, we report that the nuclear SET/TAF-Iß:Cc polyconformational ensemble is able to activate PP2A. In particular, the N-end folded, globular region of SET/TAF-Iß (a.k.a. SET/TAF-Iß ΔC)-which exhibits an unexpected, intrinsically highly dynamic behavior-is sufficient to be recognized by Cc in a diffuse encounter manner. Cc-mediated blocking of PP2A inhibition is deciphered using an integrated structural and computational approach, combining small-angle X-ray scattering, electron paramagnetic resonance, nuclear magnetic resonance, calorimetry and molecular dynamics simulations.

Structural and biochemical characterization of the relaxosome auxiliary proteins encoded on the Bacillus subtilis plasmid pLS20.

Bacterial conjugation is an important route for horizontal gene transfer. The initial step in this process involves a macromolecular protein-DNA complex called the relaxosome, which in plasmids consists of the origin of transfer (oriT) and several proteins that prepare the transfer. The relaxosome protein named relaxase introduces a nick in one of the strands of the oriT to initiate the process. Additional relaxosome proteins can exist. Recently, several relaxosome proteins encoded on the Bacillus subtilis plasmid pLS20 were identified, including the relaxase, named RelpLS20, and two auxiliary DNA-binding factors, named Aux1pLS20 and Aux2pLS20. Here, we extend this characterization in order to define their function. We present the low-resolution SAXS envelope of the Aux1pLS20 and the atomic X-ray structure of the C-terminal domain of Aux2pLS20. We also study the interactions between the auxiliary proteins and the full-length RelpLS20, as well as its separate domains. The results show that the quaternary structure of the auxiliary protein Aux1pLS20 involves a tetramer, as previously determined. The crystal structure of the C-terminal domain of Aux2pLS20 shows that it forms a tetramer and suggests that it is an analog of TraMpF of plasmid F. This is the first evidence of the existence of a TraMpF analog in gram positive conjugative systems, although, unlike other TraMpF analogs, Aux2pLS20 does not interact with the relaxase. Aux1pLS20 interacts with the C-terminal domain, but not the N-terminal domain, of the relaxase RelpLS20. Thus, the pLS20 relaxosome exhibits some unique features despite the apparent similarity to some well-studied G- conjugation systems.

Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression.

Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53 in vitro and in vivo, representing a viable therapeutic strategy for cancer treatment. Using phage display techniques, we previously identified a potent peptide activator of P53, termed PMI (TSFAEYWNLLSP), with binding affinities for both MDM2 and MDMX in the low nanomolar concentration range. Here we report an ultrahigh affinity, dual-specificity peptide antagonist of MDM2 and MDMX obtained through systematic mutational analysis and additivity-based molecular design. Functional assays of over 100 peptide analogs of PMI using surface plasmon resonance and fluorescence polarization techniques yielded a dodecameric peptide termed PMI-M3 (LTFLEYWAQLMQ) that bound to MDM2 and MDMX with K d values in the low picomolar concentration range as verified by isothermal titration calorimetry. Co-crystal structures of MDM2 and of MDMX in complex with PMI-M3 were solved at 1.65 and 3.0 Å resolution, respectively. Similar to PMI, PMI-M3 occupied the P53-binding pocket of MDM2/MDMX, which was dominated energetically by intermolecular interactions involving Phe3, Tyr6, Trp7, and Leu10. Notable differences in binding between PMI-M3 and PMI were observed at other positions such as Leu4 and Met11 with MDM2, and Leu1 and Met11 with MDMX, collectively contributing to a significantly enhanced binding affinity of PMI-M3 for both proteins. By adding lysine residues to both ends of PMI and PMI-M3 to improve their cellular uptake, we obtained modified peptides termed PMI-2K (KTSFAEYWNLLSPK) and M3-2K (KLTFLEYWAQLMQK). Compared with PMI-2K, M3-2K exhibited significantly improved antitumor activities in vitro and in vivo in a P53-dependent manner. This super-strong peptide inhibitor of the P53-MDM2/MDMX interactions may become, in its own right, a powerful lead compound for anticancer drug development, and can aid molecular design of other classes of P53 activators as well for anticancer therapy.

Calmodulin complexes with brain and muscle creatine kinase peptides.

Calmodulin (CaM) is a ubiquitous Ca2+ sensing protein that binds to and modulates numerous target proteins and enzymes during cellular signaling processes. A large number of CaM-target complexes have been identified and structurally characterized, revealing a wide diversity of CaM-binding modes. A newly identified target is creatine kinase (CK), a central enzyme in cellular energy homeostasis. This study reports two high-resolution X-ray structures, determined to 1.24 â€‹Å and 1.43 â€‹Å resolution, of calmodulin in complex with peptides from human brain and muscle CK, respectively. Both complexes adopt a rare extended binding mode with an observed stoichiometry of 1:2 CaM:peptide, confirmed by isothermal titration calorimetry, suggesting that each CaM domain independently binds one CK peptide in a Ca2+-depended manner. While the overall binding mode is similar between the structures with muscle or brain-type CK peptides, the most significant difference is the opposite binding orientation of the peptides in the N-terminal domain. This may extrapolate into distinct binding modes and regulation of the full-length CK isoforms. The structural insights gained in this study strengthen the link between cellular energy homeostasis and Ca2+-mediated cell signaling and may shed light on ways by which cells can 'fine tune' their energy levels to match the spatial and temporal demands.

YPIBP: A repository for phosphoinositide-binding proteins in yeast.

Phosphoinositides (PIs) are a family of eight lipids consisting of phosphatidylinositol (PtdIns) and its seven phosphorylated forms. PIs have important regulatory functions in the cell including lipid signaling, protein transport, and membrane trafficking. Yeast has been recognized as a eukaryotic model system to study lipid-protein interactions. Hundreds of yeast PI-binding proteins have been identified, but this research knowledge remains scattered. Besides, the complete PI-binding spectrum and potential PI-binding domains have not been interlinked. No comprehensive databases are available to support the lipid-protein interaction research on phosphoinositides. Here we constructed the first knowledgebase of Yeast Phosphoinositide-Binding Proteins (YPIBP), a repository consisting of 679 PI-binding proteins collected from high-throughput proteome-array and lipid-array studies, QuickGO, and a rigorous literature mining. The YPIBP also contains protein domain information in categories of lipid-binding domains, lipid-related domains and other domains. The YPIBP provides search and browse modes along with two enrichment analyses (PI-binding enrichment analysis and domain enrichment analysis). An interactive visualization is given to summarize the PI-domain-protein interactome. Finally, three case studies were given to demonstrate the utility of YPIBP. The YPIBP knowledgebase consolidates the present knowledge and provides new insights of the PI-binding proteins by bringing comprehensive and in-depth interaction network of the PI-binding proteins. YPIBP is available at http://cosbi7.ee.ncku.edu.tw/YPIBP/.

A fragment-based approach to assess the ligandability of ArgB, ArgC, ArgD and ArgF in the L-arginine biosynthetic pathway of Mycobacterium tuberculosis.

The L-arginine biosynthesis pathway consists of eight enzymes that catalyse the conversion of L-glutamate to L-arginine. Arginine auxotrophs (argB/argF deletion mutants) of Mycobacterium tuberculosis are rapidly sterilised in mice, while inhibition of ArgJ with Pranlukast was found to clear chronic M. tuberculosis infection in a mouse model. Enzymes in the arginine biosynthetic pathway have therefore emerged as promising targets for anti-tuberculosis drug discovery. In this work, the ligandability of four enzymes of the pathway ArgB, ArgC, ArgD and ArgF is assessed using a fragment-based approach. We identify several hits against these enzymes validated with biochemical and biophysical assays, as well as X-ray crystallographic data, which in the case of ArgB were further confirmed to have on-target activity against M. tuberculosis. These results demonstrate the potential for more enzymes in this pathway to be targeted with dedicated drug discovery programmes.

Surfactants - Compounds for inactivation of SARS-CoV-2 and other enveloped viruses.

We provide here a general view on the interactions of surfactants with viruses, with a particular emphasis on how such interactions can be controlled and employed for inhibiting the infectivity of enveloped viruses, including coronaviruses. The aim is to provide to interested scientists from different fields, including chemistry, physics, biochemistry, and medicine, an overview of the basic properties of surfactants and (corona)viruses, which are relevant to understanding the interactions between the two. Various types of interactions between surfactant and virus are important, and they act on different components of a virus such as the lipid envelope, membrane (envelope) proteins and nucleocapsid proteins. Accordingly, this cannot be a detailed account of all relevant aspects but instead a summary that bridges between the different disciplines. We describe concepts and cover a selection of the relevant literature as an incentive for diving deeper into the relevant material. Our focus is on more recent developments around the COVID-19 pandemic caused by SARS-CoV-2, applications of surfactants against the virus, and on the potential future use of surfactants for pandemic relief. We also cover the most important aspects of the historical development of using surfactants in combatting virus infections. We conclude that surfactants are already playing very important roles in various directions of defence against viruses, either directly, as in disinfection, or as carrier components of drug delivery systems for prophylaxis or treatment. By designing tailor-made surfactants, and consequently, advanced formulations, one can expect more and more effective use of surfactants, either directly as antiviral compounds or as part of more complex formulations.

Endogenous modulators of neurotrophin signaling: Landscape of the transient ATP-NGF interactions.

The Nerve Growth Factor (NGF) neurotrophin acts in the maintenance and growth of neuronal populations. Despite the detailed knowledge of NGF's role in neuron physiology, the structural and mechanistic determinants of NGF bioactivity modulated by essential endogenous ligands are still lacking. We present the results of an integrated structural and advanced computational approach to characterize the extracellular ATP-NGF interaction. We mapped by NMR the interacting surface and ATP orientation on NGF and revealed the functional role of this interaction in the binding to TrkA and p75NTR receptors by SPR. The role of divalent ions was explored in conjunction with ATP. Our results pinpoint ATP as a likely transient molecular modulator of NGF signaling, in health and disease states.

Emerging peptide antibiotics with therapeutic potential.

This review covers some of the recent progress in the field of peptide antibiotics with a focus on compounds with novel or established mode of action and with demonstrated efficacy in animal infection models. Novel drug discovery approaches, linear and macrocyclic peptide antibiotics, lipopeptides like the polymyxins as well as peptides addressing targets located in the plasma membrane or in the outer membrane of bacterial cells are discussed.

Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression

Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53

Small molecules targeting the innate immune cGAS‒STING‒TBK1 signaling pathway.

Multiple cancer immunotherapies including chimeric antigen receptor T cell and immune checkpoint inhibitors (ICIs) have been successfully developed to treat various cancers by motivating the adaptive anti-tumor immunity. Particularly, the checkpoint blockade approach has achieved great clinic success as evidenced by several U.S. Food and Drug Administration (FDA)-approved anti-programmed death receptor 1/ligand 1 or anti-cytotoxic T lymphocyte associated protein 4 antibodies. However, the majority of cancers have low clinical response rates to these ICIs due to poor tumor immunogenicity. Indeed, the cyclic guanosine monophosphate-adenosine monophosphate synthase‒stimulator of interferon genes‒TANK-binding kinase 1 (cGAS‒STING‒TBK1) axis is now appreciated as the major signaling pathway in innate immune response across different species. Aberrant signaling of this pathway has been closely linked to multiple diseases, including auto-inflammation, virus infection and cancers. In this perspective, we provide an updated review on the latest progress on the development of small molecule modulators targeting the cGAS‒STING‒TBK1 signaling pathway and their preclinical and clinical use as a new immune stimulatory therapy. Meanwhile, highlights on the clinical candidates, limitations and challenges, as well as future directions in this field are also discussed. Further, small molecule inhibitors targeting this signaling axis and their potential therapeutic use for various indications are discussed as well.

Structural insights into human Arginase-1 pH dependence and its inhibition by the small molecule inhibitor CB-1158.

Arginase-1 is a manganese-dependent metalloenzyme that catalyzes the hydrolysis of L-arginine into L-ornithine and urea. Arginase-1 is abundantly expressed by tumor-infiltrating myeloid cells that promote tumor immunosuppression, which is relieved by inhibition of Arginase-1. We have characterized the potencies of the Arginase-1 reference inhibitors (2S)-2-amino-6-boronohexanoic acid (ABH) and N ω-hydroxy-nor-L-arginine (nor-NOHA), and studied their pH-dependence and binding kinetics. To gain a better understanding of the structural changes underlying the high pH optimum of Arginase-1 and its pH-dependent inhibition, we determined the crystal structure of the human Arginase-1/ABH complex at pH 7.0 and 9.0. These structures revealed that at increased pH, the manganese cluster assumes a more symmetrical coordination structure, which presumably contributes to its increase in catalytic activity. Furthermore, we show that binding of ABH involves the presence of a sodium ion close to the manganese cluster. We also studied the investigational new drug CB-1158 (INCB001158). This inhibitor has a low-nanomolar potency at pH 7.4 and increases the thermal stability of Arginase-1 more than ABH and nor-NOHA. Moreover, CB-1158 displays slow association and dissociation kinetics at both pH 9.5 and 7.4, as indicated by surface plasmon resonance. The potent character of CB-1158 is presumably due to its increased rigidity compared to ABH as well as the formation of an additional hydrogen-bond network as observed by resolution of the Arginase-1/CB-1158 crystal structure.

Pharmaceutical modulation of the proteolytic profile of Transforming Growth Factor Beta induced protein (TGFBIp) offers a new avenue for treatment of TGFBI-corneal dystrophy.

Corneal dystrophies are a group of genetically inherited disorders with mutations in the TGFBI gene affecting the Bowman's membrane and the corneal stroma. The mutant TGFBIp is highly aggregation-prone and is deposited in the cornea. Depending on the type of mutation the protein deposits may vary (amyloid, amorphous powdery aggregate or a mixed form of both), making the cornea opaque and thereby decreases visual acuity. The aggregation of the mutant protein is found to be specific with a unique aggregation mechanism distinct to the cornea. The proteolytic processing of the mutant protein is reported to be different compared to the WT protein. The proteolytic processing of mutant protein gives rise to highly amyloidogenic peptide fragments. The current treatment option, available for patients, is tissue replacement surgery that is associated with high recurrence rates. The clinical need for a simple treatment option for corneal dystrophy patients has become highly essential either to prevent the protein aggregation or to dissolve the preformed aggregates. Here, we report the screening of 2500 compounds from the Maybridge RO3 fragment library using weak affinity chromatography (WAC). The primary hits from WAC were validated by 15N-HSQC NMR assays and specific regions of binding were identified. The recombinant mutant proteins (4th FAS-1 domain of R555W and H572R) were subjected to limited proteolysis by trypsin together with the lead compounds identified by NMR assays. The lead compounds (MO07617, RJF00203 and, BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the R555W mutant and compounds (RJF00203 and BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the H572R mutant. Thus the lead compounds reported here upon further validation and/or modification might be proposed as a potential treatment option to prevent/delay aggregation by inhibiting the formation of amyloidogenic peptides in TGFBI-corneal dystrophy.

Anticancer drug discovery by targeting cullin neddylation.

Protein neddylation is a post-translational modification which transfers the ubiquitin-like protein NEDD8 to a lysine residue of the target substrate through a three-step enzymatic cascade. The best-known substrates of neddylation are cullin family proteins, which are the core component of Cullin-RING E3 ubiquitin ligases (CRLs). Given that cullin neddylation is required for CRL activity, and CRLs control the turn-over of a variety of key signal proteins and are often abnormally activated in cancers, targeting neddylation becomes a promising approach for discovery of novel anti-cancer therapeutics. In the past decade, we have witnessed significant progress in the field of protein neddylation from preclinical target validation, to drug screening, then to the clinical trials of neddylation inhibitors. In this review, we first briefly introduced the nature of protein neddylation and the regulation of neddylation cascade, followed by a summary of all reported chemical inhibitors of neddylation enzymes. We then discussed the structure-based targeting of protein-protein interaction in neddylation cascade, and finally the available approaches for the discovery of new neddylation inhibitors. This review will provide a focused, up-to-date and yet comprehensive overview on the discovery effort of neddylation inhibitors.

Creatine kinase/α-crystallin interaction functions in cataract development.

Creatine kinase (CK) is an energy storage enzyme that plays an important role in energy metabolism. CK/phosphocreatine functions as an energy buffer and links ATP production sites with ATP utilization sites. Several key mutations in the αA-crystallin (cryaa) and αB-crystallin (cryab) genes have been linked with autosomal-dominant, hereditary human cataracts. The cryaa-R49C mutation was identified in a four-generation Caucasian family. We previously identified an increase in the quantity of CK complexed with α-crystallin in the lenses of knock-in mice expressing the cryaa-R49C mutation using proteomic analyses. Increased levels of CK in postnatal cataractous lenses may indicate increased ATP requirements during early cataract development. To gain a further understanding of the relationship between CK and α-crystallin, we investigated whether α-crystallin interacts with and forms complexes with CK, in vitro. Isothermal titration calorimetry (ITC) showed that each CK dimer bound to 28 α-crystallin subunits, with a Kd of 3.3 × 10-7 M, and that the interaction between α-crystallin and CK was endothermic, thermodynamically favorable, and entropy-driven. High-salt concentrations did not affect the interaction between CK and α-crystallin, suggesting that the interaction between CK and α-crystallin is primarily hydrophobic. Gel permeation chromatography (GPC) detected water-soluble α-crystallin and CK complexes, as determined by increased light scattering after complex formation. In addition, CK and α-crystallin formed partially-water-insoluble, high-molecular-mass complexes. Enzyme-linked immunosorbent assay (ELISA)-based enzymatic activity analyses of lens homogenates showed a 17-fold increase in CK activity in the postnatal lenses of cryaa-R49C knock-in mice. These studies indicate that the interaction between α-crystallin and CK is functionally important and that increased CK levels may be necessary to meet the increased ATP demands of ATP-dependent functions in cataractous lenses.

Real time calorimetric characterisation of clay - drug complex dispersions and particles.

Isothermal titration calorimetry (ITC) along with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDX) and high-performance liquid chromatography (HPLC) were employed to investigate the process of adsorption of propranolol hydrochloride (PPN) onto magnesium aluminium silicate (MAS) and to characterise the MAS-PPN particles formed upon complexation. The composition of MAS was confirmed by infrared (IR) spectroscopy and a calcimeter. The calorimetric results confirmed the binding between PPN and MAS at various pHs and temperatures. The overall change in enthalpy was found to be exothermic with a comparatively small entropic contribution to the total change in Gibbs free energy. These findings suggest that the binding process was enthalpically driven and entropically unfavourable (lower affinity) suggesting hydrogen bonding and electrostatic interactions dominating the interaction. The variation of pH and temperature did not have a great impact on the thermodynamics of the binding process, as observed from the similarity in enthalpy (ΔH), entropy (ΔS) or Gibbs free energy (ΔG). A slight reduction in the binding affinity (Ka) with varing pH and temperature was however observed. SEM/EDX studies showed the occurrence of changes in the microstructural properties of MAS following complexation which may explain the potential of MAS-PPN complexes for controlled drug release promoting pharmaceutical innovation.