Crystallographic fragment screen of the c-di-AMP-synthesizing enzyme CdaA from Bacillus subtilis.

Crystallographic fragment screening has become a pivotal technique in structure-based drug design, particularly for bacterial targets with a crucial role in infectious disease mechanisms. The enzyme CdaA, which synthesizes an essential second messenger cyclic di-AMP (c-di-AMP) in many pathogenic bacteria, has emerged as a promising candidate for the development of novel antibiotics. To identify crystals suitable for fragment screening, CdaA enzymes from Streptococcus pneumoniae, Bacillus subtilis and Enterococcus faecium were purified and crystallized. Crystals of B. subtilis CdaA, which diffracted to the highest resolution of 1.1 Å, were used to perform the screening of 96 fragments, yielding data sets with resolutions spanning from 1.08 to 1.87 Å. A total of 24 structural hits across eight different sites were identified. Four fragments bind to regions that are highly conserved among pathogenic bacteria, specifically the active site (three fragments) and the dimerization interface (one fragment). The coordinates of the three active-site fragments were used to perform an in silico drug-repurposing screen using the OpenEye suite and the DrugBank database. This screen identified tenofovir, an approved drug, that is predicted to interact with the ATP-binding region of CdaA. Its inhibitory potential against pathogenic E. faecium CdaA has been confirmed by ITC measurements. These findings not only demonstrate the feasibility of this approach for identifying lead compounds for the design of novel antibacterial agents, but also pave the way for further fragment-based lead-optimization efforts targeting CdaA.

Enhancing the apo protein tyrosine phosphatase non-receptor type 2 crystal soaking strategy through inhibitor-accessible binding sites.

Protein tyrosine phosphatase non-receptor type 2 (PTPN2) has recently been recognized as a promising target for cancer immunotherapy. Despite extensive structural and functional studies of other protein tyrosine phosphatases, there is limited structural understanding of PTPN2. Currently, there are only five published PTPN2 structures and none are truly unbound due to the presence of a mutation, an inhibitor or a loop (related to crystal packing) in the active site. In this report, a novel crystal packing is revealed that resulted in a true apo PTPN2 crystal structure with an unbound active site, allowing the active site to be observed in a native apo state for the first time. Key residues related to accommodation in the active site became identifiable upon comparison with previously published PTPN2 structures. Structures of PTPN2 in complex with an established PTPN1 active-site inhibitor and an allosteric inhibitor were achieved through soaking experiments using these apo PTPN2 crystals. The increased structural understanding of apo PTPN2 and the ability to soak in inhibitors will aid the development of future PTPN2 inhibitors.

Ice recrystallization inhibition activity of pulse protein hydrolysates after immobilized metal affinity separation.

Creating molecules capable of inhibiting ice recrystallization is an active research area aiming to improve the freeze-thaw characteristics of foods and biomedical materials. Peptide mixtures have shown promise in preventing freezing-induced damage, but less is known about the relationship between their amino acid compositions and ice recrystallization inhibition (IRI) activities. In this article, we used Ni2+ immobilized metal affinity chromatography (IMAC) to fractionate pulse protein hydrolysates, created by Alcalase and trypsin, into mixtures lacking and enriched in His, and Cys residues. The aim of this study was to fractionate pulse protein hydrolysates based on their amino acid compositions and evaluate their resulting physicochemical and IRI characteristics. Ni2+ IMAC fractionation induced IRI activity in all of the evaluated soy, chickpea, and pea protein hydrolysates regardless of their amino acid composition. Ni2+ IMAC fractionation produced chemically distinct fractions of peptides, differing by their molecular weights, amino acid composition, and IRI activities. The resulting peptide mixtures' molecular weight, amino acid composition, secondary structure, and sodium ion levels were found to have no correlation with their IRI activities. Thus, we demonstrate for the first time the ability of Ni2+ IMAC fractionation to induce IRI activity in hydrolyzed pulse proteins.

Insight into the Manipulation Mechanism of Polymorphic Transformation by Polymers: A Case of Cimetidine.

PURPOSE: Employing polymer additives is an effective strategy to realize the manipulation of polymorphic transformation. However, the manipulation mechanism is still not clear, which limit the precise selection of polymeric excipients and the development of pharmaceutical formulations. METHODS: The solubility of cimetidine (CIM) in acetonitrile/water mixtures were measured. And the polymorphic transformation from CIM form A to form B with the addition of different polymers was monitored by Raman spectroscopy. Furthermore, the manipulation effect of polymers was determined based on the results of experiments and molecular simulations. RESULTS: The solubility of form A is consistently higher than that of form B, which indicate that form B is the thermodynamically stable form within the examined temperature range. The presence of polyvinylpyrrolidone (PVP) of a shorter chain length could have a stronger inhibitory effect on the phase transformation process of metastable form, whereas polyethylene glycol (PEG) had almost no impact. The nucleation kinetics experiments and molecular dynamic simulation results showed that only PVP molecules could significantly decrease the nucleation rate of CIM, due to the ability of reducing solute molecular diffusion and solute-solute molecular interaction. A combination of crystal growth rate measurements and calculations of the interaction energies between PVP and the crystal faces of CIM indicate that smaller molecular weight PVP can suppress crystal growth more effectively. CONCLUSION: PVP K16-18 has more impact on the stabilization of CIM form A and inhibition of the phase transformation process. The manipulation mechanism of polymer additives in the polymorphic transformation of CIM was proposed.

Isophorone-based crystallization-induced-emission sensors detect proteome aggregation in live cells and tissues with breast cancer.

BACKGROUND: Protein misfolding and aggregation can lead to various diseases. Recent studies have shed light on the aggregated protein in breast cancer pathology, which suggests that it is crucial to design chemical sensors that visualize protein aggregates in breast cancer, especially in clinical patient-derived samples. However, most reported sensors are constrained in cultured cell lines. RESULTS: In this work, we present the development of two isophorone-based crystallization-induced-emission fluorophores for detecting proteome aggregation in breast cancer cell line and tissues biopsied from diseased patients, designated as A1 and A2. These probes exhibited viscosity sensitivity and recovered their fluorescence strongly at crystalline state. Moreover, A1 and A2 exhibit selective binding capacity and strong fluorescence for various aggregated proteins. Utilizing these probes, we detect protein aggregation in stressed breast cancer cells, xenograft mouse model of human breast cancer and clinical patient-derived samples. Notably, the fluorescence intensity of both probes light up in tumor tissues. SIGNIFICANCE: The synthesized isophorone-based crystallization-induced-emission fluorophores, A1 and A2, enable sensitive detection of protein aggregation in breast cancer cells and tissues. In the future, aggregated proteins are expected to become indicators for early diagnosis and clinical disease monitoring of breast cancer.

Utilization of bamboo biochar as a multi-functional filler of flexible poly(L-lactide)-b-poly(ethylene glycol)-b-poly(L-lactide) bioplastic.

Biodegradable poly(L-lactide)-b-poly(ethylene glycol)-b-poly(L-lactide) (PLLA-PEG-PLLA) triblock copolymer could potentially be used in bioplastic applications because it is more flexible than PLLA. However, investigations into modifying PLLA-PEG-PLLA with effective fillers are still required. In this work, bamboo biochar (BC) was used as an eco-friendly and cost-effective filler for the flexible PLLA-PEG-PLLA. The influences of BC addition on crystallization properties, thermal stability, hydrophilicity, and mechanical properties of the PLLA-PEG-PLLA were explored and compared to those of the PLLA. The PLLA-PEG-PLLA matrix and BC filler were found to have strong interfacial adhesion and good phase compatibility, while the PLLA/BC composites displayed weak interfacial adhesion and poor phase compatibility. For the PLLA-PEG-PLLA, the addition of BC induced a nucleation effect that was characterized by a decrease in the cold crystallization temperature from 76 to 71-75 °C and an increase in the crystallinity from 18.6 to 21.8-24.0%; however, this effect was not observed for the PLLA. When compared to pure PLLA-PEG-PLLA, the PLLA-PEG-PLLA/BC composites displayed greater thermal stability, tensile stress, and Young's modulus. Temperature at maximum decomposition rate (Td,max) of PLLA end-blocks increased from 315 to 319-342 °C. Ultimate tensile stress of PLLA-PEG-PLLA matrix improved from 14.5 to 16.2-22.6 MPa and Young's modulus increased from 220 to 280-340 MPa. Based on the findings, the crystallizability, thermal stability, and mechanical properties of the flexible PLLA-PEG-PLLA bioplastic were all enhanced by the use of BC as a multi-functional filler.

Modeling the Structure of Ketoprofen-Poly(vinylpyrrolidone) Amorphous Solid Dispersions with Empirical Potential Structure Refinements of X-ray Scattering Data.

The metastability of amorphous formulations poses barriers to their safe and widespread commercialization. The propensity of amorphous solid dispersions (ASDs) to crystallize is directly linked to their molecular structure. Amorphous structures are inherently complex and thus difficult to fully characterize by experiments, which makes structural simulations an attractive route for investigating which structural characteristics correlate with ASD stability. In this study, we use empirical potential structure refinement (EPSR) to create molecular models of ketoprofen-poly(vinylpyrrolidone) (KTP/PVP) ASDs with 0-75 wt % drug loading. The EPSR technique uses X-ray total scattering measurements as constraints, yielding models that are consistent with the X-ray data. We perform several simulations to assess the sensitivity of the EPSR approach to input parameters such as intramolecular bond rotations, PVP molecule length, and PVP tacticity. Even at low drug loading (25 wt %), ∼40% of KTP molecules participate in KTP-KTP hydrogen bonding. The extent of KTP-PVP hydrogen bonding does not decrease significantly at higher drug loadings. However, the models' relative uncertainties are too large to conclude whether ASDs' lower stabilities at high drug loadings are due to changes in drug-excipient hydrogen bonding or a decrease in steric hindrance of KTP molecules. This study illustrates how EPSR, combined with total scattering measurements, can be a powerful tool for investigating structural characteristics in amorphous formulations and developing ASDs with improved stability.

Actinium chelation and crystallization in a macromolecular scaffold.

Targeted alpha therapy (TAT) pairs the specificity of antigen targeting with the lethality of alpha particles to eradicate cancerous cells. Actinium-225 [225Ac; t1/2 = 9.920(3) days] is an alpha-emitting radioisotope driving the next generation of TAT radiopharmaceuticals. Despite promising clinical results, a fundamental understanding of Ac coordination chemistry lags behind the rest of the Periodic Table due to its limited availability, lack of stable isotopes, and inadequate systems poised to probe the chemical behavior of this radionuclide. In this work, we demonstrate a platform that combines an 8-coordinate synthetic ligand and a mammalian protein to characterize the solution and solid-state behavior of the longest-lived Ac isotope, 227Ac [t1/2 = 21.772(3) years]. We expect these results to direct renewed efforts for 225Ac-TAT development, aid in understanding Ac coordination behavior relative to other +3 lanthanides and actinides, and more broadly inform this element's position on the Periodic Table.

Effect of biochar-derived DOM on contrasting redistribution of chromate during Schwertmannite dissolution and recrystallization.

Biochar-derived dissolved organic matter (BDOM), is extensively involved in the recrystallization of minerals and the speciation alteration of associated toxic metals. This study investigates how BDOM extracted from tobacco petiole (TP) or tobacco stalk (TS) biochar influences the speciation repartitioning of Cr(VI) in environments impacted by acid mine drainage (AMD), focusing on interactions with secondary minerals during Schwertmannite (Sch) dissolution and recrystallization. TP-BDOM, rich in lignin-like substances, slowed down the Cr-Sch dissolution and Cr release under acidic conditions compared to TS-BDOM. TP-BDOM's higher O/C component exerts a delayed impact on Cr-Sch stability and Cr(VI) reduction. In-situ ATR-FTIR and 2D-COS analysis showed that carboxylic and aromatic N-OH groups in BDOM could interact with Cr-Sch surfaces, affecting sulfate and Cr(VI) release. It was also observed that slight recrystallization occurred from Cr-Sch to goethite, along with increased Cr incorporation into secondary minerals within TS-BDOM. This enhances our understanding of BDOM's role in Cr(VI) speciation changes in AMD-contaminated sites.

Self-assembled thymol-betaine co-crystals with controlled release and hygroscopic properties as green preservatives for aflatoxin prevention.

Mycotoxins are representative contaminants causing food losses and food safety problems worldwide. Thymol can effectively inhibit pathogen infestation and aflatoxin accumulation during grain storage, but high volatility limits its application. Here, a thymol-betaine co-crystal system was synthesized through grinding-induced self-assembly. The THY-TMG co-crystal exhibited excellent thermal stability with melting point of 91.2 °C owing to abundant intermolecular interactions. Remarkably, after 15 days at 30 °C, the release rate of thymol from co-crystal was only 55%, far surpassing that of pure thymol. Notably, the co-crystal demonstrated the ability to bind H2O in the environment while controlling the release of thymol, essentially acting as a desiccant. Moreover, the co-crystals effectively inhibited the growth of Aspergillus flavus and the biosynthesis of aflatoxin B1. In practical terms, the THY-TMG co-crystal was successful in preventing AFB1 contamination and nutrients loss in peanuts, thereby prolonging their shelf-life under conditions of 28 °C and 70% RH.

Regulation of fat crystals in water-in-oil emulsions by high-intensity ultrasound: Crystal size and tracing of droplet distribution.

In this paper, two emulsion systems with high and low solid fat contents were prepared from 20 % water phase and 80 % oil phase by adjusting the palm oil/palm stearin/soybean oil ratio. Different ultrasonic power and time were used for the pretreatment of emulsion with different solid fat content, and the application characteristics of ultrasonic in W/O emulsions were explored and evaluated. Directly using high-intensity ultrasound to prepare fatty emulsions would weaken the hardness and storage modulus G' of the samples. Although ultrasound reduced the size of fat crystals in emulsions, the interaction between water droplets and fat crystals needs to be considered. After ultrasonic treatment, water droplets were difficult to immobilize on the crystal surface and thus acted as an active filler to stabilize the emulsion together with the fat crystal network. In high solid fat emulsion systems, an increase in ultrasound power (from 100 W to 200 W) could more affect the crystallization behavior of fats than an increase in ultrasound duration (from 30 s to 60 s), and the distribution of crystals and droplets was more uniform. In the low solid fat emulsion system, the texture of the sample after ultrasonic treatment was softer, and the surface was more delicate and smoother. However, the higher ultrasonic intensity (200 W) was not conducive to the preparation of the spread. Although the ultrasound with excessive intensity promoted the formation of small crystals, it would also lead to the aggregation of small crystals. These small crystals cannot form a uniform crystal network, which increases the fluidity of emulsions.

Mechanical properties of wax-oleogels: Assessing their potential to mimic commercial margarine functionality under small and large deformations.

Utilizing waxes to gel oils presents a viable approach for diminishing trans and saturated fat levels in commercial fats such as margarines. This technique ensures that oleogels mimic traditional fats in terms of rheological properties, oil-binding capacity, and overall structure. Our study employed cooling-shear rates to finely adjust physical characteristics, evaluating rheology via SAOS-LAOS, oil retention, and crystal structure of wax oleogels, compared against commercial margarines as benchmarks. Findings indicate that wax oleogels, under specific cooling/shear conditions, exhibit softer yet more ductile-like behavior, akin to margarine, while retaining oil effectively. This similarity is evidenced through Lissajous curves and plastic dissipation ratio during yielding, reflecting a ductile yielding response characterized by square-like Lissajous curves and a plastic dissipation ratio index approximating one. Although these crystallization conditions influence the mechanical properties of wax oleogels, they do not alter oil losses or wax characteristics.

Production of Omega-3 Fatty Acid Concentrates from Common Kilka Oil: Optimization of the Urea Complexation Process.

There has been an increase in interest in the application of ω-3 PUFAs, especially EPA and DHA, in the development of various food products owing to their myriad health benefits. However, most fish oils do not contain more than 30% combined levels of EPA and DHA. In this study, through the urea complexation procedure, the production of EPA and DHA concentrate in their free fatty acids (FFAs) form was achieved from an enzymatic oil extracted from common kilka (Clupeonella cultriventris caspia). To gain the maximum value of EPA and DHA, the response surface methodology (RSM), which is an effective tool to categorize the level of independent variables onto the responses of an experimental process, was also used. Different variables including the urea-fatty acids (FAs) ratio (in the range of 2-6, w/w), the temperature of crystallization (in the range of -24-8 °C), and the time of crystallization (in the range of 8-40 h) were investigated by response surface methodology (RSM) for maximizing the EPA and DHA contents. Following the model validation, the levels of the variables at which the maximum desirability function (0.907 score) was obtained for response variables were 5:1 (urea-FAs ratio), -9 °C (the temperature of crystallization), and 24 h (the time of crystallization). Under these optimal conditions, increases of 2.2 and 4.4 times in the EPA and DHA concentrations were observed, respectively, and an increase in the concentrations of EPA and DHA from 5.39 and 13.32% in the crude oil to 12.07 and 58.36% in the ω-3 PUFA concentrates were observed, respectively. These findings indicate that the urea complexation process is efficient at optimizated conditions.

Effect of mPEG-PLGA on Drug Crystallinity and Release of Long-Acting Injection Microspheres: In Vitro and In Vivo Perspectives.

PURPOSE: Traditional progesterone (PRG) injections require long-term administration, leading to poor patient compliance. The emergence of long-acting injectable microspheres extends the release period to several days or even months. However, these microspheres often face challenges such as burst release and incomplete drug release. This study aims to regulate drug release by altering the crystallinity of the drug during the release process from the microspheres. METHODS: This research incorporates methoxy poly(ethylene glycol)-b-poly(lactide-co-glycolide) (mPEG-PLGA) into poly(lactide-co-glycolide) (PLGA) microspheres to enhance their hydrophilicity, thus regulating the release rate and drug morphology during release. This modification aims to address the issues of burst and incomplete release in traditional PLGA microspheres. PRG was used as the model drug. PRG/mPEG-PLGA/PLGA microspheres (PmPPMs) were prepared via an emulsification-solvent evaporation method. Scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), and differential scanning calorimetry (DSC) were employed to investigate the presence of PRG in PmPPMs and its physical state changes during release. RESULTS: The addition of mPEG-PLGA altered the crystallinity of the drug within the microspheres at different release stages. The crystallinity correlated positively with the amount of mPEG-PLGA incorporated; the greater the amount, the faster the drug release from the formulation. The bioavailability and muscular irritation of the long-acting injectable were assessed through pharmacokinetic and muscle irritation studies in Sprague-Dawley (SD) rats. The results indicated that PmPPMs containing mPEG-PLGA achieved low burst release and sustained release over 7 days, with minimal irritation and self-healing within this period. PmPPMs with 5% mPEG-PLGA showed a relative bioavailability (Frel) of 146.88%. IN CONCLUSION: In summary, adding an appropriate amount of mPEG to PLGA microspheres can alter the drug release process and enhance bioavailability.

Drosophila immune cells transport oxygen through PPO2 protein phase transition.

Insect respiration has long been thought to be solely dependent on an elaborate tracheal system without assistance from the circulatory system or immune cells1,2. Here we describe that Drosophila crystal cells-myeloid-like immune cells called haemocytes-control respiration by oxygenating Prophenoloxidase 2 (PPO2) proteins. Crystal cells direct the movement of haemocytes between the trachea of the larval body wall and the circulation to collect oxygen. Aided by copper and a neutral pH, oxygen is trapped in the crystalline structures of PPO2 in crystal cells. Conversely, PPO2 crystals can be dissolved when carbonic anhydrase lowers the intracellular pH and then reassembled into crystals in cellulo by adhering to the trachea. Physiologically, larvae lacking crystal cells or PPO2, or those expressing a copper-binding mutant of PPO2, display hypoxic responses under normoxic conditions and are susceptible to hypoxia. These hypoxic phenotypes can be rescued by hyperoxia, expression of arthropod haemocyanin or prevention of larval burrowing activity to expose their respiratory organs. Thus, we propose that insect immune cells collaborate with the tracheal system to reserve and transport oxygen through the phase transition of PPO2 crystals, facilitating internal oxygen homeostasis in a process that is comparable to vertebrate respiration.

Highly Soluble Dacarbazine Multicomponent Crystals Less Prone to Photodegradation.

Dacarbazine (DTIC) is a widely prescribed oncolytic agent to treat advanced malignant melanomas. Nevertheless, the drug is known for exhibiting low and pH-dependent solubility, in addition to being photosensitive. These features imply the formation of the inactive photodegradation product 2-azahypoxanthine (2-AZA) during pharmaceutical manufacturing and even drug administration. We have focused on developing novel DTIC salt/cocrystal forms with enhanced solubility and dissolution behaviors to overcome or minimize this undesirable biopharmaceutical profile. By cocrystallization techniques, two salts, two cocrystals, and one salt-cocrystal have been successfully prepared through reactions with aliphatic carboxylic acids. A detailed structural study of these new multicomponent crystals was conducted using X-ray diffraction (SCXRD, PXRD), spectroscopic (FT-IR and 1H NMR), and thermal (TG and DSC) analyses. Most DTIC crystal forms reported display substantial enhancements in solubility (up to 19-fold), with faster intrinsic dissolution rates (from 1.3 to 22-fold), contributing positively to reducing the photodegradation of DTIC in solution. These findings reinforce the potential of these new solid forms to enhance the limited DTIC biopharmaceutical profile.

Unlocking the potential of flavonoid-based co-crystal and co-amorphous systems.

Flavonoids are polyphenolic compounds that have multiple benefits in treating various life-threatening diseases. Despite their diverse pharmacological activities, the market potential of flavonoids is hampered due to their poor solubility and low bioavailability after oral administration. The current review highlights the role of co-crystals and co-amorphous systems (CAMs) in enhancing the solubility, permeability, bioavailability, and therapeutic efficacy of flavonoids. It also explains the significance of flavonoid-based co-formers in the formation of co-crystals and CAMs with other APIs to improve their efficacy. Future perspectives, patented formulations, commercial medications (including their phases of clinical trials), and challenges associated with the use of flavonoid-based co-crystals and CAMs are also mentioned in the review.

Melt-spun polylactide/ethylene vinyl alcohol copolymer fiber.

Polylactide/ethylene vinyl alcohol copolymer (PLA/EVOH) blends and fibers with different weight ratios were prepared by melt blending, and two-step melt spinning, respectively. PLA and EVOH in PLA/EVOH blends were immiscible. When EVOH content was ≤60 %, EVOH with the average diameter of about 3 µm was dispersed in PLA matrix uniformly. The dual continuous phases could be observed in PLA/EVOH blend with 70 wt% EVOH. When the EVOH content was ≥80 %, the spherical PLA phase with the diameter of 0.25 to 1 µm was dispersed in EVOH matrix. The introduction of EVOH as nucleating agent could promote the crystallization of PLA. Both PLA and EVOH components in PLA/EVOH blends formed individual crystal phases. The viscosity of PLA/EVOH blend with 5 % EVOH was lower than that of neat PLA. The viscosity of PLA/EVOH blends with the EVOH content of ≥10 % was much higher than that of neat PLA, which showed obvious shear thinning behavior. With the increase of EVOH content, the shear thinning behavior became obvious and the critical shear rate decreased gradually. The drawn PLA/EVOH fibers with the tensile strength of ≥16 cN/tex exhibited good mechanical properties. In addition, the introduction of EVOH could improve the hydrophilicity of PLA fibers.

High-throughput structure determination of an intrinsically disordered protein using cell-free protein crystallization.

Intrinsically disordered proteins (IDPs) play a crucial role in various biological phenomena, dynamically changing their conformations in response to external environmental cues. To gain a deeper understanding of these proteins, it is essential to identify the determinants that fix their structures at the atomic level. Here, we developed a pipeline for rapid crystal structure analysis of IDP using a cell-free protein crystallization (CFPC) method. Through this approach, we successfully demonstrated the determination of the structure of an IDP to uncover the key determinants that stabilize its conformation. Specifically, we focused on the 11-residue fragment of c-Myc, which forms an α-helix through dimerization with a binding partner protein. This fragment was strategically recombined with an in-cell crystallizing protein and was expressed in a cell-free system. The resulting crystal structures of the c-Myc fragment were successfully determined at a resolution of 1.92 Å and we confirmed that they are identical to the structures of the complex with the native binding partner protein. This indicates that the environment of the scaffold crystal can fix the structure of c-Myc. Significantly, these crystals were obtained directly from a small reaction mixture (30 µL) incubated for only 72 h. Analysis of eight crystal structures derived from 22 mutants revealed two hydrophobic residues as the key determinants responsible for stabilizing the α-helical structure. These findings underscore the power of our CFPC screening method as a valuable tool for determining the structures of challenging target proteins and elucidating the essential molecular interactions that govern their stability.