Correlations of Crystal Structure and Solubility in Organic Salts: The Case of the Antiplasmodial Drug Piperaquine

Five organic salts of the antiplasmodial drug piperaquine (PQ, C29H32Cl2N6) were synthesized and characterized by X-ray diffraction methods. The corresponding solubilities in water and acetic acid solutions were evaluated in the 20-50 \ub0C (293-323 K) T range by UV-vis spectroscopy, with the aim of elucidating how they depend on chemical, structural, and thermodynamic factors. Experiments were complemented by DFT calculations, both in vacuo and in the solid state, to estimate changes in thermodynamic state functions related to the solvation process. It is demonstrated that solubility is mainly governed by the electronic and chemical properties of the anion, while lattice energies and packing effects, including in-crystal conformational changes of the drug, play a less important role. PQ salts generally conform to the predictions of hard and soft acid and bases (HSAB) theory, as less soluble compounds bear ions of comparable hardness, and vice versa. A remarkable exception is the PQ hydrogen sulfate salt, whose poor solubility can be ascribed to an exceptionally stable crystal lattice. Other factors, such as entropic effects related to solid-state disorder, can influence the response of solubility to temperature

Change in Trust in US Government Health Agencies for Cancer Information in the COVID-19 Era

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Study of the key interactions in the self-recognition of the antimalarial drug chloroquine

Malaria is a parasitic disease that causes thousands of deaths every year, especially in undeveloped countries. The Plasmodium protozoa, responsible of the infection, kill human red blood cells by digesting hemoglobin. Many compounds have been employed in the last century against malaria, but nowadays the increasing resistance of Plasmodium is becoming a very serious problem. New drugs are required and to this end it is desirable to quantitatively understand the role of different functional groups in determining effective pharmacophores. This work focuses on chloroquine (CQ), a 4-aminoquinoline antiplasmodial whose effectiveness is now hampered by evolved parasite resistance. It is accepted that CQ interferes with a crucial detoxification process of the parasite [1], namely the inhibition of heme bio-crystallization, but several details of this process still remain rather obscure. In the acidic digestive vacuole of Plasmodium, CQ is supposed to interact in its diprotonated form directly with the monomeric heme in two possible ways: 1) \u3c0-\u3c0 stacking interactions between quinoline ring and heme proto-porphyrin [2] or 2) a direct Fe-N quinoline coordinative bond, supported by strong charge-assisted hydrogen bonds (CAHBs) between the tertiary amine of CQ and the propionate groups of heme [3-4]. In this work, the self-recognition of chloroquine diphosphate dihydrate salt was studied both theoretically and experimentally. High-resolution single crystal X-ray data were collected at low temperature (103 K) and complemented by quantum simulations with CRYSTAL14 [5] at the B3LYP/6-31G(p,d) theory level. The salt crystalizes in a P21/c structure, with phosphate ions forming infinite chains parallel to the b axis. CQ molecules and phosphates are connected through strong N-H\u2022\u2022\u2022O CAHBs, while a \u3c0-\u3c0 interaction is present between the quinoline rings (see figure). The topological analysis of the primary charge density, performed according with the Quantum Theory of Atoms in Molecules [6], along with the ab-initio energy decomposition, show that the coulombic interactions between the charged hydrocarbon chain of CQ and the phosphate ions seem to provide the dominant features in the molecular self-recognition, while the \u3c0-\u3c0 stacking between the quinoline moieties has just an ancillary role. These evidences suggest that, in agreement with our previous DFT/EXAFS results [3], the protonated tertiary amine of CQ is an essential component of the drug pharmacophore. [1] A. F. G. Slater, W. J. Swiggard, B. R. Orton, W. D. Flitter, D. E. Goldberg. A. Cerami and G. B. Henderson, Proc. Natl. Acad. Sci. USA 1991, 88, 325 [2] M. S. Walckzak, K. Lawniczak-Jablonska, A. Wolska, A. Sienkiewicz, L. Suarez, A. J. Kosar and D. S. Bohle, J. Phys. Chem. B 2011, 115, 1145 [3] G. Macetti, S. Rizzato, F. Beghi, L. Silvestrini and L. Lo Presti, Physica Scripta 2016, 91, 023001 [4] A. C. De Dios, R. Tycko, L. M. B. Ursos and P. D. Roepe, J. Phys. Chem. A 2003, 107, 5821 [5] R. Dovesi, V. R. Saunders, C. Roetti, R. Orlando, C. M. Zicovich-Wilson, F. Pascale, B. Civalleri, K. Doll, N. M. Harrison, I. J. Bush, P. D\u2019Arco, M. Llunell, M. Caus\ue0 and Y. No\uebl, CRYSTAL14 2014 CRYSTAL14 User's Manual. University of Torino, Torino [6] R. F. W. Bader, Atoms in molecules. A quantum theory 1990, Oxford University Press. Oxford, U.K

Intermolecular Recognition of the Antimalarial Drug Chloroquine : A Quantum Theory of Atoms in Molecules–Density Functional Theory Investigation of the Hydrated Dihydrogen Phosphate Salt from the 103 K X-ray Structure

The relevant noncovalent interaction patterns responsible for intermolecular recognition of the antiplasmodial chloroquine (CQ) in its bioactive diprotonated form, CQH22+, are investigated. Chloroquine dihydrogen phosphate hydrated salt (P21/c) was crystallized by gel diffusion. A high-resolution single-crystal X-ray diffraction experiment was performed at 103(2) K, and a density functional theory model for the in-crystal electron density was derived, allowing the estimation of the interaction energies in relevant molecular pairs. H2PO4\u2013 ions form infinite chains parallel to the monoclinic axis, setting up strong NH\ub7\ub7\ub7O charge-assisted hydrogen bonds (CAHBs) with CQH22+. Couples of facing protonated quinoline rings are packed in a \u3c0\ub7\ub7\ub7\u3c0 stacked arrangement, whose contribution to the interaction energy is very low in the crystal and completely overwhelmed by Coulomb repulsion between positive aromatic rings. This questions the ability of CQ in setting up similar stacking interactions with the positively charged Fe-protoporphyrin moiety of the heme substrate in solution. When the heme/CQ adduct incorporates a Fe\u2013N coordinative bond, stronger \u3c0\ub7\ub7\ub7\u3c0 interactions are instead established due to the lacking of net electrostatic repulsions. Yet, CAHBs among the protonated tertiary amine of CQ and the propionate group of heme still provide the leading stabilizing effect. Implications on possible modifications/improvements of the CQ pharmacophore are discussed

Experimental and theoretical study of the mechanism of action of the antimalarial drug chloroquine

Malaria is due to the Plasmodium protozoon. The parasite infects human red blood cells, where it digests hemoglobin, in the end releasing free heme (Fe-protoporphyrin IX, FePPIX) in the cytosol. There, FePPIX could produce reactive oxygen species which are toxic to the parasite. As a defense mechanism, the protozoon deactivates FePPIX by promoting its biocrystallization into hemozoin, a triclinic harmless solid. It is largely accepted that 4-aminoquinoline (AQ) drugs, and in particular the low-cost compound chloroquine (CQ), interfere with this detoxification process [1], but no unequivocal evidences on their mechanism of action still exist [2,3]. Moreover, emerging parasite resistance made CQ ineffective in the last decades. In this context, understanding the mechanism of action of chloroquine is a key point to develop novel cheap CQ-based compounds, exploitable in large-scale health campaigns, able to thwart the parasite resistance. In our very recent work [4], evidences of the existence of a direct Fe \u2013 N coordinative bond between CQ and heme in solution have been obtained. DFT calculations highlighted that charge-assisted hydrogen bonds (CAHBs) among the hydrocarbon chain of CQ and the propionate groups of heme play a crucial role in the molecular recognition, particularly in the presence of lipidic micelles. In this contribution we report on a high-resolution low-temperature single crystal X-ray diffraction study on the chloroquine diphosphate dihydrate salt. CQ crystallizes as P21/c, with dihydrogenphosphate ions (H2PO4\u2013) forming infinite chains parallel to the monoclinic axis. Doubly protonated CQ molecules, CQH22+, and H2PO4\u2013 are connected through strong N-H\u2022\u2022\u2022O CAHBs. A \u3c0-\u3c0 stacking interaction could be also set up between the quinoline rings, which lie parallel to the (a,c) plane to occupy as much as possible the free space between phosphate chains. From the molecular recognition viewpoint, \u3c0-\u3c0 stacking in the CQ crystal could be taken as a model for the \u3c0-\u3c0 CQ:heme interaction in solution, described in the literature as a possible way of interaction between the drug and its substrate, while negatively charged the phosphate ions behave as propionate groups in heme. The study of the CQ self-recognition energies through the analysis of the primary charge density confirm the hypothesis that the coulombic interactions between CQ and the phosphate are the real dominant ones, while the stacking between the quinoline moieties has just an ancillary role. These evidences further confirm that the protonated tertiary amine of CQ is an essential component of the drug pharmacophore. [1] A. F. G. Slater, W. J. Swiggard, B. R. Orton, W. D. Flitter, D. E. Goldberg. A. Cerami, G. B. Henderson Proc. Natl. Acad. Sci. USA 1991, 88, 325-329. [2] M. S. Walckzak, K. Lawniczak-Jablonska, A. Wolska, A. Sienkiewicz, L. Suarez, A. J. Kosar, D. S. Bohle J. Phys. Chem. B, 2011, 115, 1145-1150. [3] A. C. De Dios, R. Tycko, L. M. B. Ursos, P. D. Roepe J. Phys. Chem. A, 2003, 107, 5821-5825. [4] G. Macetti, S. Rizzato, F. Beghi, L. Silvestrini, L. Lo Presti Physica Scripta 2016, 91, 023001, 1-13

Correlations among solubility and crystal structure: a crystallographic and spectroscopic study of the antimalarial drug piperaquine

Malaria is caused by the Plasmodium protozoa, which infect human through the bite of Anopheles mosquitoes. After entering the erythrocytes, Plasmodium digests the hemoglobin, releasing free toxic heme (Fe(III)-protoporphyrin-IX) into its digestive vacuole (DV). The dimerization of the free heme and its subsequent sequestration into triclinic hemozoin crystals is an effective way for the parasite to attain its detoxification. 4-Aminoquinoline (4-AQ) type drugs are believed to interfere with this process, either by hampering the dimerization of the heme in solution or by inhibiting the growth of the hemozoin crystals.[1] Piperaquine (PQ) is a 4-AQ drug, nowadays used as a phosphate salt in expensive ACTs (Artemisinin Combined Therapies) for the treatment of malaria in areas where drug-resistant parasites are present.[2] The understanding of the intermolecular interactions that are involved in the molecular action of these drugs is essential for the design of novel, effective and cheap alternatives. Purposes of the present work are (i) to study the self-recognition of PQ in the solid state, to identify the leading non-covalent interactions that determine how the charged drug recognizes its environment, and (ii) to correlate structural and energetic aspects of the crystal packing with the measured solubility of PQ crystals. To these ends, we report for the first time the structures, together with the corresponding crystallization methods from aqueous solvents, of various PQ4+ salts with H2PO4-, NO3\u2013, HSO4\u2013, Br\u2013 and SO3CF3- anions. The crystals were prepared starting from neutral PQ, that was dissolved in concentrated solutions of the corresponding inorganic acid. Crystals were then grown by slow evaporation of solvent. The structures were solved by single-crystal X-ray diffraction in the 120 K \u2013 RT T range. The electrostatic nature of the dominant interactions was confirmed with the use of A. Gavezzotti\u2019s AA-CLP method,[3] while the main stabilizing contacts were highlighted using Hirshfeld surface fingerprint plots.[4] It turns out that the structure-determining interactions are (i) charge-assisted hydrogen bonds (CAHBs) among the charged N-H donors and the negatively charged counterions and, possibly, (ii) \u3c0-\u3c0 interactions between PQ quinoline rings. Similar interactions were also found to be relevant in the molecular recognition pattern of Chloroquine phosphate,[5,6] another important 4-AQ class drug, and can therefore play an important role in the heme-drug recognition process. Structure-solubility relations in the PQ salts were explored in the 293\u2013323 K T range by means of UV-Vis Spectroscopy in water. Correlations with DFT estimates of the lattice energies by solid-state quantum mechanical calculations were rationalized in terms of relevant packing features and discussed in the context of investigating the possible utility of the newly synthesized crystal structures in practical pharmaceutical formulations. Acknowledgments: This research was funded by the Development Plan of Athenaeum (Universit\ue0 degli Studi di Milano) \u2013 Line 2, Action B, project NOVAQ (Understanding structure-function relationships in 4-aminoquinoline drugs: an experimental and theoreti-cal route toward novel antimalarials), n\uba PSR2015-1716FDEMA_08 1 Olafson et al., Proc. Natl. Acad. Sci. 2017, 114(29), 7531 2 Davis et al., Drugs 2005, 65(1), 75 3 Gavezzotti, A. New J. Chem. 2011, 35(7), 1360 4 McKinnon et al., Acta Crystallographica Section B: Structural Science. 2004, 60, 627 5 Macetti et al., Cryst. Growth Des. 2016, 16 (10), 6043 6 Macetti et al., Phys. Scr. 2016, 91 (2), 2300

On the interplay among non-covalent interactions and activity of 4-aminoquinoline antimalarials: a crystallographic and spectroscopic study

Malaria is due to protozoa of the genus Plasmodium, which infect human red blood cells and digest the host hemoglobin. Degradation of the latter in the acidic food vacuole (pH ~ 5) releases free hematin (hydroxylated Fe protoporphyrin-IX, FePPIX(OH)), which is toxic to the parasite [1]. Therefore, Plasmodium deactivates hematin by promoting its crystallization into harmless pale yellow P1bar crystals of beta-hematin. 4-aminoquinoline drugs (AQ), such as chloroquine (CQ) and piperaquine (PQ), interfere with this detoxification process, either by coordinating free heme in solution [2], or by poisoning fastest-growing crystal faces of beta-hematin [3]. However, there is no general consensus on the structure of the AQ/heme complex [4], which depends on various chemical variables (aqueous/lipidic environment, pH). We here aim at quantitatively disclosing the chemical physics underlying the pharmacophoric features of CQ and PQ in the context of predicting which chemical modifications should be applied on the AQ scaffold to enhance the drug functionality against the biochemical resistance mechanism evolved by Plasmodium [5,6]. EXAFS spectroscopy in solution across the Fe Kalpha absorption edge (~ 7.1 keV) explored the first shell coordination geometry of iron in hematin, both in the presence and in the absence of AQ systems. Differences in the signal were related to the possible occurrence of a direct Fe\u2013N coordinative bond involving the quinoline nitrogen atom, which might coexist with other possible (e.g. pi\ub7\ub7\ub7pi stacked) adduct geometries [5] (Fig. 1). Quantum mechanical DFT calculations showed that an aliphatic tertiary NH+ amino group might also be a crucial part of the pharmacophore (Fig. 1), as it is able to set up strong charge-assisted hydrogen bonds with proprionate groups of hematin. This complies well with single-crystal X-ray diffraction outcomes on the CQ dihydrogen phosphate salt at 103 K[6], where H2PO4\u2013 ions form hydrogen-bonded pillars which strongly interact with positively charged chloroquine molecules. Comparison of the CQ crystal structure with those of various hydrated salts of PQ (NO3\u2013, SO42\u2013, H2PO4\u2013), grown by advanced sol-gel methods, disclosed subtle analogies and differences in the non-covalent interaction networks of the two drugs, which are also related to their solubilities. [1] L. Ko\u159en\ufd, M. Oborn\uedk, J. Luke\u161, PLoS Pathog. 2013, 9(1), e1003088. [2] D.C. Warhurst J.C. Craig, K.S. Raheem, Biochem. Pharmacol. 2007, 73, 1910. [3] M.S.Walczak, K. Lawniczak-Jablonska, A. Wolska, A. Sienkiewicz, L. Suarez, A.J. Kosar, D.S. Bohle J. Phys. Chem. B 2011, 115, 1145. [4] J. Gildenhuys, T. Roex, T.J. Egan, K.A. De Villiers, J. Am. Chem. Soc. 2013, 135, 1037. [5] G. Macetti, S. Rizzato, F. Beghi, L. Silvestrini, L. Lo Presti, Physica Scripta 2016, 91, 023001. [6] G. Macetti, L. Loconte, S. Rizzato, C. Gatti, L. Lo Presti, Crystal Growth Des. 2016. 16, 6043

Functional evidence of mTORβ splice variant involvement in the pathogenesis of congenital heart defects

mTOR dysregulation has been described in pathological conditions, such as cardiovascular and overgrowth disorders. Here we report on the first case of a patient with a complex congenital heart disease and an interstitial duplication in the short arm of chromosome 1, encompassing part of the mTOR gene. Our results suggest that an intragenic mTOR microduplication might play a role in the pathogenesis of non-syndromic congenital heart defects (CHDs) due to an upregulation of mTOR/Rictor and consequently an increased phosphorylation of PI3K/AKT and MEK/ERK signaling pathways in patient-derived amniocytes. This is the first report which shows a causative role of intragenic mTOR microduplication in the etiology of an isolated complex CHD

Molecular and Functional Characterization of Three Different Postzygotic Mutations in PIK3CA-Related Overgrowth Spectrum (PROS) Patients: Effects on PI3K/AKT/mTOR Signaling and Sensitivity to PIK3 Inhibitors

BACKGROUND PIK3CA-related overgrowth spectrum (PROS) include a group of disorders that affect only the terminal portion of a limb, such as type I macrodactyly, and conditions like fibroadipose overgrowth (FAO), megalencephaly-capillary malformation (MCAP) syndrome, congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, skeletal and spinal anomalies (CLOVES) syndrome and Hemihyperplasia Multiple Lipomatosis (HHML). Heterozygous postzygotic PIK3CA mutations are frequently identified in these syndromes, while timing and tissue specificity of the mutational event are likely responsible for the extreme phenotypic variability observed. METHODS: We carried out a combination of Sanger sequencing and targeted deep sequencing of genes involved in the PI3K/AKT/mTOR pathway in three patients (1 MCAP and 2 FAO) to identify causative mutations, and performed immunoblot analyses to assay the phosphorylation status of AKT and P70S6K in affected dermal fibroblasts. In addition, we evaluated their ability to grow in the absence of serum and their response to the PI3K inhibitors wortmannin and LY294002 in vitro. RESULTS AND CONCLUSION: Our data indicate that patients' cells showed constitutive activation of the PI3K/Akt pathway. Of note, PI3K pharmacological blockade resulted in a significant reduction of the proliferation rate in culture, suggesting that inhibition of PI3K might prove beneficial in future therapies for PROS patients