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Here, we show that 3,5-bis[(1E)-2-(2,6-dichlorophenyl)ethenyl]-1H-pyrazole 2l depolymerizes microtubules and reduces the number of growing tips of microtubules. The fluorescence recovery after photobleaching experiment in live MCF-7 cells showed that pyrazole 2l suppresses spindle microtubule dynamics. Further, the compound inhibits chromosome movements, activates the spindle assembly checkpoint and blocks mitosis in MCF-7 cells. Pyrazole 2l treatment induced cell death in a variety of pathways. Pyrazole 2l induces cell death independent of BubR1 and p53 levels of MCF-7 cells upon microtubule depolymerization. Further, pyrazole 2l increases the interaction between NF-κB and microtubules and enhances the nuclear localization of NF-κB at its half-maximal proliferation inhibitory concentration while a high concentration of the compound reduced the nuclear localization of NF-κB. Interestingly, the compound exerted significantly stronger antiproliferative effects in cancerous cells than in non-cancerous cells. The results indicated that pyrazole 2l inhibits mitosis by targeting microtubules, induces several types of cell death stimuli and suggests its potential as a lead in developing anticancer agent.

Supplementary Table S1 from Class I β-tubulin mutations in 2-methoxyestradiol-resistant acute lymphoblastic leukemia cells: implications for drug-target interactions

Supplementary Fig. S1 legend from Class I β-tubulin mutations in 2-methoxyestradiol-resistant acute lymphoblastic leukemia cells: implications for drug-target interactions

2-Methoxyestradiol (2ME2) is a naturally occurring derivative of estradiol that has been shown to be an active small molecule that has antitumor and antiangiogenic properties. 2ME2 binds to β-tubulin near the colchicine-binding site, inhibits microtubule polymerization, and induces mitotic arrest. To improve understanding of the mechanisms of action and resistance to 2ME2, we selected leukemia cells, CCRF-CEM, that display increasing resistance to 2ME2, and three of the highly resistant sublines were chosen for detailed analysis. The 2ME2 cells selected in 7.2 to 28.8 μmol/L were found to be 47- to 107-fold resistant to 2ME2 and exhibited low levels of cross-resistance to vinblastine. Two of the lowest 2ME2-resistant sublines were significantly hypersensitive to colchicine and epothilone B, but the hypersensitive effects were lost in the highest 2ME2-resistant subline. Moreover, 2ME2-resistant cells require 10-fold higher concentrations of 2ME2 to induce G2-M cell cycle arrest and have higher amounts of tubulin polymer compared with parental cells. Gene and protein sequencing revealed four class I β-tubulin mutations, S25N, D197N, A248T, and K350N, in the 2ME2-resistant cells. The S25N mutation is within the paclitaxel-binding site, whereas A248T and K350N are within the colchicine-binding site on β-tubulin, yet the resistant cells were not cross-resistant to paclitaxel or colchicine. This strongly suggests that the mutations have induced conformational changes to the binding site that resulted in 2ME2 resistance. The 2ME2-resistant leukemia cells provide novel insights into microtubule stability and drug-target interactions. [Mol Cancer Ther 2008;7(9):3150–9]

Supplementary Fig. S1 from Class I β-tubulin mutations in 2-methoxyestradiol-resistant acute lymphoblastic leukemia cells: implications for drug-target interactions

Synthetic cannabinoid receptor agonists (SCRAs) remain one the most prevalent classes of new psychoactive substances (NPS) worldwide, and examples are generally poorly characterised at the time of first detection. We have synthesised a systematic library of amino acid-derived indole-, indazole-, and 7-azaindole-3-carboxamides related to recently detected drugs ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA, and characterised these ligands for in vitro binding and agonist activity at cannabinoid receptor subtypes 1 and 2 (CB(1) and CB(2)), and in vivo cannabimimetic activity. All compounds showed high affinity for CB(1) (K(i) 0.299–538 nM) and most at CB(2) (K(i) = 0.912–2190 nM), and most functioned as high efficacy agonists of CB(1) and CB(2) in a fluorescence-based membrane potential assay and a βarr2 recruitment assay (NanoBiT®), with some compounds being partial agonists in the NanoBiT® assay. Key structure–activity relationships (SARs) were identified for CB(1)/CB(2) binding and CB(1)/CB(2) functional activities; (1) for a given core, affinities and potencies for tert-leucinamides (ADB-) > valinamides (AB-) ≫ phenylalaninamides (APP-); (2) for a given amino acid side-chain, affinities and potencies for indazoles > indoles ≫ 7-azaindoles. Radiobiotelemetric evaluation of ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA in mice demonstrated that ADB-BUTINACA and ADB-P7AICA were cannabimimetic at 0.1 mg kg(−1) and 10 mg kg(−1) doses, respectively, as measured by pronounced decreases in core body temperature. APP-BUTINACA failed to elicit any hypothermic response up to the maximally tested 10 mg kg(−1) dose, yielding an in vivo potency ranking of ADB-BUTINACA > ADB-P7AICA > APP-BUTINACA.

Over 200 synthetic cannabinoid receptor agonists (SCRAs) have been identified as new psychoactive substances. Effective monitoring and characterization of SCRAs are hindered by the rapid pace of structural evolution. Ahead of possible appearance on the illicit drug market, new SCRAs were synthesized to complete a systematic library of cumyl-indole- (

Context The demand of palliative care is increasing due to the aging population and treatment hesitancy or intentional avoidance compromises symptom management. Objectives To identify patient beliefs associated with medication hesitancy by using the theory of planned behavior (TPB) namely, attitudes, subjective norms, behavioral intention, and perceived behavioral control associated with medication hesitancy or intentional noncompliance by avoidance. Methods The Preferred Reporting Items for Systematic Reviews and Meta-Analysis guideline was followed to conduct a systematic literature search involving the CINAHL, Embase, MEDLINE, and PsycINFO databases from inception until March 2022. Hand-searched articles from reference lists and gray literature were included. Thematic analysis was conducted on qualitative data and triangulated with quantitative data. Results About 554 articles were retrieved from the literature search and 17 articles were included based on the eligibility criteria. Three subthemes that were identified under TPB constructs were attitude: negative attitude toward medications, passive attitude toward illness and inaccurate information about disease or medication; one subtheme was identified under subjective norms: perceived negative opinions from others; and one subtheme was identified under perceived behavioral control: perception of manageable symptoms. Quantitative data provided triangulation of qualitative findings related to fear of addiction and side effects, feelings of hopelessness, unclear direction and information, social stigma, endurable symptoms, and illness as determinants for medication avoidance. Significance of results This systematic review highlighted some patient beliefs related to medication hesitancy or avoidance. Clinicians should take patient beliefs and concerns into consideration when creating treatment regimens for people receiving palliative care to optimize medication adherence and the quality of care.

The charge density distribution in a novel cocrystal (1) complex of 1,3-dimethylxanthine (theophylline) and propanedioic acid (malonic acid) has been determined. The molecules crystallize in the triclinic, centrosymmetric space group P1̅, with four independent molecules (Z = 4) in the asymmetric unit (two molecules each of theophylline and malonic acid). Theophylline has a notably high hygroscopic nature, and numerous cocrystals have shown a significant improvement in stability to humidity. A charge density study of the novel polymorph has identified interesting theoretical results correlating the stability enhancement of theophylline via cocrystallization. Topological analysis of the electron density highlighted key differences (up to 17.8) in Laplacian (∇2ρ) between the experimental (EXP) and single-point (SP) models, mainly around intermolecular-bonded carbonyls. Further investigation via molecular electrostatic potential maps reaffirmed that the charge redistribution enhanced intramolecular hydrogen bonding, predominantly for N(2′) and N(2) (61.2 and 61.8 kJ mol–1, respectively). An overall weaker lattice energy of the triclinic form (−126.1 kJ mol–1) compared to that of the monoclinic form (−133.8 kJ mol–1) suggests a lower energy threshold to overcome to initiate dissociation. Future work via physical testing of the novel cocrystal in both dissolution and solubility will further solidify the correlation between theoretical and experimental results.

Antibacterial resistance is a prominent issue with monotherapy often leading to treatment failure in serious infections. Many mechanisms can lead to antibacterial resistance including deactivation of antibacterial agents by bacterial enzymes. Enzymatic drug modification confers resistance to β-lactams, aminoglycosides, chloramphenicol, macrolides, isoniazid, rifamycins, fosfomycin and lincosamides. Novel enzyme inhibitor adjuvants have been developed in an attempt to overcome resistance to these agents, only a few of which have so far reached the market. This review discusses the different enzymatic processes that lead to deactivation of antibacterial agents and provides an update on the current and potential enzyme inhibitors that may restore bacterial susceptibility.

This study investigated the effect of 2-methylimidazole (2-MIM) addition on the fluorescence of ethyl-7-hydroxy-2-oxo-2H-chromene-3-carboxylate using low-cost density functional theory (DFT) and Time-Dependent DFT calculations on single crystal X-ray geometries of ethyl-7-hydroxy-2-oxo-2H-chromene-3-carboxylate hydrate (1), 2-MIM (2), and the 1 : 1 co-crystal of (1) and (2), (3). At low concentrations (1 : 1–1 : 10) of 2-MIM, the fluorophore shows a decrease in the fluorescence intensity, but at higher concentrations (above 1 : 10) the fluorescence excitation maximum shifted from 354 nm to 405 nm, with a significant emission intensity increase. The changed excitation and emission profile at high concentrations is due to the deprotonation of the coumarin's phenolic group, which was confirmed by the increased shielding of the aromatic protons in the titration 1H NMR spectra. The experimental fluorescence data between the 1 : 1 and 1 : 10 ratios agreed with the theoretical fluorescence data, with a redshift and decreased intensity when comparing (1) and (3). The data indicated that combining the fluorophore with 2-MIM increased levels of vibronic coupling between 2-MIM and the fluorophore decreasing de-excitation efficiency. These increased vibronic changes were due to charge transfer between the fluorophore and 2-MIM in (3). The subtle movement of the proton, H(5) toward N(2′) (0.07 Å) caused a significant decrease in fluorescence due to electron density distribution (EDD) changes. This was identified by comparison of the EDD in the excited (S1) and ground (S0) states plotted as an isosurface of EDD difference. For the higher concentrations, an alternative excitation pathway was explored by modifying the crystal geometry of (3) based on 1H NMR spectroscopy data to resemble excitoplexes. Theses excitoplex geometries reflected the fluorescence profile of the fluorophore with high concentrations of 2-MIM; there were dramatic changes in the theoretical fluorescence pathway, which was 100% vibronic coupling compared to 15.31% in the free fluorophore. At this concentration, the de-excitation pathway causes remodelling of the lactone ring via stretching/breaking the C[double bond, length as m-dash]O bond in the S1 causing increased fluorescence by movement of the transition dipole moment. These results reflect previous studies, but the methods used are less experimentally and computationally expensive. This study is among the first to explain charge transfer fluorescence using crystalline geometries. This study will be of interest to the fields of crystal engineering and fluorescence spectroscopy.

The pharmaceutical agent theophylline (THEO) is primarily used as a bronchodilator and is commercially available in both tablet and liquid dosage forms. THEO is highly hygroscopic, reducing its stability, overall shelf-life, and therefore usage as a drug. THEO and dicarboxylic acid cocrystals were designed by Trask et al. in an attempt to decrease the hygroscopic behaviour of THEO; cocrystallisation of THEO with malonic acid (MA) did not improve the hygroscopic stability of THEO in simulated atmospheric humidity testing. The current study employed high-resolution X-ray crystallography, and Density Functional Theory (DFT) calculations to examine the electron density distribution (EDD) changes between the cocrystal and its individual components. The EED changes identified the reasons why the THEO:MA cocrystal did not alter the hygroscopic profile of THEO. The cocrystal was equally porous, with atomic packing factors (APF) similar to those of THEO 0.73 vs. 0.71, respectively. The THEO:MA (1) cocrystal structure is held together by an array of interactions; a heterogeneous synthon between the imidazole and a carboxylic fragment stabilising the asymmetric unit, a pyrimidine-imidazole homosynthon, and an aromatic cycle stack between two THEO moieties have been identified, providing 9.7-12.9 kJ mol−1 of stability. These factors did not change the overall relative stability of the cocrystal relative to its individual THEO and MA components, as shown by cocrystal (1) and THEO being equally stable, with calculated lattice energies within 2.5 kJ mol−1 of one other. The hydrogen bond analysis and fragmented atomic charge analysis highlighted that the formation of (1) combined both the EDD of THEO and MA with no net chemical change, suggesting that the reverse reaction — (1) back to THEO and MA — is of equal potential, ultimately producing THEO hydrate formation, in agreement with the work of Trask et al. These results highlight that a review of the EDD change associated with a chemical reaction can aid in understanding cocrystal design. In addition, they indicate that cocrystal design requires further investigation before becoming a reliable process, with particular emphasis on identifying the appropriate balance of synthon engineering, weak interactions, and packing dynamics.

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS). They commonly comprise

GABAϱ receptors are distinctive GABAergic receptors from other ionotropic GABAA and metabotropic GABAB receptors in their pharmacological, biochemical, and electrophysiological properties. Although GABA-ϱ1 receptors are the most studied in this subfamily, GABA-ϱ2 receptors are widely distributed in the brain and are considered a potential target for treating neurological disorders such as stroke. The structure of GABA-ϱ2 receptors and their pharmacological features are poorly studied. We generated the first homology model of GABA-ϱ2 channel, which predicts similar major interactions of GABA with the binding-site residues in GABA-ϱ1 and GABA-ϱ2 channels. We also investigated the pharmacological properties of several GABA analogues on the activity of GABA-ϱ2 receptors. In comparison to their pharmacological effect on GABA-ϱ1 receptors, the activation effect of these ligands and their potentiation/inhibition impact on GABA response have interestingly shown inter-selectivity between the two GABA-ϱ receptors. Our results suggest that several GABA analogues can be used as research tools to study the distinctive physiology of GABA-ϱ1 and GABA-ϱ2 receptors. Furthermore, their partial agonist effect may hold promise for the future discovery of selective modulatory agents on GABAA receptors.

The charge density distribution in a novel cocrystal (1) complex of 1,3-dimethylxanthine (theophylline) and propanedioic acid (malonic acid) has been determined. The molecules crystallize in the triclinic, centrosymmetric space group

Synthetic cannabinoid receptor agonists (SCRAs) remain a prolific class of new psychoactive substances (NPS) and continue to expand rapidly. Despite the recent identification of hydroxybenzotriazole (HOBt) containing SCRAs in synthetic cannabis samples, there is currently no information regarding the pharmacological profile of these NPS with respect to human CB1 and CB2 receptors. In the current study, a series consisting of seven HOBt indole-, indazole-, and 7-azaindole-carboxylates bearing a range of N-alkyl substituents were synthesized and pharmacologically evaluated. Competitive binding assays at CB1 and CB2 demonstrated that all analogues except a 2-methyl-substituted derivative had low affinity for CB1 (Ki = 3.80-43.7 μM) and CB2 (Ki = 2.75-18.2 μM). A fluorometric functional assay revealed that 2-methylindole- and indole-derived HOBt carboxylates were potent and efficacious agonists of CB1 (EC50 = 12.0 and 63.7 nM; Emax = 118 and 120%) and CB2 (EC50 = 10.9 and 321 nM; Emax = 91 and 126%). All other analogues incorporating indazole and 7-azaindole cores and bearing a range of N1-substituents showed relatively low potency for CB1 and CB2. Additionally, a reporter assay monitoring β-arrestin 2 (βarr2) recruitment to the receptor revealed that the 2-methylindole example was the most potent and efficacious at CB1 (EC50 = 131 nM; Emax = 724%) and the most potent at CB2 (EC50 = 38.2 nM; Emax = 51%). As with the membrane potential assay, the indazole and other indole HOBt carboxylates were considerably less potent at both receptors, and analogues comprising a 7-azaindole core showed little activity. Taken together, these data suggest that NNL-3 demonstrates little CB1 receptor activity and is unlikely to be psychoactive in humans. NNL-3 is likely an unintended SCRA manufacturing byproduct. However, the synthesis of NNL-3 analogues proved simple and general, and some of these showed potent cannabimetic profiles in vitro, indicating that HOBt esters of this type may represent an emerging class of SCRA NPS.

CYCLODEXTRINS HAVE BEEN EMPLOYED AS DELIVERY AGENTS FOR LIPOPHILIC ANION TRANSPORTERS, WHICH ALLOW THEIR INCORPORATION INTO LIPID BILAYERS WITHOUT USING AN ORGANIC SOLVENT OR PRE-INCORPORATION.

Two polymorphic forms of caffeine (CAF)–glutaric acid (GLU) cocrystals have been studied via high-resolution X-ray crystallography and Bader’s quantum theory of atoms in molecules (QTAIM). For both the monoclinic, 1, and triclinic, 2, systems the experimental charge density distributions of the 1:1 ratio of CAF and GLU polymorphs have been determined and compared. Previous studies have determined that 1 is less stable than 2, in relative humidity (RH) testing. A topological analysis of the electron density distribution (EDD) revealed little difference between the two polymorph internal systems. The packing densities (0.76 vs 0.74) and lattice energies (−101.1 vs −107.1 kJ mol–1) of 1 and 2, respectively, are nearly equivalent, implying that the differences in hygroscopicity between the two polymorphs are not due to crystal lattice porosity or stability. A topological analysis of the number and strength of hydrogen bonds for 1 and 2 revealed nine hydrogen bonds in both polymorphs. “Classical” (O–H···X) hydrogen bonds were similarly present in both polymorphs, stabilizing the cocrystals. However, the sum of the stability produced from the “nonclassical” (C–H···X) bonds is higher in 2: −27.6 vs −38.2 kJ mol–1 for 1 and 2, respectively. One of the nine hydrogen bonds in 1 and 2 varies from the others, caused by the torsional rotation of the aliphatic carbon chain in GLU. This bond is critical for packing stabilization, creating a parallelogram-like packing arrangement in 2 in comparison to ribboning in 1. A Hirshfeld surface analysis found that the percentages of O–H···X hydrogen bonds were nearly identical in 1 and 2 (23.9% vs 22.1%); however, the H···H contacts were higher in 2 (61.4% vs 65.8% for 1 and 2, respectively), suggesting that more hydrogen-based contacts require competitive displacement by water in the hydration of 2 in comparison to 1. Additionally, a stabilizing aromatic cycle stack between CAF molecules is present in 2 due to the varied parallelogram packing arrangement, which was absent in 1; this provided ∼11.3 kJ mol–1 of stability to the system of 2. The solid-state entropies and molecular dipole moments (MDMs) of 1 and 2 supported the relative stability of the individual polymorphs, with 1 having a higher entropy and dipole moment in comparison to 2 (123.2 vs 112.8 J K–1 mol–1 and 7.45 and 4.93 D for 1 and 2, respectively), implying that it has the potential to hydrate more rapidly. These findings are in good agreement with previous experimental RH stability studies, giving further insight into the information gained from thermally averaged ground-state crystal electron density data.

Carbamazepine (CBZ) is used in the treatment of multiple neurological conditions. Although efficacious, its potential has been limited by its poor solubility, which means that patients are required to take very large doses to gain the desired effect. Co-crystals have been proposed as a means of improving the physicochemical properties of pharmaceutical compounds while maintaining their efficacy. CBZ cocrystallized with saccharin (SAC) and nicotinamide (NIC) have previously been studied, with the CBZ-SAC crystal being more soluble than the commercially available product Tegretol, which only contains CBZ, while the nicotinamide cocrystal was found to be less soluble. High-resolution X-ray crystallography has been carried out on the CBZ-SAC cocrystal and its individual constituents to determine which features of the electron density distribution contribute to the differing physical properties. The number of hydrogen bonds found for the CBZ, SAC, and CBZ-SAC systems were 8, 5, and 10, respectively. Homosynthons (interactions between a pair of identical functional groups) are the primary bonding motif in CBZ and SAC, while a heterosynthon is also present in the cocrystal. Molecular electrostatic potential (MEP) maps show that cocrystallization results in changes in distribution around the carboxamide group, thus accommodating heterosynthon formation and leading to subsequent charge redistribution across the CBZ molecule. Additional lattice energy calculations were not able to provide a definitive answer as to which system was most stable. Solid state entropy calculations revealed that the CBZ-SAC cocrystal had a higher entropy, providing explanations for the lower melting point and improved dissolution profile previously described. These investigations at an electronic level help to explain the greater solubility of the CBZ-SAC cocrystal compared to CBZ alone.

Activation of the CB2 receptor is an attractive therapeutic strategy for the treatment of a wide range of inflammatory diseases. However, receptor subtype selectivity is necessary in order to circumvent the psychoactive effects associated with activation of the CB1 receptor. We aimed to use potent, non-selective synthetic cannabinoids designer drugs to develop selective CB2 receptor agonists. Simple structural modifications such as moving the amide substituent of 3-amidoalkylindole synthetic cannabinoids to the 2-position and bioisosteric replacement of the indole core to the 7-azaindole scaffold are shown to be effective and general strategies to impart receptor subtype selectivity. 2-Amidoalkylindole 16 (EC50 CB1 > 10 μM, EC50 CB2 = 189 nM) and 3-amidoalkyl-7-azaindole 21 (EC50 CB1 > 10 μM, EC50 = 49 nM) were found to be potent and selective agonists with favourable physicochemical properties. Docking studies were used to elucidate the molecular basis for the observed receptor subtype selectivity for these compounds.

Development of neuroinflammation agents targeting the translocator protein (TSPO) has been hindered by a common single nucleotide polymorphism (A147T) at which TSPO ligands commonly lose affinity. To this end, carbazole acetamide scaffolds were synthesized and structure activity relationships elaborated to explore the requirements for high-affinity binding to both TSPO wild type (WT) and the polymorphic TSPO A147T. This study reports high binding affinity and nondiscriminating TSPO ligands.

Ensuring adequate conifer regeneration after high severity wildfires is a common objective for ecologists and forest managers. In the Klamath region of Oregon and California, a global hotspot of botanical biodiversity, concerns over regeneration have led to post-fire management on many sites, which involves salvage logging followed by site preparation, conifer planting, and manual shrub release. To quantify the impacts of post-fire management, we sampled 62 field sites that burned at high severity nearly 20 years ago in the Klamath-Siskiyou Mountain bioregion, stratifying by management and aspect. We measured cover of shrubs and trees and density and frequency of trees and used Nonmetric Multidimensional Scaling to compare community composition, plant community assemblage based on regenerative traits, and density of tree species between aspect and management. On average, shrub cover exceeded the cover of conifers, hardwoods or grasses, regardless of management history or aspect. The average number of species was lower and resprouting species were less abundant on south aspect sites; seed banking species were most abundant on north aspects. Post-fire management was associated with greater cover of seed banking and nitrogen-fixing species but it did not affect diversity. Management had no impact on Douglas-fir regeneration, the main species of concern in the region. Regeneration of ponderosa pine was higher on sites with post-fire management, but only on south slopes. The frequency of Douglas-fir was associated with aspect, while the frequency of ponderosa pine was associated with management. Overall, our study demonstrates the important role that aspect plays in determining the effectiveness of management after high severity wildfires. Indeed, the effect of aspect on site conditions often overwhelmed the ability of management to influence community composition (including different regenerative strategies), species diversity, and regeneration. Managed sites differed from unmanaged sites in several diverse ways with varied implications for longer-term forest development. Managed sites had taller dominant conifers, which suggests that post-fire management could hasten the period needed to achieve fire resistance. Managed sites were similar in plant community composition but had more homogeneous structure--e.g., managed sites had fewer snags, which are an important habitat feature for several bird species in the region. Finally, management was not associated with conifer regeneration success on north slopes, suggesting that interventions may not be needed uniformly across post-fire landscape. New policies of ecosystem-based management in the Klamath region should consider the important interactions between aspect and post-fire management, and tailor management practices based on specific objectives and landscape context.

New treatment strategies are required for cryptococcosis, a leading mycosis in HIV-AIDS patients. Following the identification of Cryptococcus proteins differentially expressed in response to fluconazole, we targeted farnesyl pryrophosphate synthetase (FPPS), an enzyme in the squalene biosynthesis pathway, using nitrogenous bisphosphonates. We hypothesized that these would disrupt squalene synthesis and thereby produce synergy with fluconazole, which acts on a downstream pathway that requires squalene. The susceptibilities of 39 clinical isolates from 6 different species of Cryptococcus were assessed for bisphosphonates and fluconazole, used both independently and in combination. Effective fluconazole-bisphosphonate combinations were then assessed for fungicidal activity, efficacy against biofilms, and ability to resolve cryptococcosis in an invertebrate model. The nitrogenous bisphosphonates risedronate, alendronate, and zoledronate were antifungal against all strains tested. Zoledronate was the most effective (geometric mean MIC = 113.03 mg/liter; risedronate = 378.49 mg/liter; alendronate = 158.4 mg/liter) and was broadly synergistic when combined with fluconazole, with a fractional inhibitory concentration index (FICI) of ≤0.5 in 92% of isolates. Fluconazole and zoledronate in combination were fungicidal in a time-kill assay, inhibited Cryptococcus biofilms, prevented the development of fluconazole resistance, and resolved infection in a nematode model. Supplementation with squalene eliminated bisphosphonate-mediated synergy, demonstrating that synergy was due to the inhibition of squalene biosynthesis. This study demonstrates the utility of targeting squalene synthesis for improving the efficacy of azole-based antifungal drugs and suggests bisphosphonates are promising lead compounds for further antifungal development.

Hyperpigmentation is a common skin condition with serious psychosocial consequences. Decapeptide-12, a novel peptide, has been found to be safer than hydroquinone in reducing content of melanin, with efficacy up to more than 50% upon 16 weeks of twice daily treatment. However, the peptide suffers from limited transcutaneous penetration due to its hydrophilicity and large molecular weight. Therefore, decapeptide-12 was modified by adding a palmitate chain in an attempt to overcome this limitation. We also tested the effects of chemical penetration enhancers and microneedles to deliver two peptides through skin. Enhanced skin permeation was found using an in vitro human skin permeation model. Moreover, we examined peptide retention of different formulations in skin. Our data showed that palm-peptides in microneedle patch was the most effective.

The 18 kDa translocator protein (TSPO) is a target for the development of imaging agents to detect neuroinflammation. The clinical utility of second-generation TSPO ligands has been hindered by the presence of a polymorphism, rs6971, which causes a non-conservative substitution of alanine for threonine at amino acid residue 147 (TSPO A147T). Given the complex nature of TSPO binding, and the lack of non-discriminating high-affinity ligands at both wild type and A147T forms of TSPO, a series of novel TSPO ligands containing various heterocyclic scaffolds was developed to explore the pharmacophoric drivers of affinity loss at TSPO A147T. In general, N-benzyl-N-methyl-substituted amide ligands showed increased affinity at TSPO A147T, and a pyrazolopyrimidine acetamide containing this motif displayed low nanomolar binding affinities to both TSPO forms.

The orthosteric binding site of GABA-gated ion channels has been widely explored. Many residues in the binding site of GABA were studied. The interactions due to the binding of GABA into the binding site drive channel activation and determine the potency and efficacy of GABA response. The combined effect of a competitive ligand and GABA on GABA-ρ1 receptors has been poorly studied. Here, we used point mutations, molecular modeling, and electrophysiological studies to explore the role of two hydrophilic residues (Serine 168 and Serine 243) of the GABA-ρ1 receptors in response to the binding of GABA and other studied ligands. Our results suggested that Ser168 residue stabilizes either closed state or open conformation depending on the other determinant interactions of each state. On the other hand, Ser243 residue is predicted to form different inter-subunit interactions with residues in the adjacent subunit at different states of the channel. Our current findings enlighten us to reasonably explain the additive/inhibitive effects of applying a competitive ligand with GABA simultaneously. Understanding the mixed effect of potentiation and inhibition would facilitate the discovery of new drugs to work as a direct GABA's activity modulators with more selectivity at various subunits forming GABA-gated ion channels.

On deforested or degraded land, planting mixtures of native forest tree species facilitates establishment of incoming tree seedling species (species recruitment) by rapidly re-establishing canopy cover. However, delayed colonization of plantations by large-seeded, zoochorous (LSZ) tree species can affect the species composition, structure, and function of the developing forest. The objective of this study was to investigate whether microsites limit establishment (germination and early seedling survival) of LSZ tree species in three 13-year-old, seasonally dry, tropical forest restoration plantations in northern Thailand. We conducted a seed sowing experiment that combined five LSZ tree species with four microsite treatments in a split-plot design. All five tree species were previously absent from the understory of the plantations, despite seed sources being present in nearby natural forest. The four treatments simulated potential microsites that naturally-dispersed seeds may encounter. They included seed deposition on the surface of soil and on leaf litter, as well as seed burial beneath soil and beneath leaf litter. We fenced the experimental areas to prevent seed predation and focus on the environmental effects of microsites on the different stages of seedling establishment. Following seed sowing, we measured germination and seedling survival, mean height, and stem diameter over 26 months. The microsites treatments did not significantly affect germination and seedling survival, providing strong evidence that establishment of the tested species was not limited by the applied microsite environments. Furthermore, although there were significant differences in germination and survival among species, relatively high overall germination (37%) and 26-month seedling survival (58.5%), irrespective of microsite treatment, suggested that environmental conditions in the 13-year-old plantations generally met the requirements for germination and early seedling establishment. Microsite treatments also did not correlate with seedling height or stem diameter at 20 months, suggesting that initial germination microsites have no effect on seedling growth and robustness. Taken together, the results of this investigation support the hypothesis that seedlings of LSZ tree species are slow to colonize tropical forest restoration plantations because of seed limitations resulting from inadequate seed dispersal or low seed availability. The successful establishment of LSZ seedlings from sown seeds in this experiment suggests that direct seeding beneath the canopy of restoration plantations may be an effective way to offset seed dispersal limitations in restoration plantations. Furthermore, the absence of microsite treatment effects suggests broadcast sowing of seeds may be a simple way to recolonize closed-canopy restoration plantations with LSZ tree species.

Background The prevalence of azole resistance in Aspergillus fumigatus is uncertain in Australia. Azole exposure may select for resistance. We investigated the frequency of azole resistance in a large number of clinical and environmental isolates. Methods A. fumigatus isolates [148 human, 21 animal and 185 environmental strains from air (n = 6) and azole-exposed (n = 64) or azole-naive (n = 115) environments] were screened for azole resistance using the VIPcheck™ system. MICs were determined using the Sensititre™ YeastOne YO10 assay. Sequencing of the Aspergillus cyp51A gene and promoter region was performed for azole-resistant isolates, and cyp51A homology protein modelling undertaken. Results Non-WT MICs/MICs at the epidemiological cut-off value of one or more azoles were observed for 3/148 (2%) human isolates but not amongst animal, or environmental, isolates. All three isolates grew on at least one azole-supplemented well based on VIPcheck™ screening. For isolates 9 and 32, the itraconazole and posaconazole MICs were 1 mg/L (voriconazole MICs 0.12 mg/L); isolate 129 had itraconazole, posaconazole and voriconazole MICs of >16, 1 and 8 mg/L, respectively. Soil isolates from azole-exposed and azole-naive environments had similar geometric mean MICs of itraconazole, posaconazole and voriconazole (P > 0.05). A G54R mutation was identified in the isolates exhibiting itraconazole and posaconazole resistance, and the TR34/L98H mutation in the pan-azole-resistant isolate. cyp51A modelling predicted that the G54R mutation would prevent binding of itraconazole and posaconazole to the haem complex. Conclusions Azole resistance is uncommon in Australian clinical and environmental A. fumigatus isolates; further surveillance is indicated.

Hyperpigmentation is a common skin condition with serious psychosocial consequences. Decapeptide-12, a novel peptide, has been found to be safer than hydroquinone in reducing melanin content, with efficacy up to more than 50% upon 16 weeks of twice-daily treatment. However, the peptide suffers from limited transcutaneous penetration due to its hydrophilicity and high molecular weight. Therefore, decapeptide-12 was modified by adding a palmitate chain in an attempt to overcome this limitation. Molecular docking results showed that the two peptides exhibited similar biological activity towards tyrosinase. We also tested the effect of chemical penetration enhancers and microneedles to deliver the two peptides into and through skin, using an in vitro human skin permeation method. It was shown that the palm-peptide achieved the best skin retention owing to the increased lipophilicity. In addition, skin permeation of the palm-peptides was enhanced by the chemical skin penetration enhancers, namely, oleic acid and menthol. Skin permeation of the native peptide was enhanced by the microneedle patch but not the chemical skin penetration enhancers. Cutaneous absorption of the palm-peptides was estimated to have achieved its therapeutic concentration within skin. The combinatory approach of using molecular modification, chemical penetration enhancement, and microneedle patch proves to be useful to enhanceskin permeation of the peptides.

The framework species method (FSM) of forest restoration involves planting mixtures of native forest trees to catalyze or complement natural regeneration and accelerate recovery of a diverse understory in tropical forests. Data on naturally regenerating tree seedlings, however, is sparse, and we lack an understanding of how environmental factors affect seedling persistence and growth. We investigated relationships between microsite light availability and two-year survival and growth of naturally-recruited tree seedlings in 11- to 14-year-old FSM trial plots, established to restore seasonally dry tropical forest (SDTF) in the highlands of northern Thailand. We also explored relationships among seedling survival, growth, successional guild and leaf habit. We sampled seedlings belonging to 13 tree species, representing various successional guilds (pioneer, intermediate and late-successional) and leafing habits (evergreen and deciduous). They included seedlings of “overstory species,” species planted to establish the plots, and “re-colonizing species,” species recruited into the plots by seed-dispersal from outside. Seventy-two percent of sampled seedlings survived two years. Ten species had high two-year survival (≥69%); however, three deciduous pioneer species had low survival ( R m 2 = 0.230 ) or RGRD ( R m 2 = 0.138 ) suggesting that there are additional, unconsidered factors important to growth. High two-year seedling survival indicated that within 14 years of implementing the FSM, the understory environment had become adequate for regeneration of a wide range of tree species. Regeneration of re-colonizing species confirmed previous reports that the FSM fosters regeneration of species-diverse tree communities. The results suggested that thinning or gap creation, to increase light levels, may accelerate seedling growth, but are not essential for regeneration of the studied species, and that enrichment planting or direct seeding of intermediate and late-successional species would help to accelerate ecosystem diversification. Introducing such species just before canopy closure and before planted overstory trees become reproductive may provide them with a competitive advantage over seedlings of overstory species.

Adamantanyl benzamide 1 was identified as a potent P2X7R antagonist but failed to progress further due to poor metabolic stability. We describe the synthesis and SAR of a series of bioisosteres of benzamide 1 to explore improvements in the pharmacological properties of this lead. Initial efforts investigated a series of heteroaromatic bioisosteres, which demonstrated improved physicochemical properties but reduced P2X7R antagonism. Installation of bioisosteric fluorine on the adamantane bridgeheads was well tolerated and led to a series of bioisosteres with improved physicochemical properties and metabolic stability. Trifluorinated benzamide 34 demonstrated optimal physicochemical parameters, superior metabolic stability (ten times longer than lead benzamide 1), and an improved physicokinetic profile and proved effective in the presence of several known P2X7R polymorphisms.

Over 44 million people live with Alzheimer's disease (AD) worldwide. Currently, only symptomatic treatments are available for AD and no cure exists. Considering the lack of effective treatments for AD due to its multi-factorial pathology, development of novel multi-target-directed drugs are desirable. Herein, we report the development of a novel series of thiosemicarbazones derived from 1-benzylpiperidine, a pharmacophore within the acetylcholinesterase inhibitor, Donepezil. These thiosemicarbazones were designed to target five major AD hallmarks, including: low acetylcholine levels, dysfunctional autophagy, metal dys-homeostasis, protein aggregation and oxidative stress. Of these thiosemicarbazones, pyridoxal 4-N-(1-benzylpiperidin-4-yl)thiosemicarbazone (PBPT) emerged as the lead compound. This agent demonstrated the most promising multi-functional activity by exhibiting very low anti-proliferative activity, substantial iron chelation efficacy, inhibition of copper-mediated amyloid-β aggregation, inhibition of oxidative stress, moderate acetylcholinesterase inhibitory activity and autophagic induction. These diverse properties highlight the potential of the lead ligand, PBPT, as a promising multi-functional agent for AD treatment.

Stimulated reporting occurs when patients and healthcare professionals are influenced or "stimulated" by media publicity to report specific drug-related adverse reactions, significantly biasing pharmacovigilance analyses. Among countries where the non-benzodiazepine hypnotic drug zolpidem is marketed, the United States experienced a comparable surge of media reporting during 2006-2009 linking the above drug with the development of complex neuropsychiatric sleep-related behaviors. However, the effect of this stimulated reporting in the United States Food and Drug Administration Adverse Event Reporting System has not been explored.Using disproportionality analyses, reporting odds ratios for zolpidem exposure and the following adverse events; parasomnia, movement-based parasomnia, nonmovement-based parasomnia, amnesia, hallucination, and suicidality were determined and compared to all other medications in the database, followed by specific comparison to the benzodiazepine hypnotic class, year-by-year from 2003 to 2012.Odds ratios were increased significantly during and after the period of media publicity for parasomnias, movement-based parasomnias, amnesias and hallucinations. We also observed that zolpidem adverse drug reaction (ADR) reports have higher odds for parasomnias, movement-based parasomnias, amnesias, hallucinations, and suicidality compared to all other drugs, even before the media publicity cluster.Although our results indicate that zolpidem reports have higher odds for the ADR of interest even before the media publicity cluster, negative media coverage greatly exacerbated the reporting of these adverse reactions. The effect of such reporting must be borne in mind when decisions around drugs which have been the subject of intense media publicity are made by health professionals or regulatory bodies.

Natural samples of the substituted basic Cu(II) chloride series, Cu4–xMx2+(OH)6Cl2(M = Zn, Ni, or Mg) were investigated by single-crystal X-ray diffraction in order to elucidate compositional boundaries associated with paratacamite and its congeners. The compositional ranges examined are Cu3.65Zn0.35(OH)6Cl2 – Cu3.36Zn0.64(OH)6Cl2 and Cu3.61Ni0.39(OH)6Cl2 – Cu3.13Ni0.87(OH)6Cl2, along with a single Mg-bearing phase. The majority of samples studied have trigonal symmetry (R3̄m) analogous to that of herbertsmithite (Zn) and gillardite (Ni), with a ≈ 6.8, c ≈ 14.0 Å. Crystallographic variations for these samples caused by composition are compared with both published and new data for the R3̄m sub-cell of paratacamite, paratacamite-(Mg) and paratacamite-(Ni). The observed trends suggest that the composition of end-members associated with the paratacamite congeners depend upon the nature of the substituting cation.

The microtubule-binding taxanes, docetaxel and cabazitaxel, are administered intravenously for the treatment of castration-resistant prostate cancer (CRPC) as the oral administration of these drugs is largely hampered by their low and highly variable bioavailabilities. Using a simple, rapid, and environmentally friendly microwave-assisted protocol, we have synthesized a number of 3,5-bis(styryl)pyrazoles 2a-l, thus allowing for their screening for antiproliferative activity in the androgen-independent PC3 prostate cancer cell line. Surprisingly, two of these structurally simple 3,5-bis(styryl)pyrazoles (2a and 2l) had concentrations which gave 50% of the maximal inhibition of cell proliferation (GI50) in the low micromolar range in the PC3 cell line and were thus selected for extensive further biologic evaluation (apoptosis and cell cycle analysis, and effects on tubulin and microtubules). Our findings from these studies show that 3,5-bis[(1E)-2(2,6-dichlorophenyl)ethenyl]-1H-pyrazole 2l 1) caused significant effects on the cell cycle in PC3 cells, with the vast majority of treated cells in the G2/M phase (89%); 2) induces cell death in PC3 cells even after the removal of the compound; 3) binds to tubulin [dissociation constant (Kd) 0.4 ± 0.1 μM] and inhibits tubulin polymerization in vitro; 4) had no effect upon the polymerization of the bacterial cell division protein FtsZ (a homolog of tubulin); 5) is competitive with paclitaxel for binding to tubulin but not with vinblastine, crocin, or colchicine; and 6) leads to microtubule depolymerization in PC3 cells. Taken together, these results suggest that 3,5-bis(styryl)pyrazoles warrant further investigation as lead compounds for the treatment of CRPC. SIGNIFICANCE STATEMENT: The taxanes are important components of prostate cancer chemotherapy regimens, but their oral administration is hampered by very low and highly variable oral bioavailabilities resulting from their poor absorption, poor solubility, high first-pass metabolism, and efficient efflux by P-glycoprotein. New chemical entities for the treatment of prostate cancer are thus required, and we report here the synthesis and investigation of the mechanism of action of some bis(styryl)pyrazoles, demonstrating their potential as lead compounds for the treatment of prostate cancer.

The past decade has seen an increase in aspergillosis in humans and animals due to Aspergillus viridinutans species complex members. Azole resistance is common to these infections, carrying a poor prognosis. cyp51A gene mutations are the main cause of acquired azole resistance in Aspergillus fumigatus. This study aimed to determine if the azole-resistant phenotype in A. viridinutans complex members is associated with cyp51A mutations or extrolite profiles., The past decade has seen an increase in aspergillosis in humans and animals due to Aspergillus viridinutans species complex members. Azole resistance is common to these infections, carrying a poor prognosis. cyp51A gene mutations are the main cause of acquired azole resistance in Aspergillus fumigatus. This study aimed to determine if the azole-resistant phenotype in A. viridinutans complex members is associated with cyp51A mutations or extrolite profiles. The cyp51A gene of clinical and environmental isolates was amplified using novel primers, antifungal susceptibility was tested using the Clinical and Laboratory Standards Institute methodology, and extrolite profiling was performed using agar plug extraction. Very high azole MICs were detected in 84% of the isolates (31/37). The MICs of the newer antifungals luliconazole and olorofim (F901318) were low for all isolates. cyp51A sequences revealed 113 nonsynonymous mutations compared to the sequence of wild-type A. fumigatus. M172A/V and D255G, previously associated with A. fumigatus azole resistance, were common among all isolates but were not correlated with azole MICs. Two environmental isolates with nonsusceptibility to itraconazole and high MICs of voriconazole and isavuconazole harbored G138C, previously associated with azole-resistant A. fumigatus. Some novel mutations were identified only among isolates with high azole MICs. However, cyp51A homology modeling did not cause a significant protein structure change for these mutations. There was no correlation between extrolite patterns and susceptibility. For A. viridinutans complex isolates, cyp51A mutations and the extrolites that they produced were not major causes of antifungal resistance. Luliconazole and olorofim show promise for treating azole-resistant infections caused by these cryptic species.