Discovering the Solid-State Secrets of Lorlatinib by NMR Crystallography: To Hydrogen Bond or not to Hydrogen Bond.

Lorlatinib is an active pharmaceutical ingredient (API) used in the treatment of lung cancer. Here, an NMR crystallography analysis is presented whereby the single-crystal X-ray diffraction structure (CSD: 2205098) determination is complemented by multinuclear (1H, 13C, 14/15N, 19F) magic-angle spinning (MAS) solid-state NMR and gauge-including projector augmented wave (GIPAW) calculation of NMR chemical shifts. Lorlatinib crystallises in the P21 space group, with two distinct molecules in the asymmetric unit cell, Z' = 2. Three of the four NH2 hydrogen atoms form intermolecular hydrogen bonds, N30-H…N15 between the two distinct molecules and N30-H…O2 between two equivalent molecules. This is reflected in one of the NH21H chemical shifts being significantly lower, 4.0 ppm compared to 7.0 ppm. Two-dimensional 1H-13C, 14N-1H and 1H (double-quantum, DQ)-1H (single-quantum, SQ) MAS NMR spectra are presented. The 1H resonances are assigned and specific HH proximities corresponding to the observed DQ peaks are identified. The resolution enhancement at a 1H Larmor frequency of 1 GHz as compared to 500 or 600 MHz is demonstrated.

Elucidation of microbial lignin degradation pathways using synthetic isotope-labelled lignin.

Pathways by which the biopolymer lignin is broken down by soil microbes could be used to engineer new biocatalytic routes from lignin to renewable chemicals, but are currently not fully understood. In order to probe these pathways, we have prepared synthetic lignins containing 13C at the sidechain ß-carbon. Feeding of [ß-13C]-labelled DHP lignin to Rhodococcus jostii RHA1 has led to the incorporation of 13C label into metabolites oxalic acid, 4-hydroxyphenylacetic acid, and 4-hydroxy-3-methoxyphenylacetic acid, confirming that they are derived from lignin breakdown. We have identified a glycolate oxidase enzyme in Rhodococcus jostii RHA1 which is able to oxidise glycolaldehyde via glycolic acid to oxalic acid, thereby identifying a pathway for the formation of oxalic acid. R. jostii glycolate oxidase also catalyses the conversion of 4-hydroxyphenylacetic acid to 4-hydroxybenzoylformic acid, identifying another possible pathway to 4-hydroxybenzoylformic acid. Formation of labelled oxalic acid was also observed from [ß-13C]-polyferulic acid, which provides experimental evidence in favour of a radical mechanism for α,ß-bond cleavage of ß-aryl ether units.

Exploring the Potential of Multinuclear Solid-State 1 H, 13 C, and 35 Cl Magnetic Resonance To Characterize Static and Dynamic Disorder in Pharmaceutical Hydrochlorides.

Crystallographic disorder, whether static or dynamic, can be detrimental to the physical and chemical stability, ease of crystallization and dissolution rate of an active pharmaceutical ingredient. Disorder can result in a loss of manufacturing control leading to batch-to-batch variability and can lengthen the process of structural characterization. The range of NMR active nuclei makes solid-state NMR a unique technique for gaining nucleus-specific information about crystallographic disorder. Here, we explore the use of high-field 35 Cl solid-state NMR at 23.5 T to characterize both static and dynamic crystallographic disorder: specifically, dynamic disorder occurring in duloxetine hydrochloride (1), static disorder in promethazine hydrochloride (2), and trifluoperazine dihydrochloride (3). In all structures, the presence of crystallographic disorder was confirmed by 13 C cross-polarization magic-angle spinning (CPMAS) NMR and supported by GIPAW-DFT calculations, and in the case of 3, 1 H solid-state NMR provided additional confirmation. Applying 35 Cl solid-state NMR to these compounds, we show that higher magnetic fields are beneficial for resolving the crystallographic disorder in 1 and 3, while broad spectral features were observed in 2 even at higher fields. Combining the data obtained from 1 H, 13 C, and 35 Cl NMR, we show that 3 exhibits a unique case of disorder involving the + N-H hydrogen positions of the piperazinium ring, driving the chloride anions to occupy three distinct sites.

Hepatoprotective Role Of N-Hexane Fraction And Crude Methanolic Extract Of Withania Coagulans In Balb C Mice.

Background: Withania coagulans is one of the most important medicinal herbs due to its wide range of biological activities. The aim of this study was to compare the hepatoprotective activity of crude methanolic extract versus n- hexane fraction of fruit of Withania coagulans in CCl4 induced liver toxicity. Methods: This study was done on 36 Balb c mice in Department of Anatomy of Khyber Medical College Peshawar. Results: The mean serum of Group 1(control Group) was 33.41±1.82U/L, for Group 2 (CCl4 treated Group) was 89.01±7.51 U/L, for Group 3(low dose Group) was 49.91±3.48 U/L and for Group 4 (High dose Group) was 50.86±4.87 U/L. There was significant difference in the readings of Group1 and Group 2 which indicated CCL4 induced hepatotoxicity in two groups. There was significant difference in the values of Group 3 and Group 4 (p value .000), showing the hepatotoxicity in these Groups was further enhanced. The mean AST at the end of six weeks for Group1 was 26.80±3.21U/L, for Group 2 was 149.01±13.63U/L, for Group 3 (including both low doses) was 70.81±7.92U/L and for Group 4 (High doses group) was 51.01±11.05U/. Conclusion: Withania coagulans both fractions have hepatoprotective effect against CCL4 induced hepatic toxicity in high and low doses in Balb c mice.

Combining X-ray and NMR Crystallography to Explore the Crystallographic Disorder in Salbutamol Oxalate.

Salbutamol is an active pharmaceutical ingredient commonly used to treat respiratory distress and is listed by the World Health Organization as an essential medicine. Here, we establish the crystal structure of its oxalate form, salbutamol oxalate, and explore the nature of its crystallographic disorder by combined X-ray crystallography and 13C cross-polarization (CP) magic-angle spinning (MAS) solid-state NMR. The *C-OH chiral center of salbutamol (note that the crystal structures are a racemic mixture of the two enantiomers of salbutamol) is disordered over two positions, and the tert-butyl group is rotating rapidly, as revealed by 13C solid-state NMR. The impact of crystallization conditions on the disorder was investigated, finding variations in the occupancy ratio of the *C-OH chiral center between single crystals and a consistency across samples in the bulk powder. Overall, this work highlights the contrast between investigating crystallographic disorder by X-ray diffraction and solid-state NMR experiment, and gauge-including projector-augmented-wave (GIPAW) density functional theory (DFT) calculations, with their combined use, yielding an improved understanding of the nature of the crystallographic disorder between the local (i.e., as viewed by NMR) and longer-range periodic (i.e., as viewed by diffraction) scale.

Evaluation of Alpha-Amylase Inhibitory, Antioxidant, and Antimicrobial Potential and Phytochemical Contents of Polygonum hydropiper L.

Polygonum hydropiper L. is a traditionally used medicinal plant. The present study was designed to explore the α-amylase inhibitory, antioxidant, and antimicrobial activities of Polygonum hydropiper L. Polarity-based solvent extracts (n-hexane, acetone, chloroform, methanol, ethanol, and water) of Polygonum hydropiper leaves and stem were used. Antioxidant activity was assessed by free radical scavenging assay (FRAP) and 2,2-diphenylpicrylhydrazyl (DPPH) free radical scavenging activity methods. Quantitative phytochemical analyses suggested that the stem of Polygonum hydropiper L. contains higher levels of bioactive compounds than its leaves (p < 0.05). The results suggested that stem-derived extracts of Polygonum hydropiper L. are more active against bacterial species, including two Gram-positive and three Gram-negative strains. Moreover, our results showed that the bioactive compounds of Polygonum hydropiper L. significantly inhibit α-amylase activity. Finally, we reported the polarity-based solvent extracts of Polygonum hydropiper L. and revealed that the stem, rather than leaves, has a high antioxidant potential as measured by FRAP and DPPH assay with IC50 values of 1.38 and 1.59 mg/mL, respectively. It may also be deducted from the data that the Polygonum hydropiper L. could be a significant candidate, which should be subjected to further isolation and characterization, to be used as an antidiabetic, antimicrobial and antioxidant resource in many industries, like food, pharmaceuticals and cosmetics.

Lumbosacral Transitional Vertebrae In Patients Visiting Tertiary Care Hospital Of Khyber Pakhtukhwa: A Radiological Study.

BACKGROUND: Lumbosacral transitional vertebra is a common anatomical variant of the spine that possibly is related to low back pain. The objective is to determine the frequency of Lumbosacral Transitional Vertebrae on lumbosacral radiographs in patients with low back pain visiting Neurosurgery OPD of Khyber Teaching Hospital Peshawar Khyber Pakhtunkhwa (KP). METHODS: This cross-sectional study was conducted on radiographs of lumbosacral spine in patients visiting for low back pain to the Neurosurgery OPD of Khyber Teaching Hospital Peshawar. A total of 300 anteroposterior (AP) and lateral lumbosacral plain radiographs were studied for the variation in lumbosacral spinal segment. The transition was classified according to the scheme used by Castellvi. RESULTS: Among a total of 300 patients, normal anatomy of lumbosacral segments was observed in 215 patients (72%). About 85 cases (28%) showed Lumbosacral Transitional Vertebrae. The relationship of vertebral variation with age and gender was not observed. With regard to morphology, 29 (34.1%) patients had Castellvi type I, 26 cases (30.5%) were observed with Castellvi type II and 12 patients (14.1%) had type III while 18 cases (21.2%) had type IV transitional morphology. CONCLUSIONS: Lumbosacral Transitional Vertebrae are commonly found in patient with low back pain. Identification of Lumbosacral Transitional Vertebrae is clinically important to prevent an intervention at an incorrect vertebral level.

An Integrated Systems Biology and Network-Based Approaches to Identify Novel Biomarkers in Breast Cancer Cell Lines Using Gene Expression Data.

Breast cancer is the most common cause of death in women worldwide. Approximately 5%-10% of instances are attributed to mutations acquired from the parents. Therefore, it is highly recommended to design more potential drugs and drug targets to eradicate such complex diseases. Network-based gene expression profiling is a suggested tool for discovering drug targets by incorporating various factors such as disease states, intensities based on gene expression as well as protein-protein interactions. To find prospective biomarkers in breast cancer, we first identified differentially expressed genes (DEGs) statistical methods p-value and false discovery rate were initially used. Of the total 82 DEGs, 67 were upregulated while the remaining 17 were downregulated. Sub-modules and hub genes include VEGFA with the highest degree, followed by 15 CCND1 and CXCL8 with 12-degree score was found. The survival analysis revealed that all the hub genes have important role in the development and progression of breast cancer. Enrichment analysis revealed that most of these genes are involved in signaling pathways and in the extracellular spaces. We also identified transcription factors and kinases, which regulate proteins in the DEGs PPI. Finally, drugs for each hub genes were identified. These results further expanded the knowledge regarding important biomarkers in breast cancer.

In silico T-cell and B-cell Epitope Based Vaccine Design Against Alphavirus Strain of Chikungunya.

BACKGROUND: Chikungunya an arbovirus, is transmitted to humans by the bite of Aedes mosquito. The virus occurrences have been reported in Southeast Asian countries including Pakistan. Its symptoms include typical febrile illness and arthralgic syndrome. The virus has not decisively proved to be life-threatening. METHODS: The attempt was to design T-cell and B-cell epitope-based vaccine for Chikungunya. The proteome of chikungunya was retrieved, antigenic proteins were identified and T-cell epitopes and B-cell epitopes were predicted. Interacting HLA alleles were also identified. The final analysis was done to confirm that predicted T-cell epitopes and B-cell epitopes can be used as a vaccine. RESULTS: About 32 T-cell epitopes and a 10mer B-cell epitope were identified. Both T-cell and Bcell epitopes demonstrated strong interactions with HLA alleles. The predicted T-cell and B-cell epitopes were docked with respective HLA alleles. The docking analysis showed that the predicted respective epitopes best fit into the binding pockets of the alleles. CONCLUSION: On the basis of this computational analysis, it is suggested that these predicted epitopes can be used as a remedy against Alphavirus strain of chikungunya. Further laboratory experiments can be conducted to determine the efficacy and stability of this work.