Design, synthesis, molecular modeling, in vitro and in vivo biological evaluation of potent anthranilamide derivatives as dual P-glycoprotein and CYP3A4 inhibitors.

Paclitaxel (PTX) is considered the blockbuster chemotherapy treatment for cancer. Paclitaxel's (PTX) oral administration has proven to be extremely difficult, mostly because of its susceptibility to intestinal P-glycoprotein (P-gp) and cytochrome P450 (CYP3A4). The concurrent local inhibition of intestinal P-gp and CYP3A4 is a promising approach to improve the oral bioavailability of paclitaxel while avoiding potential unfavorable side effects of their systemic inhibition. Herein, we report the rational design and evaluation of novel dual potent inhibitors of P-gp and CYP3A4 using an anthranilamide derivative tariquidar as a starting point for their structural optimizations. Compound 14f, bearing N-imidazolylbenzyl side chain, was found to have potent and selective P-gp (EC50 = 28 nM) and CYP3A4 (IC50 = 223 nM) inhibitory activities with low absorption potential (Papp (A-to-B) <0.06). In vivo, inhibitor 14f improved the oral absorption of paclitaxel by 6 times in mice and by 30 times in rats as compared to vehicle, while 14f itself remained poorly absorbed. Compound 14f, possessing dual P-gp and CYP3A4 inhibitory activities, offered additional enhancement in paclitaxel oral absorption compared to tariquidar in mice. Evaluating the CYP effect of 14f on oral absorption of paclitaxel requires considering the variations in CYP expression between animal species. This study provides further medicinal chemistry advice on strategies for resolving concerns with the oral administration of chemotherapeutic agents.

Patterns and outcomes of acute toxicological cases before, during, and after COVID-19 lockdown in Sohag University hospitals, Egypt.

Background: Toxicologists manage poisoning by preventing, detecting, and treating it, which requires continuous data collection and analysis of toxicological hazards. Aim of the work: The study aims to report and compare the pattern and outcome of acute toxicological cases admitted to Sohag University Hospitals during the COVID-19 lockdown (2020-2021) with the year before (2019) and the year after (2022). Methods: This comparative study reviewed the sociodemographic and clinical data in the medical records. The study showed that Sohag University Hospitals received 670 toxicological cases between 2019 and 2022; 105 cases in 2019, 347 cases in 2020-2021, and 218 cases in 2022. Results: Most of patients were below seven years with no sex differences. Accidental poisoning was the most frequent toxicity. The oral route was the most common in the three studied periods. During the lockdown, metal phosphide was the most frequent (19.0%), while therapeutic agents were the most reported after the lockdown (23.9%). The delay time showed a significant difference between the studied periods (p-value < 0.001). In the three studied periods, complete recovery was achieved in more than 70% of cases; however, the mortality rate and the rate of complications during the lockdown period (10.4% and 9.5%, respectively) were almost twice those of the year before and the year after the pandemic with significant odds ratio of mortality during pandemic (OR) 0.07 CI 95% (0.02, 0.11). Conclusion: The pandemic had a bad impact on outcomes as showed the highest percentage of mortality compared to before and after COVID-19 periods.

Design, synthesis, molecular modelling and biological evaluation of novel 6-amino-5-cyano-2-thiopyrimidine derivatives as potent anticancer agents against leukemia and apoptotic inducers.

Herein, a novel series of 6-amino-5-cyano-2-thiopyrimidines and condensed pyrimidines analogues were prepared. All the synthesized compounds (1a-c, 2a-c, 3a-c, 4a-r and 5a-c) were evaluated for in vitro anticancer activity by the National Cancer Institute (NCI; MD, USA) against 60 cell lines. Compound 1c showed promising anticancer activity and was selected for the five-dose testing. Results demonstrated that compound 1c possessed broad spectrum anti-cancer activity against the nine cancerous subpanels tested with selectivity ratio ranging from 0.7 to 39 at the GI50 level with high selectivity towards leukaemia. Mechanistic studies showed that Compound 1c showed comparable activity to Duvelisib against PI3Kδ (IC50 = 0.0034 and 0.0025 µM, respectively) and arrested cell cycle at the S phase and displayed significant increase in the early and late apoptosis in HL60 and leukaemia SR cells. The necrosis percentage showed a significant increase from 1.13% to 3.41% in compound 1c treated HL60 cells as well as from 1.51% to 4.72% in compound 1c treated leukaemia SR cells. Also, compound 1c triggered apoptosis by activating caspase 3, Bax, P53 and suppressing Bcl2. Moreover, 1c revealed a good safety profile against human normal lung fibroblast cell line (WI-38 cells). Molecular analysis of Duvelisib and compound 1c in PI3K was performed. Finally, these results suggest that 2-thiopyrimidine derivative 1c might serve as a model for designing novel anticancer drugs in the future.

Down-regulation of hepatic expression of GHR/STAT5/IGF-1 signaling pathway fosters development and aggressiveness of HCV-related hepatocellular carcinoma: Crosstalk with Snail-1 and type 2 transforming growth factor-beta receptor.

BACKGROUND AND AIMS: So far, few clinical trials are available concerning the role of growth hormone receptor (GHR)/signal transducer and activator of transcription 5 (STAT5)/insulin like growth factor-1 (IGF-1) axis in hepatocarcinogenesis. The aim of this study was to evaluate the hepatic expression of GHR/STAT5/IGF-1 signaling pathway in hepatocellular carcinoma (HCC) patients and to correlate the results with the clinico-pathological features and disease outcome. The interaction between this signaling pathway and some inducers of epithelial-mesenchymal transition (EMT), namely Snail-1 and type 2 transforming growth factor-beta receptor (TGFBR2) was studied too. MATERIAL AND METHODS: A total of 40 patients with HCV-associated HCC were included in this study. They were compared to 40 patients with HCV-related cirrhosis without HCC, and 20 healthy controls. The hepatic expression of GHR, STAT5, IGF-1, Snail-1 and TGFBR2 proteins were assessed by immunohistochemistry. RESULTS: Compared with cirrhotic patients without HCC and healthy controls, cirrhotic patients with HCC had significantly lower hepatic expression of GHR, STAT5, and IGF-1proteins. They also displayed significantly lower hepatic expression of TGFBR2, but higher expression of Snail-1 versus the non-HCC cirrhotic patients and controls. Serum levels of alpha-fetoprotein (AFP) showed significant negative correlations with hepatic expression of GHR (r = -0.31; p = 0.029) and STAT5 (r = -0.29; p = 0.04). Hepatic expression of Snail-1 also showed negative correlations with GHR, STAT5, and IGF-1 expression (r = -0.55, p = 0.02; r = -0.472, p = 0.035, and r = -0.51, p = 0.009, respectively), whereas, hepatic expression of TGFBR2 was correlated positively with the expression of all these proteins (r = 0.47, p = 0.034; 0.49, p = 0.023, and r = 0.57, p<0.001, respectively). Moreover, we reported that decreased expression of GHR was significantly associated with serum AFP level>100 ng/ml (p = 0.048), increased tumor size (p = 0.02), vascular invasion (p = 0.002), and advanced pathological stage (p = 0.01). Similar significant associations were found between down-regulation of STAT5 expression and AFP level > 100 ng/ml (p = 0.006), vascular invasion (p = 0.009), and advanced tumor stage (p = 0.007). Also, attenuated expression of IGF-1 showed a significant association with vascular invasion (p < 0.001). Intriguingly, we detected that lower expression of GHR, STAT5 and IGF-1 were considered independent predictors for worse outcome in HCC. CONCLUSION: Decreased expression of GHR/STAT5/IGF-1 signaling pathway may have a role in development, aggressiveness, and worse outcome of HCV-associated HCC irrespective of the liver functional status. Snail-1 and TGFBR2 as inducers of EMT may be key players. However, large prospective multicenter studies are needed to validate these results.

Neuropilin-2 axis in regulating secretory phenotype of neuroendocrine-like prostate cancer cells and its implication in therapy resistance.

Neuroendocrine (NE)-like tumors secrete various signaling molecules to establish paracrine communication within the tumor milieu and to create a therapy-resistant environment. It is important to identify molecular mediators that regulate this secretory phenotype in NE-like cancer. The current study highlights the importance of a cell surface molecule, Neuropilin-2 (NRP2), for the secretory function of NE-like prostate cancer (PCa). Our analysis on different patient cohorts suggests that NRP2 is high in NE-like PCa. We have developed cell line models to investigate NRP2's role in NE-like PCa. Our bioinformatics, mass spectrometry, cytokine array, and other supporting experiments reveal that NRP2 regulates robust secretory phenotype in NE-like PCa and controls the secretion of factors promoting cancer cell survival. Depletion of NRP2 reduces the secretion of these factors and makes resistant cancer cells sensitive to chemotherapy in vitro and in vivo. Therefore, targeting NRP2 can revert cellular secretion and sensitize PCa cells toward therapy.

Novel pyrrolopyrimidine derivatives: design, synthesis, molecular docking, molecular simulations and biological evaluations as antioxidant and anti-inflammatory agents.

Current medical approaches to control the Covid-19 pandemic are either to directly target the SARS-CoV-2 via innovate a defined drug and a safe vaccine or indirectly target the medical complications of the virus. One of the indirect strategies for fighting this virus has been mainly dependent on using anti-inflammatory drugs to control cytokines storm responsible for severe health complications. We revealed the discovery of novel fused pyrrolopyrimidine derivatives as promising antioxidant and anti-inflammatory agents. The newly synthesised compounds were evaluated for their in vitro anti-inflammatory activity using RAW264.7 cells after stimulation with lipopolysaccharides (LPS). The results revealed that 3a, 4b, and 8e were the most potent analogues. Molecular docking and simulations of these compounds against COX-2, TLR-2 and TLR-4 respectively was performed. The former results were in line with the biological data and proved that 3a, 4b and 8e have potential antioxidant and anti-inflammatory effects.

Discovery and Characterization of Potent Dual P-Glycoprotein and CYP3A4 Inhibitors: Design, Synthesis, Cryo-EM Analysis, and Biological Evaluations.

Targeted concurrent inhibition of intestinal drug efflux transporter P-glycoprotein (P-gp) and drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4) is a promising approach to improve oral bioavailability of their common substrates such as docetaxel, while avoiding side effects arising from their pan inhibitions. Herein, we report the discovery and characterization of potent small molecule inhibitors of P-gp and CYP3A4 with encequidar (minimally absorbed P-gp inhibitor) as a starting point for optimization. To aid in the design of these dual inhibitors, we solved the high-resolution cryo-EM structure of encequidar bound to human P-gp. The structure guided us to prudently decorate the encequidar scaffold with CYP3A4 pharmacophores, leading to the identification of several analogues with dual potency against P-gp and CYP3A4. In vivo, dual P-gp and CYP3A4 inhibitor 3a improved the oral absorption of docetaxel by 3-fold as compared to vehicle, while 3a itself remained poorly absorbed.

Design, Synthesis, Molecular Modeling and Antitumor Evaluation of Novel Indolyl-Pyrimidine Derivatives with EGFR Inhibitory Activity.

Scaffolds hybridization is a well-known drug design strategy for antitumor agents. Herein, series of novel indolyl-pyrimidine hybrids were synthesized and evaluated in vitro and in vivo for their antitumor activity. The in vitro antiproliferative activity of all compounds was obtained against MCF-7, HepG2, and HCT-116 cancer cell lines, as well as against WI38 normal cells using the resazurin assay. Compounds 1-4 showed broad spectrum cytotoxic activity against all these cancer cell lines compared to normal cells. Compound 4g showed potent antiproliferative activity against these cell lines (IC50 = 5.1, 5.02, and 6.6 µM, respectively) comparable to the standard treatment (5-FU and erlotinib). In addition, the most promising group of compounds was further evaluated for their in vivo antitumor efficacy against EAC tumor bearing mice. Notably, compound 4g showed the most potent in vivo antitumor activity. The most active compounds were evaluated for their EGFR inhibitory (range 53-79%) activity. Compound 4g was found to be the most active compound against EGFR (IC50 = 0.25 µM) showing equipotency as the reference treatment (erlotinib). Molecular modeling study was performed on compound 4g revealed a proper binding of this compound inside the EGFR active site comparable to erlotinib. The data suggest that compound 4g could be used as a potential anticancer agent.

Polyphenolic Profile of Callistemon viminalis Aerial Parts: Antioxidant, Anticancer and In Silico 5-LOX Inhibitory Evaluations.

Five new compounds viz kaempferol 3-O-(4″-galloyl)-ß-d-glucopyranosyl-(1‴→6″)-O-ß-d-glucopyranoside (1), kaempferol 3-O-ß-d-mannuronopyranoside (2), kaempferol 3-O-ß-d-mannopyranoside (3), quercetin 3-O-ß-d-mannuronopyranoside (4), 2, 3 (S)- hexahydroxydiphenoyl]-d-glucose (5) along with fifteen known compounds were isolated from 80% aqueous methanol extract (AME) of C. viminalis. AME and compounds exerted similar or better antioxidant activity to ascorbic acid using DPPH, O2-, and NO inhibition methods. In addition, compounds 16, 4, and 7 showed cytotoxic activity against MCF-7 cell lines while 3, 7 and 16 exhibited strong activity against HepG2. An in silico analysis using molecular docking for polyphenolic compounds 2, 3, 7, 16 and 17 against human stable 5-LOX was performed and compared to that of ascorbic acid and quercetin. The binding mode as well as the enzyme-inhibitor interactions were evaluated. All compounds occupied the 5-LOX active site and showed binding affinity greater than ascorbic acid or quercetin. The data herein suggest that AME, a source of polyphenols, could be used against oxidative-stress-related disorders.

Design, synthesis, and evaluation of a novel benzamidine-based inhibitor of VEGF-C binding to Neuropilin-2.

The Neuropilin (Nrp) family of cell surface receptors have key physiological and pathological functions. Nrp2 is of particular interest due to its involvement in tumor metastasis. Currently, peptide and small molecule inhibitors that target Nrp utilize arginine-based molecules which have limitations due to high inherent flexibility and issues related to stability. Further, there are no known small molecule inhibitors specific for Nrp2. Recent molecular insights identify a key ligand binding region in the b1 domain of Nrp2 responsible for binding the C-terminus of its cognate ligand VEGF-C. Based on this, we report the discovery of a novel benzamidine-based inhibitor that functions through competitive inhibition of VEGF-C binding to Nrp2. Further, we have explored inhibitor functionality and selectivity by defining its structure-activity relationship (SAR) providing valuable insights on this benzamidine-based family of Nrp2 inhibitors. This study provides the basis for further development of a potent and specific small molecule inhibitor that competitively targets pathological Nrp2 function.

Predictive role of IL-17A/IL-10 ratio in persistent asthmatic patients on vitamin D supplement.

Asthma is an airway inflammatory disorder. Vitamin (Vit) D is a potent immuno-modulator. It suppresses Interleukin (IL)-17 and induces IL-10. This study aims to investigate the role of IL-17A and IL-10 in predicting asthma control in case of Vit D supplementation. Seventy-nine patients enrolled in this study (42 patients received Vit D supplement and 37 patients did not receive the supplement). The enrolled patients were assessed at the beginning of this study and after 3 months. At the end of the study, there was a significant improvement in pulmonary function parameters in the Vit D supplemented group when compared to both the baseline values and the non-supplemented group. There was a significant decrease in serum IL-17A levels and a significant increase in serum IL-10 levels in comparison with the baseline values (p < 0.0001). The highest correlation of FEV1% improvement percentage was associated with the baseline IL-17A/IL-10 ratio (r = 0.65; p < 0.0001). The IL-17A/IL-10 ratio at a cutoff ≥ 2.66 had a sensitivity of 72.2% and a specificity of 83.3%. The IL-17A/IL-10 ratio had an adjusted odds ratio = 4.66 (p = 0.04). Vit D supplementation reduces the serum IL-17A levels and elevates the serum IL-10 levels in persistent asthmatic patients. So, Vitamin D can be used as an adjunct therapy side by side with the conventional asthma therapy. The IL-17A/IL-10 ratio seems to be a possible predictive biomarker for asthma improvement in patients depending on Vit D supplementation.

Design, Synthesis, Molecular Modeling, and Biological Evaluation of Novel Thiouracil Derivatives as Potential Antithyroid Agents.

Hyperthyroidism is the result of uncontrolled overproduction of the thyroid hormones. One of the mostly used antithyroid agents is 6-n-propyl-2-thiouracil (PTU). The previously solved X-ray crystal structure of the PTU bound to mammalian lactoperoxidase (LPO) reveals that the LPO-PTU binding site is basically a hydrophobic channel. There are two hydrophobic side chains directed towards the oxygen atom in the C-4 position of the thiouracil ring. In the current study, the structural activity relationship (SAR) was performed on the thiouracil nucleus of PTU to target these hydrophobic side chains and gain more favorable interactions and, in return, more antithyroid activity. Most of the designed compounds show superiority over PTU in reducing the mean serum T4 levels of hyperthyroid rats by 3% to 60%. In addition, the effect of these compounds on the levels of serum T3 was found to be comparable to the effect of PTU treatment. The designed compounds in this study showed a promising activity profile in reducing levels of thyroid hormones and follow up experiments will be needed to confirm the use of the designed compounds as new potential antithyroid agents.

Modulating hydrogen-bond basicity within the context of protein-ligand binding: A case study with thrombin inhibitors that reveals a dominating role for desolvation.

Understanding subtle aspects of hydrogen bonding is a challenging but crucial task to improve our ability to design ligands with high affinity for protein hosts. To gain a deeper understanding of these aspects, we investigated a series of thrombin inhibitors in which the basicity of the ligand's group that accepts an H-bond from Gly216 was modulated via bioisosterism; e.g., a C=O acceptor was made electron deficient or rich via bioisosteric replacements of the adjacent moiety. Although the ligand's binding affinity was anticipated to improve when the H-bond basicity is increased (due to stronger H-bonding with the protein), we herein present data that unexpectedly revealed an opposite trend. This trend was attributed to a dominating role played by desolvation in determining the relative binding affinity. For example, a decrease in the H-bond basicity reduces the desolvation penalty and, as experimentally observed, improves the binding affinity, given that the reduction in the desolvation penalty dominates the change in the net contribution of the ligand's interactions with the protein. The current study, therefore, reveals that desolvation can be a major underlying cause for the apparently counterintuitive structure-activity relationship (SAR) outcomes, and indicates that understanding this factor can improve our ability to predict binding affinity and to design more potent ligands.

Ligand binding cooperativity: Bioisosteric replacement of CO with SO2 among thrombin inhibitors.

Ligand-protein binding is a complex process that involves the formation of number of non-covalent interactions, e.g. H-bonds and hydrophobic interactions, between the ligand and the protein host. Upon binding, ligand functional groups can act synergistically (positive cooperativity) to improve the overall ligand binding affinity beyond what would be expected from their individual contributions. In this study, using thrombin as a protein model system, we evaluated the effect of the bioisosteric replacement of a carbonyl functionality with a sulphonyl functionality on positive cooperativity between their H-bonds with thrombin and hydrophobic binding in the adjacent S3 pocket. The positive cooperativity observed was greatly reduced when replacing the carbonyl group with a sulphonyl group. Evaluating how bioisosteric replacements affect cooperativity is important for making better informed ligand optimization SAR decisions.

Binding cooperativity between a ligand carbonyl group and a hydrophobic side chain can be enhanced by additional H-bonds in a distance dependent manner: A case study with thrombin inhibitors.

One of the underappreciated non-covalent binding factors, which can significantly affect ligand-protein binding affinity, is the cooperativity between ligand functional groups. Using four different series of thrombin inhibitors, we reveal a strong positive cooperativity between an H-bond accepting carbonyl functionality and the adjacent P3 hydrophobic side chain. Adding an H-bond donating amine adjacent to the P3 hydrophobic side chain further increases this positive cooperativity thereby improving the Ki by as much as 546-fold. In contrast, adding an amidine multiple H-bond/salt bridge group in the distal S1 pocket does not affect this cooperativity. An analysis of the crystallographic B-factors of the ligand groups inside the binding site indicates that the strong cooperativity is mainly due to a significant mutual reduction in the residual mobility of the hydrophobic side chain and the H-bonding functionalities that is absent when the separation distance is large. This type of cooperativity is important to encode in binding affinity prediction software, and to consider in SAR studies.

Calcium-channel activation and matrix protein upregulation in bone cells in response to mechanical strain.

Femur-derived osteoblasts cultured from rat femora were loaded with Fluo-3 using the AM ester. A quantifiable stretch was applied and [Ca(2+)]i levels monitored by analysis of fluorescent images obtained using an inverted microscope and laser scanning confocal imaging system. Application of a single pulse of tensile strain via an expandable membrane resulted in immediate increase in [Ca(2+)]i in a proportion of the cells, followed by a slow and steady decrease to prestimulation levels. Application of parathyroid hormone (10(-6) M) prior to mechanical stimulation potentiated the load-induced elevation of [Ca(2+)]i. Mechanically stimulating osteoblasts in Ca(2+)-free media or in the presence of either nifedipine (10 microM; L-type Ca(2+)-channel blocker) or thapsigargin (1 microM; depletes intracellular Ca(2+) stores) reduced strain-induced increases in [Ca(2+) ]i. Furthermore, strain-induced increases in [Ca(2+)]i were enhanced in the presence of Bayer K 8644 (500 nm), an agonist of L-type calcium channels. The effects of mechanical strain with and without inhibitors and agonists are described on the total cell population and on single cell responses. Application of strain and strain in the presence of the calcium-channel agonist Bay K 8644 to periosteal-derived osteoblasts increased levels of the extracellular matrix proteins osteopontin and osteocalcin within 24 h postload. This mechanically induced increase in osteopontin and osteocalcin was inhibited by the addition of the calcium-channel antagonist, nifedipine. Our results suggest an important role for L-type calcium channels and a thapsigargin-sensitive component in early mechanical strain transduction pathways in osteoblasts.

Increased reactive oxygen species in familial amyotrophic lateral sclerosis with mutations in SOD1.

Amyotrophic lateral sclerosis (ALS) is a paralytic disorder characterized by degeneration of large motor neurons of the brain and spinal cord. A subset of ALS is inherited (familial ALS, FALS) and is associated with more than 70 different mutations in the SOD1 gene. Here we report that lymphoblast cell lines derived from FALS patients with 16 different mutations in SOD1 gene exhibit significant increase of intracellular reactive oxygen species (ROS) compared with sporadic ALS (SALS) and normal controls (spouses of ALS patients). The ROS generation did not correlate with SOD1 activity. Further, cells incubated with vitamin C, catalase or the flavinoid quercetin significantly reduced ROS in all groups. The catalase inhibitor 3-amino-1,2,4-triazole resulted in a ten-fold increase of ROS in all groups. Neither L-nitroarginine, a nitric oxide synthase inhibitor or vitamin E altered the ROS levels. Thus, these studies suggest that hydrogen peroxide (H(2)O(2)) is a major ROS elevated in FALS lymphoblasts and it may contribute to the degeneration of susceptible cells. Further, we postulate a mechanism by which increased H(2)O(2) could be generated by mutant SOD1.

Hormonal regulation of [Ca(2+)](i) in periosteal-derived osteoblasts: effects of parathyroid hormone, 1,25(OH)(2)D(3) and prostaglandin E(2).

The effects of hormonal modulators of osteoblast function, parathyroid hormone, 1,25(OH)(2)D(3) and prostaglandins on [Ca(2+)](i) in periosteal-derived osteoblasts from rat femurs have been investigated. Our results show that application of parathyroid hormone PTH (10(-5) M) and prostaglandin E(2) (PGE(2)) (4 microM) result in a rapid heterogeneous elevation in [Ca(2+)](i) that, in the case of PTH, is dependent on both extracellular and intracellular sources of calcium. Variable responses to treatments have been found within populations of cells. The PGE(2) response is dose dependent. Treatment with 1,25(OH)(2)D(3) (10(-8) M) induces a brief (60-90 sec) elevation in [Ca(2+)](i) that is almost totally abolished in EGTA-buffered Ca(2+)-free medium. Interactive effects of multiple hormone treatments have been observed. Pretreatment with 1,25(OH)(2)D(3) results in near-total inhibition of the PTH and PGE(2) responses. In conclusion, modulation of [Ca(2+)](i) appears to play a role not only in the direct effects of osteotropic hormones on osteoblasts but also in the synergistic and antagonistic effects between circulating hormones.