Cytotoxicity and comparative binding mechanism of piperine with human serum albumin and α-1-acid glycoprotein

<div><p>Human serum albumin (HSA) and α-1-acid glycoprotein (AGP) (acute phase protein) are the plasma proteins in blood system which transports many drugs. To understand the pharmacological importance of piperine molecule, here, we studied the anti-inflammatory activity of piperine on mouse macrophages (RAW 264.7) cell lines, which reveals that piperine caused an increase in inhibition growth of inflammated macrophages. Further, the fluorescence maximum quenching of proteins were observed upon binding of piperine to HSA and AGP through a static quenching mechanism. The binding constants obtained from fluorescence emission were found to be <i>K</i>_piperine= 5.7 ± .2 × 10⁵ M⁻¹ and <i>K</i>_piperine = 9.3± .25 × 10⁴ M⁻¹ which correspond to the free energy of −7.8 and −6.71 kcal M⁻¹at 25 °C for HSA and AGP, respectively. Further, circular dichrosim studies revealed that there is a marginal change in the secondary structural content of HSA due to partial destabilization of HSA–piperine complexes. Consequently, inference drawn from the site-specific markers (phenylbutazone, site I marker) studies to identify the binding site of HSA noticed that piperine binds at site I (IIA), which was further authenticated by molecular docking and molecular dynamic (MD) studies. The binding constants and free energy corresponding to experimental and computational analysis suggest that there are hydrophobic and hydrophilic interactions when piperine binds to HSA. Additionally, the MD studies have showed that HSA–piperine complex reaches equilibration state at around 3 ns, which prove that the HSA–piperine complex is stable in nature.</p></div

Spectroscopic evaluation of synthesized 5β-dihydrocortisol and 5β-dihydrocortisol acetate binding mechanism with human serum albumin and their role in anticancer activity

<p>Our study focus on the biological importance of synthesized 5β-dihydrocortisol (Dhc) and 5β-dihydrocortisol acetate (DhcA) molecules, the cytotoxic study was performed on breast cancer cell line (MCF-7) normal human embryonic kidney cell line (HEK293), the IC₅₀ values for MCF-7 cells were 28 and 25 μM, respectively, whereas no toxicity in terms of cell viability was observed with HEK293 cell line. Further experiment proved that Dhc and DhcA induced 35.6 and 37.7% early apoptotic cells and 2.5, 2.9% late apoptotic cells, respectively, morphological observation of cell death through TUNEL assay revealed that Dhc and DhcA induced apoptosis in MCF-7 cells. The complexes of HSA–Dhc and HSA–DhcA were observed as static quenching, and the binding constants (<i>K</i>) was 4.7 ± .03 × 10⁴ M⁻¹ and 3.9 ± .05 × 10⁴ M⁻¹_, and their binding free energies were found to be −6.4 and −6.16 kcal/mol, respectively. The displacement studies confirmed that lidocaine 1.4 ± .05 × 10⁴ M⁻¹ replaced Dhc, and phenylbutazone 1.5 ± .05 × 10⁴ M⁻¹ replaced by DhcA, which explains domain I and domain II are the binding sites for Dhc and DhcA. Further, FT-IR, synchronous spectroscopy, and CD results revealed that the secondary structure of HSA was altered in the presence of Dhc and DhcA. Furthermore, the atomic force microscopy and transmission electron microscopy showed that the dimensions like height and molecular size of the HSA–Dhc and HSA–DhcA complex were larger compared to HSA alone. Detailed analysis through molecular dynamics simulations also supported greater stability of HSA–Dhc and HSA–DhcA complexes, and root-mean-square-fluctuation interpreted the binding site of Dhc as domain IB and domain IIA for DhcA. This information is valuable for further development of steroid derivative with improved pharmacological significance as novel anti-cancer drugs.</p

Development of quinazolinone and vanillin acrylamide hybrids as multi-target directed ligands against Alzheimer’s disease and mechanistic insights into their binding with acetylcholinesterase

In view of Multi-Target Directed Ligand (MTDL) approach in treating Alzheimer’s Disease (AD), a series of novel quinazolinone and vanillin cyanoacetamide based acrylamide derivatives (9a-z) were designed, synthesized, and assessed for their activity against a panel of selected AD targets including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), amyloid β protein (Aβ), and also 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and neuroprotective activities. Five of the target analogs 9e, 9h, 9 l, 9t and 9z showed elevated AChE inhibitory activity with IC50 values of 1.058 ± 0.06, 1.362 ± 0.09, 1.434 ± 0.10, 1.015 ± 0.10, 1.035 ± 0.02 µM respectively, high inhibition selectivity against AChE over BChE and good DPPH radical scavenging activity. Enzyme kinetic studies of the potent hybrids in the series disclosed their mixed inhibition approach. Active analogs were found to be non-toxic on SK-N-SH cell lines and have excellent neuroprotective effects against H2O2-induced cell death. Strong modulating affinities on Aβ aggregation process were observed for most active compounds since; they irretrievably interrupted the morphology of Aβ42 fibrils, increased the aggregates and declined the Aβ-induced toxicity in neurons. From the fluorescence emission studies, the binding constants (K) were determined as 2.5 ± 0.021x103, 2.7 ± 0.015x103, 3.7 ± 0.020x103, 2.4 ± 0.013x104, and 5.0 ± 0.033x103 M−1 and binding free energies as −5.82 ± 0.033, −6.07 ± 0.042, −6.26 ± 0.015, −7.71 ± 0.024, and −6.29 ± 0.026 kcal M−1 for complexes of AChE–9e, 9h, 9 l, 9t and 9z, respectively. Moreover, the CD analysis inferred the limited modifications in the AChE secondary structure when it binds to 9e, 9h, 9 l, 9t and 9z. On the basis of docking studies against AChE, the most active congeners were well oriented in the enzyme’s active site by interacting with both catalytic active site (CAS) and peripheral anionic site (PAS). In summary, these quinazolinone and vanillin acrylamide hybrid analogs can be used as promising molecular template to further explore their in vivo efficiency in the development of lead compound to treat AD. Communicated by Ramaswamy H. Sarma</p