Daniel Pushparaju Yeggoni

Binding and molecular dynamics studies of 7-hydroxycoumarin derivatives with human serum albumin and its pharmacological importance

Human serum albumin (HSA) is one of the most widely studied proteins and is an important plasma protein responsible for binding and transport of many exogenous and endogenous drugs. Coumarin derivatives play a critical role as anticancer, antidiabetic, anticoagulant, and analgesic agents. Here we have studied the cytotoxic activity of 7-hydroxycoumarin derivatives (7HC-1, 7HC-2, and 7HC-3) on mouse macrophage (RAW 264.7) cell lines. These studies revealed that 7-hydroxycoumarin derivatives caused an increased inhibition in growth of inflamed macrophages in a concentration-dependent manner with an IC50 of 78, 63, and 50 μM. Further studies, using fluorescence, circular dichroism spectroscopy, molecular docking, and molecular dynamics methods, show binding of 7HC (umbelliferone) derivatives with HSA at physiological pH 7.2. The binding constant of 7HC derivatives with HSA obtained from fluorescence emission was found to be K7HC-1 = 4.6 ± 0.01 × 10(4) M(-1), K7HC-2 = 1.3 ± 0.01 × 10(4) M(-1), and K7HC-3 = 7.9 ± 0.01 × 10(4) M(-1) which corresponds to -6.34 kcal/mol, -5.58 kcal/mol, and -6.65 kcal/mol of free energy. In contrast, the binding of these coumarin derivatives (7HC-1, 7HC-2, and 7HC-3) was almost negligible with α-1-glycoprotein (AGP). Circular dichroism (CD) studies revealed a decreased α-helix content with an increase in the β-sheets and random coils in HSA upon interaction with coumarin derivatives, suggesting a partial unfolding of the HSA secondary structure. Site probe studies with phenylbutazone (Site I) and ibuprofen (Site II) indicated that 7HC derivatives specifically bind to sub domains IIIA and IIIB of HSA which is further corroborated by molecular dynamics and docking studies suggesting that binding is specific in nature. The values of free energies and binding constants coincide for both experimental and in silico analysis and suggest that there are hydrophobic interactions when coumarin derivatives bind to HSA. Molecular dynamics studies showed that the HSA-coumarin complex reaches an equilibration state at around 3.5 ns which indicates that the HSA-coumarin complexes were stable. Thus these interactions play a central role in development of coumarin derivative-inspired drugs.

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

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 Kpiperine = 5.7 ± .2 × 105 M−1 and Kpiperine = 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.

A comparative binding mechanism between human serum albumin and α-1-acid glycoprotein with corilagin: biophysical and computational approach

The binding of corilagin with plasma serum proteins like human serum albumin (HSA) and α-1-acid glycoprotein (AGP) was investigated under physiological conditions. To understand the pharmacological importance of the corilagin molecule, anti-inflammatory activity on mouse macrophages (RAW 264.7) cell lines was studied. This study reveals that corilagin caused an increase in inhibition growth of inflamed macrophages in concentration-dependent manner with an IC50 value of 66 μM. Further, intrinsic fluorescence of HSA and AGP was quenched upon titration of corilagin, and the binding constants obtained from fluorescence emission was found to be Kcorilagin 4.2 ± 0.02 × 105 M−1 which corresponds to the free energy of −7.6 kcal M−1 at 25 °C for a HSA–corilagin complex. Interestingly, corilagin showed binding with AGP, an acute phase protein, and the binding constant was found to be Kcorilagin = 1.5 ± 0.01 × 104 M−1 and its free energy was −5.6 kcal M−1 at 25 °C. Further, the average binding distance, r, between the donor (HSA) and acceptor (corilagin) was calculated and found to be 1.32 nm according to Forsters theory of non-radiation energy transfer. Later, circular dichroism studies emphasized that there are marginal changes in secondary structural conformation of HSA in the presence of corilagin. Corilagin is specifically bound to site I of HSA which was proved by site specific marker, phenylbutazone. Furthermore, the binding details between corilagin and HSA revealed that corilagin was bound to subdomain IIA through multiple interactions like hydrogen bonding and hydrophobic effects. Molecular dynamic studies (MD) also suggest that binding is very precise to site I (IIA domain) on HSA. Also, MD studies showed that HSA–corilagin complex reaches equilibration state at around 4 ns, which proves that the HSA–corilagin complex is stable in nature, hence the experimental and computational results are in agreement. Thus, examining the interaction mechanism of corilagin with plasma proteins may play a critical role in developing corilagin inspired drugs.

Protein stability, conformational change and binding mechanism of human serum albumin upon binding of embelin and its role in disease control

Here, we present the inclusive binding mode of phytochemical embelin, an anticancer drug with human serum albumin (HSA) established under physiological condition. Also, to understand the pharmacological role of embelin molecule, here, we have studied the anti-cancer activity of embelin on human cervical cancer cell line (HeLa cell line), which revealed that embelin showed dose dependent inhibition in the growth of cancer cells and also induces 26.3% of apoptosis at an IC50 value of 29μM. Further, embelin was titrated with HSA and the fluorescence emission quenching of HSA due to the formation of the HSA-embelin complex was observed. The binding constant of this complex is 5.9±.01×10(4)M(-1) and the number of bound embelin molecules is approximately 1.0. Consequently, molecular displacement and computational docking experiments show that the embelin is binding to subdomain IB to HSA. Further evidence from microTOF-Q mass spectrometry showed an increase in mass from 66,563Da to 66,857Da observed for free HSA and HSA+embelin complex, signifying that there is robust binding of embelin with HSA. In addition, the variations of HSA secondary structural elements in presence of embelin were confirmed by circular dichroism which indicates partial unfolding of protein. Furthermore, the transmission electron micrographs established that complex formation leads to aggregation of HSA plus embelin. Molecular dynamics simulations revealed that the stability of the HSA-embelin complexes and results suggests that at around 3500ps the complex reaches equilibration state which clearly contributes to the understanding of the stability of the HSA-embelin complexes.

Elucidating the binding interaction of andrographolide with the plasma proteins: biophysical and computational approach

The present study focuses on the interactions of andrographolide (ANDR) with plasma proteins, human serum albumin (HSA), and α-1-acid glycoprotein (AGP), and their biological importance. To understand the pharmacological role of ANDR, its anticancer activity was studied on a breast cancer cell line (MCF-7); it showed a dose-dependent inhibition of growth, and its IC50 value was found to be 55 μM. Furthermore, to evaluate the binding mechanism of AGP and HSA with andrographolide, fluorescence emission quenching was observed as a static mechanism upon the binding of ANDR to plasma proteins. Additionally, active HSA sensor chip surfaces were prepared through an amine-coupling reaction protocol, and the equilibrium association constants for ANDR–HSA were then determined by surface plasmon resonance (SPR) analysis. The association constants of ANDR binding to HSA, obtained with fluorescence and SPR, were KA(AGD) = 1.85 ± 0.02 × 104 M−1 and 3.1 ± 0.04 × 103 M−1, respectively. Similarly, the ANDR binding affinity with AGP was analyzed through fluorescence and SPR, and the calculated binding association values were 1.5 ± 0.01 × 103 M−1 and 1.3 ± 0.04 × 103 M−1, respectively. Molecular displacement and in silico docking shows that ANDR binds to subdomain IIB. Consequently, circular dichroism analysis showed that there is partial perturbation in the structure of HSA upon an increase in the concentration of ANDR. Moreover, molecular dynamics simulation revealed that the stability of the HSA–ANDR complexes reached an equilibration state at around 3000 ps, which clearly indicates the rigidity and stability of the HSA–ANDR complexes. Thus, our results provide evidence that both plasma proteins (HSA and AGP) can act as carrier proteins for ANDR.

Investigation of binding mechanism of novel 8-substituted coumarin derivatives with human serum albumin and α-1-glycoprotein

Coumarin molecules have biological activities possessing lipid-controlling activity, anti-hepatitis C activity, anti-diabetic, anti-Parkinson activity, and anti-cancer activity. Here, we have presented an inclusive study on the interaction of 8-substituted-7-hydroxy coumarin derivatives (Umb-1/Umb-2) with α-1-glycoprotein (AGP) and human serum albumin (HSA) which are the major carrier proteins in the human blood plasma. Binding constants obtained from fluorescence emission data were found to be KUmb-1=3.1 ± .01 × 104 M−1, KUmb-2 = 7 ± .01 × 104 M−1, which corresponds to −6.1 and −6.5 kcal/mol of free energy for Umb-1 and Umb-2, respectively, suggesting that these derivatives bind strongly to HSA. Also these molecules bind to AGP with binding constants of KUmb-1-AGP=3.1 ± .01 × 103 M−1 and KUmb-2-AGP = 4.6 ± .01 × 103 M−1. Further, the distance, r between the donor (HSA) and acceptor (Umb-1/Umb-2) was calculated based on the Forster’s theory of non-radiation energy transfer and the values were observed to be 1.14 and 1.29 nm in Umb-1–HSA and Umb-2–HSA system, respectively. The protein secondary structure of HSA was partially unfolded upon binding of Umb-1 and Umb-2. Furthermore, site displacement experiments with lidocaine, phenylbutazone (IIA), and ibuprofen (IIIA) proves that Umb derivatives significantly bind to subdomain IIIA of HSA which is further supported by docking studies. Furthermore, Umb-1 binds to LYS402 with one hydrogen bond distance of 2.8 Å and Umb-2 binds to GLU354 with one hydrogen bond at a distance of 2.0 Å. Moreover, these molecules are stabilized by hydrophobic interactions and hydrogen bond between the hydroxyl groups of carbon-3 of coumarin derivatives.

Molecular binding mechanism of 5-hydroxy-1-methylpiperidin-2-one with human serum albumin

5-hydroxy-1-methylpiperidin-2-one (5-HMP) is a piperidine alkaloid found in various medicinal plants including seeds of Piper nigrum, Piper longum, fruit of Piper retrofractum and other medicinal plants such as Pinus ponderosa, aerial parts of Piper methysticum, stem bark of Microcos paniculata, Cassia leptophylla, rhizome of Arisaema decipiens and root of Lobelia laxiflora. This type of piperidine alkaloids reported against various diseases such as piperidine for cancer, piperamides and piperzine analogs for depressant, antioxidants, piperine for hypertensive and inflammatory problems, quinine for malarial disease, ephedrine for asthma, vincamine used as vasodilatory agent, piperine as antihyperglycemic agent. Also, 5-HMP from Tragia involucrata L. leaves for bronchoconstriction as antihistamine reported from the previous study and also aerial part of T. involucrata extracts reported against MCF-7 and KB cell line, methanol extract of T. involucrata root contains wound healing activity, T. involucrata extract possesses analgesic and anti-inflammatory (Alagar Yadav, Ramalingam, Jabamalai Raj, & Subban, 2015). Human serum albumin (HSA) is a major protein in blood plasma which contains excellent binding capacity for Ca, Na, K, hormones, bilirubin, zinc with metal binding site and it also regulates the osmotic pressure of blood. HSA is a carrier protein, abundantly (60%) present in human blood plasma contains 585 amino acids arranged as a single polypeptide chain. Among the amino acid residues, the region between 1–195, 196–383, and 384–585 resides as three major domains I, II, and III, respectively, namely Sudlow site I and II. Each domain comprises two sub-domains linked with 17 disulfide bridges with heart shape structure. When considering the binding region of HSA among the six subdomains, the principle binding region located at IIA and III A sub-domains due to the presence of Trp 214, Lys 199, Tyr 411 (Sudlow, Birkett, & Wade, 1975). Further, the major vital role of HSA is to transport and deliver the various drugs to multiple organs of the body with their good binding affinity and it also acts as receptor for fatty acid, bilirubin, warfarin, indole, benzodiazepines with the lowest binding affinity (Muller & Wollert, 1979). Most of the drugs were originated from the medicinal plants and it produced various classes of phyto-constituents including alkaloids, flavonoids, terpenoids, steroids and tannins as secondary metabolites were used as phyto-pharmaceutical drugs (Veeresham, 2012) with good binding properties on HSA. The different kinds of alkaloids and their binding affinity with HSA studied among scientific communities. In that, the piperine, Caffeine, indole, isomer of ciprofloxacin N, B, F, and I binds with HSA with binding constant of .5 × 10, 12 × 10, 4.2 × 10, 6.92 × 10, 3.87 × 10, 4.06 × 10, and 2.7 × 10 M, respectively. Likewise, various flavonoids also contain good binding properties with HSA such as quercetin, hyperin, baicalin, rutin, curcumin, and genistein. Similar binding studies were carried out in our laboratory with HSA along with different phytochemicals. For the last recent period our lab group reported the high binding affinity of HSA with different phytochemicals including trans-feruloylmaslinic acid, β – sitosterol, N-trans-p-coumaroyltyramine, betulinic acid, L-3,4 dihydroxy phenylalanine and piperine. Since 5-HMP has several important roles in curing many diseases but the understanding of bioavailability of the drug and its pharmacokinetics, HSA is a good model system. Moreover, the binding studies of 5-HMP are studied with plasma protein. Thus, the present study reveals the molecular interactions of such valuable phytochemical 5HMP with HSA by the formation of ligand–protein complexes as preliminary step of drug invention among the ADME feature (Benet, Kroetz, Sheiner, Hardman, & Limbird, 1996) and it is also an important factor that can

Probing the interaction mechanism of menthol with blood plasma proteins and its cytotoxicity activities

Probing the interaction mechanism of menthol with blood plasma proteins and its cytotoxicity activities Daniel Pushparaju Yeggoni, Aparna Rachamallu, Shreya Dubey, Argha Mitra and Rajagopal Subramanyam* Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India; National Institute of Animal Biotechnology, Axis Clinicals Building, Miyapur, Hyderabad 500049, India

New flavone-cyanoacetamide hybrids with combination of cholinergic, antioxidant, modulation β-amyloid aggregation and neuroprotection properties as innovative multifunctional therapeutic candidates for Alzheimer’s disease and unraveling their mechanism of action with acetylcholinesterase

In line with the modern multi-target-directed ligand paradigm of Alzheimers disease (AD), a series of 19 compounds composed of flavone and cyanoacetamide groups have been synthesized and evaluated as multifunctional agents against AD. Biological evaluation demonstrated that compounds 7j, 7n, 7o, 7r, and 7s exhibited excellent inhibitory potency (AChE, IC50 of 0.271 ± 0.012 to 1.006 ± 0.075 μM) and good selectivity toward acetylcholinesterase, significant antioxidant activity, good modulation effects on self-induced Aβ aggregation, low cytotoxicity, and neuroprotection in human neuroblastoma SK-N-SH cells. Further, an inclusive study on the interaction of 7j, 7n, 7o, 7r, and 7s with AChE at physiological pH 7.2 using fluorescence, circular dichroism, and molecular docking methods suggested that these derivatives bind strongly to the peripheral anionic site of AChE mostly through hydrophobic interactions. Overall, the multifunctional profiles and strong AChE binding affinity highlight these compounds as promising prototypes for further pursuit of innovative multifunctional drugs for AD.