Aparna Rachamallu

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 Kpiperineu2009=u20095.7 ± .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.

Anticancer effects of the novel 1α, 25‐dihydroxyvitamin D3 hybrid analog QW1624F2‐2 in human neuroblastoma

Vitamin D3 analogs are potential anti‐cancer agents with theoretically wide therapeutic index, but there have been limited studies directed towards human neuroblastoma. The antiproliferative ability of the novel vitamin D3 hybrid analog QW‐1624F2‐2 (QW, 1‐hydroxymethyl‐16‐ene‐24, 24‐F2‐26, 27‐bishomo‐25‐hydroxyvitamin D3) was examined in two human neuroblastoma‐derived cell‐lines. Analog QW inhibited cell‐cycle progression of IMR5 cells with accumulation in G1 phase. QW induced the differentiation of CHP134 as evidenced by increased neurite length. These effects were accompanied by decreased expression of MYCN in both the cell‐lines treated with QW. Furthermore, QW inhibited the migration of CHP134 cells in matrigel invasion assays, indicating its anti‐invasive ability. In athymic nude mice, we found that QW was less calcemic than EB1089 (1α, 25‐dihydroxy‐22, 24‐diene‐24, 26,27‐trishomovitamin D3). Systemic administration of QW in a mouse xenotransplantation model revealed that it is more effective than EB1089 in suppressing the growth of CHP134 flank tumors. In summary, the low‐calcemic hybrid analog QW showed significant anti‐tumor activity in vivo and thus exhibits potential as a novel cancer therapeutic.

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.

γδ T cell mediated immune responses in Disease and Therapy

The role of γδ T cells in immunotherapy has gained specific importance in the recent years because of their prominent function involving directly or indirectly in the rehabilitation of the diseases. γδ T cells represent a minor population of T cells that express a distinct T cell receptor (TCR) composed of γδ chains instead of αβ chains. Unlike αβ T cells, γδ T cells display a restricted TCR repertoire and recognize mostly unknown non-peptide antigens. γδ T cells act as a link between innate and adaptive immunity, because they lack precise major histocompatibility complex (MHC) restriction and seize the ability to recognize ligands that are generated during affliction. Skin epidermal γδ T cells recognize antigen expressed by damaged or stressed keratinocytes and play an indispensable role in tissue homeostasis and repair through secretion of distinct growth factors. γδ T cell based immunotherapy strategies possess great prominence in the treatment because of the property of their MHC-independent cytotoxicity, copious amount of cytokine release, and a immediate response in infections. Understanding the role of γδ T cells in pathogenic infections, wound healing, autoimmune diseases, and cancer might provide knowledge for the successful treatment of these diseases using γδ T cell based immunotherapy. Enhancing the human Vγ9Vδ2 T cells functions by administration of aminobisphosphonates like zoledronate, pamidronate, and bromohydrin pyrophosphate along with cytokines and monoclonal antibodies shows a hopeful approach for treatment of tumors and infections. The current review summarizes the role of γδ T cells in various human diseases and immunotherapeutic approaches using γδ T cells.

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.

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

Anti-inflammatory profile of Aegle marmelos (L) Correa (Bilva) with special reference to young roots grown in different parts of India

Background Aegle marmelos (Bilva) is being used in Ayurveda for the treatment of several inflammatory disorders. The plant is a member of a fixed dose combination of Dashamoola in Ayurveda. However, the usage of roots/root bark or stems is associated with sustainability concerns. Objectives The present study is aimed to compare the anti-inflammatory properties of different extracts of young roots (year wise) and mature parts of Bilva plants collected from different geographical locations in India, so as to identify a sustainable source for Ayurvedic formulation. Materials and methods A total of 191 extracts (petroleum ether, ethyl acetate, ethanol and aqueous) of roots, stems and leaves of A. marmelos (collected from Gujarat, Maharashtra, Odisha, Chhattisgarh, Karnataka and Andhra Pradesh region) were tested for anti-inflammatory effects in vitro on isolated target enzymes cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), lymphocyte proliferation assay (LPA), cytokine profiling in LPS induced mouse macrophage (RAW 264.7) cell line and in vivo carrageenan induced paw edema in mice. Results Of 191 extracts, 44 extracts showed COX-2 inhibition and 38 extracts showed COX-1 inhibition, while none showed 5-LOX inhibition. Cytokine analysis of the 44 extracts showing inhibition of COX-2 suggested that only 17 extracts modulated the cytokines by increasing the anti-inflammatory cytokine IL-2 and reducing the pro-inflammatory cytokines like IL-1β, MIP1-α and IL-6. The young (2 and 3 years) roots of Bilva plants from Gujarat and young (1 yr) roots from Odisha showed the most potent anti-inflammatory activity by suppressing the pro-inflammatory cytokines and inducing anti-inflammatory cytokines. These three extracts have also shown in vivo anti-inflammatory activity comparable to that in adult stem and root barks. Conclusion The present study reveals that young roots of Bilva plants from Gujarat and Odisha region could form a sustainable source for use in Ayurvedic formulations with anti-inflammatory activities. The present study also indicates that the region in which the plants are grown and the age of the plants play an important role in exhibiting the anti-inflammatory effect.