Molecular interaction of quercetin and its derivatives against nucleolin in breast cancer: in-silico and in-vitro study.

Nucleolin, a multifaceted RNA binding domain protein is overexpressed in various cancers leading to dysfunction of several cellular signaling pathways. Quercetin, a distinctive bioactive molecule, along with its derivatives have shown exclusive physio-chemical properties which makes them appealing choices for drug development, yet their role in targeted cancer therapy is limited. Here, the RBD domain structure of Nucleolin was modeled and stabilized by MD simulations for a period of 1000 ns. Molecular docking was performed to determine the binding capability of ligands with the target. To determine the stability of the ligand inside the binding pocket of the protein, MD simulation was performed for a period of 250 ns each for Quercetin-4'-o'-Glucoside, Quercetin_9 and Quercetin complexes. Further, in-vitro studies including cytotoxicity and RT-PCR assays were performed to validate quercetin against Nucleolin. Molecular docking and MD Simulation studies suggested a potential mechanism of interaction of Quercetin-4'-o'-Glucoside, Querectin_9 and Quercetin with Nucleolin with the binding free energy of -63.653, -58.86 and -46.9 kcal/mol, respectively. Moreover, Lys 348 and Glu379 were identified as important amino acids in ligand interaction located at the RRM2 motif of Nucleolin. In-vitro studies showed significant downregulation in Nucleolin expression by 15.18 and 2.51-fold at 48h and 72h respectively in MCF-7 cells with Quercetin (IC50 = 160 µM). Our findings suggested the potential role of specific RRM motifs in interaction with natural compounds targeting Nucleolin. This could be an effective strategy in the identification of potential molecules in targeting Nucleolin which can be further explored for developing targeted therapies for breast cancer.Communicated by Ramaswamy H. Sarma.

The effect of immediate environment on bond strength of different bond types-A valence bond study.

The ability to design catalysis largely depends on our understanding of the electrostatic effect of the surrounding on the bonds participating in the reaction. Here, we used a simplistic model of point charges (PCs) to determine a set of rules guiding how to construct PC-bond arrangement that can strengthen or weaken different chemical bonds. Using valence bond theory to calculate the in situ bond energies, we show that the effect of the PC mainly depends on the bond's dipole moment irrespective of its type (being covalent or charge shift). That is, polar bonds are getting stronger or weaker depending on the sign and location of the PC, whereas non- or weakly polar bonds become stronger or weaker depending only on the location of the PC and to a smaller extent compared with polar bonds. We also show that for polar bonds, the maximal bond strengthening and weakening effect can be achieved when the PC is placed along the bond axis, as close as possible to the more and less polarizable atom/fragment, respectively. Finally, due to the stabilizing effects of polarizability, we show that, overall, it is easier to cause bond strengthening compared with bond weakening. Particularly, for polar bonds, bond strengthening is larger than bond weakening obtained by an oppositely signed PC. These rules should be useful in the future design of catalysis in, e.g., enzyme active sites.

Mechanistic insight into the role of mevalonate kinase by a natural fatty acid-mediated killing of Leishmania donovani.

We evaluated the anti-leishmanial efficacy of different saturated medium-chain fatty acids (FAs, C8-C18) where FA containing C8 chain, caprylic acid (CA), was found to be most potent against Leishmania donovani, the causative agent for visceral leishmaniasis (VL). Different analogs of CA with C8 linear chain, but not higher, along with a carboxyl/ester group showed a similar anti-leishmanial effect. Ergosterol depletion was the major cause of CA-mediated cell death. Molecular docking and molecular dynamic simulation studies indicated the enzyme mevalonate kinase (MevK) of the ergosterol biosynthesis pathway as a possible target of CA. Enzyme assays with purified recombinant MevK and CA/CA analogs confirmed the target with a competitive inhibition pattern. Using biochemical and biophysical studies; strong binding interaction between MevK and CA/CA analogs was established. Further, using parasites with overexpressed MevK and proteomics studies of CA-treated parasites the direct role of MevK as the target was validated. We established the mechanism of the antileishmanial effect of CA, a natural product, against VL where toxicity and drug resistance with current chemotherapeutics demand an alternative. This is the first report on the identification of an enzymatic target with kinetic parameters and mechanistic insights against any organism for a natural medium-chain FA.

Penta-o-galloyl-beta-d-Glucose (PGG) inhibits inflammation in human rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis model.

O-GlcNAcylation is a reversible post-translational modification that regulates numerous cellular processes, including embryonic development as well as immune responses. However, its role in inflammation remains ambiguous. This study was designed to examine the role of O-GlcNAcylation in rheumatoid arthritis (RA) and its regulation using human RA patient-derived synovial fibroblasts (RASFs). The efficacy of penta-O-galloyl-beta-D-glucose (PGG), a potent anti-inflammatory molecule, in regulating inflammatory processes in human RASFs was also evaluated. Human synovial tissues and RASFs exhibited higher expression of O-GlcNAcylation compared to their non-diseased counterparts. Pretreatment of RASFs with Thiamet G, an inhibitor of O-GlcNAcase, markedly increased the O-GlcNAc-modified proteins and concomitantly inhibited the IL-1ß-induced IL-6 and IL-8 production in human RASFs in vitro. Pretreatment of human RASFs with PGG (0.5-10 µM) abrogated IL-1ß-induced IL-6 and IL-8 production in a dose-dependent manner. Immunoprecipitation analysis showed that PGG inhibited O-GlcNAcylation of TAB1 to reduce its association with TGF ß-activated kinase 1 (TAK1) and its autophosphorylation, an essential signaling step in IL-1ß-induced signaling pathways. Molecular docking in silico studies shows that PGG occupies the C174 position, an ATP-binding site in the kinase domain to inhibit TAK1 kinase activity. Oral administration of PGG (25 mg/kg/day) for 10 days from disease onset significantly ameliorated rat adjuvant-induced (AIA) in rats. PGG treatment reduced the phosphorylation of TAK1 in the treated joints compared to AIA joints, which correlated with the reduced disease severity and suppressed levels of serum IL-1ß, GM-CSF, TNF-α, and RANKL. These findings suggest O-GlcNAcylation as a potential therapeutic target and provide the rationale for testing PGG or structurally similar molecule for their therapeutic efficacy.

Osteogenic growth peptide is a potent anti-inflammatory and bone preserving hormone via cannabinoid receptor type 2.

The endocannabinoid system consists mainly of 2-arachidonoylglycerol and anandamide, as well as cannabinoid receptor type 1 and type 2 (CB2). Based on previous studies, we hypothesized that a circulating peptide previously identified as osteogenic growth peptide (OGP) maintains a bone-protective CB2 tone. We tested OGP activity in mouse models and cells, and in human osteoblasts. We show that the OGP effects on osteoblast proliferation, osteoclastogenesis, and macrophage inflammation in vitro, as well as rescue of ovariectomy-induced bone loss and prevention of ear edema in vivo are all abrogated by genetic or pharmacological ablation of CB2. We also demonstrate that OGP binds at CB2 and may act as both an agonist and positive allosteric modulator in the presence of other lipophilic agonists. In premenopausal women, OGP circulating levels significantly decline with age. In adult mice, exogenous administration of OGP completely prevented age-related bone loss. Our findings suggest that OGP attenuates age-related bone loss by maintaining a skeletal CB2 tone. Importantly, they also indicate the occurrence of an endogenous peptide that signals via CB2 receptor in health and disease.

Fenchone Derivatives as a Novel Class of CB2 Selective Ligands: Design, Synthesis, X-ray Structure and Therapeutic Potential.

A series of novel cannabinoid-type derivatives were synthesized by the coupling of (1S,4R)-(+) and (1R,4S)-(-)-fenchones with various resorcinols/phenols. The fenchone-resorcinol derivatives were fluorinated using Selectfluor and demethylated using sodium ethanethiolate in dimethylformamide (DMF). The absolute configurations of four compounds were determined by X-ray single crystal diffraction. The fenchone-resorcinol analogs possessed high affinity and selectivity for the CB2 cannabinoid receptor. One of the analogues synthesized, 2-(2',6'-dimethoxy-4'-(2″-methyloctan-2″-yl)phenyl)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-ol (1d), had a high affinity (Ki = 3.51 nM) and selectivity for the human CB2 receptor (hCB2). In the [35S]GTPγS binding assay, our lead compound was found to be a highly potent and efficacious hCB2 receptor agonist (EC50 = 2.59 nM, E(max) = 89.6%). Two of the fenchone derivatives were found to possess anti-inflammatory and analgesic properties. Molecular-modeling studies elucidated the binding interactions of 1d within the CB2 binding site.

Takinib Inhibits Inflammation in Human Rheumatoid Arthritis Synovial Fibroblasts by Targeting the Janus Kinase-Signal Transducer and Activator of Transcription 3 (JAK/STAT3) Pathway.

TGF ß-activated kinase 1 (TAK1) is an important participant in inflammatory pathogenesis for diseases such as rheumatoid arthritis (RA) and gouty arthritis. The central position it occupies between the mitogen activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways makes it an attractive therapeutic target. As this field has developed in recent years, several novel inhibitors have been presented as having specific activity that reduces the TAK1 function either covalently as in the case of 5Z-7-oxozeanol (5Z7O) or reversibly (NG-25). However, the mechanism through which takinib elicits its anti-inflammatory activity remains elusive. While this inhibitor shows great promise, a thorough analysis of its inhibitor function and its potential off-target effects is necessary before addressing its clinical potential or its use in inflammatory conditions. An analysis through Western blot showed an unexpected increase in IL-1ß-induced TAK1 phosphorylation-a prerequisite for and indicator of its functional potential-by takinib while simultaneously demonstrating the inhibition of the JAK/STAT pathway in human rheumatoid arthritis synovial fibroblasts (RASFs) in vitro. In THP-1 monocyte-derived macrophages, takinib again led to the lipopolysaccharide-induced phosphorylation of TAK1 without a marked inhibition of the TAK1 downstream effectors, namely, of c-Jun N-terminal kinase (JNK), phospho-c-Jun, NF-κB phospho-p65 or phospho-IκBα. Taken together, these findings indicate that takinib inhibits inflammation in these cells by targeting multiple signaling pathways, most notably the JAK/STAT pathway in human RASFs.

EGCG, a Green Tea Catechin, as a Potential Therapeutic Agent for Symptomatic and Asymptomatic SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged to be the greatest threat to humanity in the modern world and has claimed nearly 2.2 million lives worldwide. The United States alone accounts for more than one fourth of 100 million COVID-19 cases across the globe. Although vaccination against SARS-CoV-2 has begun, its efficacy in preventing a new or repeat COVID-19 infection in immunized individuals is yet to be determined. Calls for repurposing of existing, approved, drugs that target the inflammatory condition in COVID-19 are growing. Our initial gene ontology analysis predicts a similarity between SARS-CoV-2 induced inflammatory and immune dysregulation and the pathophysiology of rheumatoid arthritis. Interestingly, many of the drugs related to rheumatoid arthritis have been found to be lifesaving and contribute to lower COVID-19 morbidity. We also performed in silico investigation of binding of epigallocatechin gallate (EGCG), a well-known catechin, and other catechins on viral proteins and identified papain-like protease protein (PLPro) as a binding partner. Catechins bind to the S1 ubiquitin-binding site of PLPro, which might inhibit its protease function and abrogate SARS-CoV-2 inhibitory function on ubiquitin proteasome system and interferon stimulated gene system. In the realms of addressing inflammation and how to effectively target SARS-CoV-2 mediated respiratory distress syndrome, we review in this article the available knowledge on the strategic placement of EGCG in curbing inflammatory signals and how it may serve as a broad spectrum therapeutic in asymptomatic and symptomatic COVID-19 patients.

Suppression of monosodium urate crystal-induced inflammation by inhibiting TGF-ß-activated kinase 1-dependent signaling: role of the ubiquitin proteasome system.

Monosodium urate (MSU) crystals activate inflammatory pathways that overlap with interleukin-1ß (IL-1ß) signaling. However, the post-translational mechanisms involved and the role of signaling proteins in this activation are unknown. In the present study, we investigated the intracellular signaling mechanisms involved in MSU-induced activation of THP-1 macrophages and human nondiseased synovial fibroblasts (NLSFs) and the in vivo efficacy of an inhibitor of tumor growth factor-ß (TGF-ß)-activated kinase 1 (TAK1), 5Z-7-oxozeaenol, in MSU-induced paw inflammation in C57BL/6 mice. THP-1 macrophage activation with MSU crystals (25-200 µg/ml) resulted in the rapid and sustained phosphorylation of interleukin-1 receptor-activated kinase 1 (IRAK1 Thr209) and TAK1 (Thr184/187) and their association with the E3 ubiquitin ligase TRAF6. At the cellular level, MSU inhibited the deubiquitinases A20 and UCHL2 and increased 20s proteasomal activity, leading to a global decrease in K63-linked ubiquitination and increase in K48-linked ubiquitination in THP-1 macrophages. While MSU did not stimulate cytokine production in NLSFs, it significantly amplified IL-1ß-induced IL-6, IL-8, and ENA-78/CXCL5 production. Docking studies and MD simulations followed by TAK1 in vitro kinase assays revealed that uric acid molecules are capable of arresting TAK1 in an active-state conformation, resulting in sustained TAK1 kinase activation. Importantly, MSU-induced proinflammatory cytokine production was completely inhibited by 5Z-7-oxozeaenol but not IRAK1/4 or TRAF6 inhibitors. Administration of 5Z-7-oxozeaenol (5 or 15 mg/kg; orally) significantly inhibited MSU-induced paw inflammation in C57BL/6 mice. Our study identifies a novel post-translational mechanism of TAK1 activation by MSU and suggests the therapeutic potential of TAK1 in regulating MSU-induced inflammation.

Critical role of IL-1α in IL-1ß-induced inflammatory responses: cooperation with NF-κBp65 in transcriptional regulation.

The IL-1 cytokines are considered among the first family of cytokines that orchestrate acute and chronic inflammatory diseases. Both IL-1ß and IL-1α are members of the IL-1 family; however, their distinct roles in the inflammatory processes remain poorly understood. We explored the role of IL-1α in IL-1ß-activated signaling pathways causing synovial inflammation in rheumatoid arthritis (RA). Using synovial fibroblasts isolated from RA joints, we found that IL-1ß significantly stimulated IL-1α expression, which was selectively inhibited by blocking the NF-κB pathway. Knockdown of IL-1α using small interfering RNA abolished IL-1ß-induced pro-IL-1α and pro-IL-1ß expression and suppressed inflammation. Native and chromatin immunoprecipitation studies showed that IL-1α cooperates in NF-κBp65 binding to the distal region of IL-1α promoter and to the proximal region of IL-1ß promoter upstream of the transcription start site to stabilize their gene transcription. Molecular dynamics simulation of IL-1α or IL-1ß binding to IL-1 receptor showed distinct interaction sites that corroborate with the ability of IL-1α to differentially activate phosphorylation of signaling proteins compared with IL-1ß. Our study highlights the importance of IL-1α in mediating IL-1ß-induced inflammation in addition to maintaining its expression and providing a rationale for targeting IL-1α to minimize the role of IL-1ß in inflammatory diseases like RA.-Singh, A. K., Fechtner, S., Chourasia, M., Sicalo, J., Ahmed, S. Critical role of IL-1α in IL-1ß-induced inflammatory responses: cooperation with NF-κBp65 in transcriptional regulation.

Molecular insights into the differences in anti-inflammatory activities of green tea catechins on IL-1ß signaling in rheumatoid arthritis synovial fibroblasts.

In this study, we found that catechins found in green tea (EGCG, EGC, and EC) differentially interfere with the IL-1ß signaling pathway which regulates the expression of pro-inflammatory mediators (IL-6 and IL-8) and Cox-2 in primary human rheumatoid arthritis synovial fibroblasts (RASFs). EGCG and EGC inhibited IL-6, IL-8, and MMP-2 production and selectively inhibited Cox-2 expression. EC did not exhibit any inhibitory effects. When we looked at the expression of key signaling proteins in the IL-1ß signaling pathway, we found all the tested catechins could inhibit TAK-1 activity. Therefore, the consumption of green tea offers an overall anti-inflammatory effect. Molecular docking analysis confirms that EGCG, EGC, and EC all occupy the active site of the TAK1 kinase domain. However, EGCG occupies the majority of the TAK1 active site. In addition to TAK1 inhibition, EGCG can also inhibit P38 and nuclear NF-κB expression whereas EC and EGC were not effective inhibitors. Our findings suggest one of the main health benefits associated with the consumption of green tea are due to the activity of EGCG and EGC which are both present at higher amounts. Although EGCG is the most effective catechin at inhibiting downstream inflammatory signaling, its effectiveness could be hindered by the presence of EC. Therefore, varying EC content in green tea may reduce the anti-inflammatory effects of other potential catechins in green tea.

Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches.

OBJECTIVE: Kirsten rat sarcoma (K-Ras) protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. METHODS: In the present study, we targeted the nucleotide-binding site in the "on" and "off" state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. RESULTS: Interestingly, the designed compounds exhibit a binding preference for the "off" state over "on" state conformation of K-Ras protein. Moreover, the designed compounds' interactions are similar to guanosine diphosphate and, thus, could presumably act as a potential lead for K-Ras. The predicted drug-likeness properties of these compounds suggest that these compounds follow the Lipinski's rule of five and have tolerable absorption, distribution, metabolism, excretion and toxicity values. CONCLUSION: Thus, through the current study, we propose targeting only "off" state conformations as a promising strategy for the design of reversible inhibitors to pharmacologically inhibit distinct conformations of K-Ras protein.

Regulation of Transforming Growth Factor ß-Activated Kinase Activation by Epigallocatechin-3-Gallate in Rheumatoid Arthritis Synovial Fibroblasts: Suppression of K(63) -Linked Autoubiquitination of Tumor Necrosis Factor Receptor-Associated Factor 6.

OBJECTIVE: Transforming growth factor ß-activated kinase 1 (TAK1) is a key MAPKKK family protein in interleukin-1ß (IL-1ß), tumor necrosis factor (TNF), and Toll-like receptor signaling. This study was undertaken to examine the posttranslational modification of TAK1 and its therapeutic regulation in rheumatoid arthritis (RA). METHODS: The effect of TAK1, IL-1 receptor-associated kinase 1 (IRAK-1), and TNF receptor-associated factor 6 (TRAF6) inhibition was evaluated in IL-1ß-stimulated human RA synovial fibroblasts (RASFs). Western blotting, immunoprecipitation, and 20S proteasome assay were used to study the ubiquitination process in RASFs. The efficacy of epigallocatechin-3-gallate (EGCG), a potent antiinflammatory molecule, in regulating these processes in RASFs was evaluated. Molecular docking was performed to examine the interaction of EGCG with human TAK1, IRAK-1, and TRAF6. These findings were confirmed using a rat model of adjuvant-induced arthritis (AIA). RESULTS: Inhibition of TAK1, but not IRAK-1 or TRAF6, completely abrogated IL-1ß-induced IL-6 and IL-8 synthesis in RASFs. EGCG inhibited TAK1 phosphorylation at Thr(184/187) and occupied the C(174) position, an ATP-binding site, to inhibit its kinase activity. EGCG pretreatment also inhibited K(63) -linked autoubiquitination of TRAF6, a posttranslational modification essential for TAK1 autophosphorylation, by forming a stable H bond at the K(124) position on TRAF6. Furthermore, EGCG enhanced proteasome-associated deubiquitinase expression to rescue proteins from proteasomal degradation. Western blot analyses of joint homogenates from rats with AIA showed a significant increase in K(48) -linked polyubiquitination, TAK1 phosphorylation, and TRAF6 expression when compared to naive rats. Administration of EGCG (50 mg/kg/day) for 10 days ameliorated AIA in rats by reducing TAK1 phosphorylation and K(48) -linked polyubiquitination. CONCLUSION: Our findings provide a rationale for targeting TAK1 for the treatment of RA with EGCG.

HU-446 and HU-465, Derivatives of the Non-psychoactive Cannabinoid Cannabidiol, Decrease the Activation of Encephalitogenic T Cells.

Cannabidiol (CBD), the non-psychoactive cannabinoid, has been previously shown by us to decrease peripheral inflammation and neuroinflammation in mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). Here we have studied the anti-inflammatory effects of newly synthesized derivatives of natural (-)-CBD ((-)-8,9-dihydro-7-hydroxy-CBD; HU-446) and of synthetic (+)-CBD ((+)-8,9-dihydro-7-hydroxy-CBD; HU-465) on activated myelin oligodendrocyte glycoprotein (MOG)35-55-specific mouse encephalitogenic T cells (T(MOG) ) driving EAE/MS-like pathologies. Binding assays followed by molecular modeling revealed that HU-446 has negligible affinity toward the cannabinoid CB1 and CB2 receptors while HU-465 binds to both CB1 and CB2 receptors at the high nanomolar concentrations (Ki = 76.7 ± 5.8 nm and 12.1 ± 2.3 nm, respectively). Both, HU-446 and HU-465, at 5 and 10 µm (but not at 0.1 and 1 µm), inhibited the MOG35-55-induced proliferation of autoreactive T(MOG) cells via CB1/CB2 receptor independent mechanisms. Moreover, both HU-446 and HU-465, at 5 and 10 µm, inhibited the release of IL-17, a key autoimmune cytokine, from MOG35-55-stimulated T(MOG) cells. These results suggest that HU-446 and HU-465 have anti-inflammatory potential in inflammatory and autoimmune diseases.

CB2 cannabinoid receptor agonist enantiomers HU-433 and HU-308: An inverse relationship between binding affinity and biological potency.

Activation of the CB2 receptor is apparently an endogenous protective mechanism. Thus, it restrains inflammation and protects the skeleton against age-related bone loss. However, the endogenous cannabinoids, as well as Δ(9)-tetrahydrocannabinol, the main plant psychoactive constituent, activate both cannabinoid receptors, CB1 and CB2. HU-308 was among the first synthetic, selective CB2 agonists. HU-308 is antiosteoporotic and antiinflammatory. Here we show that the HU-308 enantiomer, designated HU-433, is 3-4 orders of magnitude more potent in osteoblast proliferation and osteoclast differentiation culture systems, as well as in mouse models, for the rescue of ovariectomy-induced bone loss and ear inflammation. HU-433 retains the HU-308 specificity for CB2, as shown by its failure to bind to the CB1 cannabinoid receptor, and has no activity in CB2-deficient cells and animals. Surprisingly, the CB2 binding affinity of HU-433 in terms of [(3)H]CP55,940 displacement and its effect on [(35)S]GTPγS accumulation is substantially lower compared with HU-308. A molecular-modeling analysis suggests that HU-433 and -308 have two different binding conformations within CB2, with one of them possibly responsible for the affinity difference, involving [(35)S]GTPγS and cAMP synthesis. Hence, different ligands may have different orientations relative to the same binding site. This situation questions the usefulness of universal radioligands for comparative binding studies. Moreover, orientation-targeted ligands have promising potential for the pharmacological activation of distinct processes.

Copper-chaperones with dicoordinated Cu(I)--unique protection mechanism.

Cu(I) dicoordination with thiolate ligands is not common. Yet, different from its homologue proteins, human copper chaperone is known to bind Cu(I) using this low coordination number while binding Cu(I) only via the two conserved Cysteine residues, Cys12 and Cys15. Based on structural analysis, this work determines that the protein possesses two distinct conformations referred to as "in" and "out" due to the relative positioning of Cys12 (one of Cu(I) binding residues). The "out" conformation, with Cys12 pointing out, imposes a buried Cu(I) position, whereas the "in" conformation with Cys12 pointing inwards results in a more exposed Cu(I) thus, available for transfer. Using QM/MM methods along with thermodynamic cycles these two conformations are shown to exhibit different coordination preference, suggesting that the protein has evolved to have a unique Cu(I) protection mechanism. It is proposed that the "out" conformation with a preference to dicoordination prevents Cu(I) interaction with external ligands and/or Cu(I) release to the solvent, whereas the "in" conformation with preference to tricoordinated Cu(I), facilitates Cu(I) transfer to target proteins, where additional ligands are involved.

A single-site mutation (F429H) converts the enzyme CYP 2B4 into a heme oxygenase: a QM/MM study.

The intriguing deactivation of the cytochrome P450 (CYP) 2B4 enzyme induced by mutation of a single residue, Phe429 to His, is explored by quantum mechanical/molecular mechanical calculations of the O-OH bond activation of the (Fe(3+)OOH)(-) intermediate. It is found that the F429H mutant of CYP 2B4 undergoes homolytic instead of heterolytic O-OH bond cleavage. Thus, the mutant acquires the following characteristics of a heme oxygenase enzyme: (a) donation by His429 of an additional NH---S H-bond to the cysteine ligand combined with the presence of the substrate retards the heterolytic cleavage and gives rise to homolytic O-OH cleavage, and (b) the Thr302/water cluster orients nascent OH(•) and ensures efficient meso hydroxylation.

The nucleotide, inhibitor, and cation binding sites of P-type II ATPases.

P-type ATPases constitute a ubiquitous superfamily of cation transport enzymes, responsible for carrying out actions of paramount importance in biology such as ion transport and expulsion of toxic ions from cells. The harmonized toggling of gates in the extra- and intracellular domains explain the phenomenon of specific cation binding in selective physiological states. A quantitative understanding of the fundamental aspects of ion transport mechanism and regulation of P-type ATPases requires detailed knowledge of thermodynamical, structural, and functional properties. Computational studies have made significant contributions to our understanding of biological ion pumps. Various 3D structures of Ca(2+) -ATPase between E1 and E2 transition states have given a impetus to the theorists to work on the Na(+) K(+) - and H(+) K(+) -ATPase to address important questions about their function. The current review delineates the importance of cation, nucleotide, and inhibitor binding domains, with a focus on the therapeutic potential and biological relevance of the three P-type II ATPases. This will give an insight into the ion selectivity and their conduction across the transmembrane helices of P-type II ATPases, which may pave the way to a range of fundamental questions about the mechanism and aid in the efforts of structure- and analog-based drug design.

Comparison of computational methods to model DNA minor groove binders.

There has been a profound interest in designing small molecules that interact in sequence-selective fashion with DNA minor grooves. However, most in silico approaches have not been parametrized for DNA ligand interaction. In this regard, a systematic computational analysis of 57 available PDB structures of noncovalent DNA minor groove binders has been undertaken. The study starts with a rigorous benchmarking of GOLD, GLIDE, CDOCKER, and AUTODOCK docking protocols followed by developing QSSR models and finally molecular dynamics simulations. In GOLD and GLIDE, the orientation of the best score pose is closer to the lowest rmsd pose, and the deviation in the conformation of various poses is also smaller compared to other docking protocols. Efficient QSSR models were developed with constitutional, topological, and quantum chemical descriptors on the basis of B3LYP/6-31G* optimized geometries, and with this ΔT(m) values of 46 ligands were predicted. Molecular dynamics simulations of the 14 DNA-ligand complexes with Amber 8.0 show that the complexes are stable in aqueous conditions and do not undergo noticeable fluctuations during the 5 ns production run, with respect to their initial placement in the minor groove region.

Aromatic-Aromatic Interactions Database, A(2)ID: an analysis of aromatic π-networks in proteins.

The geometrical arrangement of the aromatic rings of phenylalanine, tyrosine, tryptophan and histidine has been analyzed at a database level using the X-ray crystal structure of proteins from PDB in order to find out the aromatic-aromatic (π-π) networks in proteins and to understand how these aromatic rings are connected with each-other in a specific π-π network. A stringent examination of the 7848 proteins indicates that close to 89% of the proteins have occurrence of at least a network of 2π or a higher π-π network. The occurrence of π-π networks in various protein superfamilies based on SCOP, CATH and EC classifiers has also been probed in the present work. In general, we find that multidomain and membrane proteins as well as lyases show a more number of these networks. Analysis of the distribution of angle between planes of two proximal aromatic rings (ϕ) distribution indicates that at a larger cutoff distance (between centroid of two aromatic rings), above 5Å, C-H⋯π interactions (T-shaped orientation) are more prevalent, while π-π interactions (stacked orientation) are more prevalent at a smaller cutoff distance. The connectivity patterns of π-π networks propose strong propensity of finding arrangement of aromatic residues as clusters rather than linear arrangement. We have also made a public domain database "Aromatic-Aromatic Interactions Database" (A(2)ID) comprising of all types of π-π networks and their connectivity pattern present in proteins. It can be accessed by url http://203.199.182.73/gnsmmg/databases/aidb/aidb.html.