Increased Binding Efficiency Research Articles

Cheminformatics and systems pharmacology approaches to unveil the potential plant bioactives to combat COVID-19.

COVID-19 was a global pandemic in the year 2020. Several treatment options failed to cure the disease. Thus, plant-based medicines are becoming a trend nowadays due to their less side effects. Bioactive chemicals from natural sources have been utilised for centuries as treatment options for a variety of ailments. To find out the potent bioactive compounds to counteract COVID-19, we use systems pharmacology and cheminformatics. They use the definitive data and predict the possible outcomes. In this study, we collected a total of 72 phytocompounds from the medicinally important plants such as Garcinia mangostana and Cinnamomum verum, of which 13 potential phytocompounds were identified to be active against the COVID-19 infection based on Swiss Target Prediction and compound target network analysis. These phytocompounds were annotated to identify the specific human receptor that targets COVID-19-specific genes such as MAPK8, MAPK14, ACE, CYP3A4, TLR4 and TYK2. Among these, compounds such as smeathxanthone A, demethylcalabaxanthone, mangostanol, trapezifolixanthone from Garcinia mangostana and camphene from C. verum were putatively target various COVID-19-related genes. Molecular docking results showed that smeathxanthone A and demethylcalabaxanthone exhibit increased binding efficiency towards the COVID-19-related receptor proteins. These compounds also showed efficient putative pharmacoactive properties than the commercial drugs ((R)-remdesivir, favipiravir and hydroxychloroquine) used to cure COVID-19. In conclusion, our study highlights the use of cheminformatics approach to unravel the potent and novel phytocompounds against COVID-19. These phytocompounds may be safer to use, more efficient and less harmful. This study highlights the value of natural products in the search for new drugs and identifies candidates with great promise.

MOLECULAR DOCKING STUDIES OF THIAZOLOPYRIMIDINE DERIVATIVES AS ANTIMICROBIAL AGENTS

Antimicrobial agents are essential drugs for the health of humans and animals as they cure infectious diseases produced by several contagious strains (bacteria, fungi, parasites, and viruses). Antibiotics efficacy is restricted by an increased number of antibiotic-resistant bacteria. Computational chemistry is crucial in the development of novel potential therapeutics. In this study, the objective was to integrate the two separately bioactive molecules, i.e., thiazoles and pyrimidines into one molecule to produce compounds with better pharmacological activity. Various thiazolo-pyrimidine derivatives were planned through a suitable synthetic scheme and were docked on DNA gyrase subunit B and dihydrofolate reductase, which are established targets for microbial infection. The results were studied and the findings were compared to two known medications as well as potential inhibitors. The result recognizes few thiazolo-pyrimidine derivatives with increased binding efficiency leading to enhanced potency.

Genetics of HLA Peptide Presentation and Impact on Outcomes in HLA-Matched Allogeneic Hematopoietic Cell Transplantation

Minor histocompatibility antigens (mHAs), recipient-derived peptide epitopes presented on the cell surface, are known to mediate graft-versus-host disease (GVHD); however, there are no current methods to associate mHA features with GVHD risk. This deficiency is due in part to the lack of technological means to accurately predict, let alone confirm, the tremendous number of potential mHAs in each individual transplant. Previous studies have shown that different HLA molecules present varying fractions of candidate peptide epitopes; however, the genetic "distance" between HLA-matched donors and recipients is relatively constrained. From these 2 observations, it is possible that the HLA type for a donor-recipient pair (DRP) would provide a surrogate measurement of the number of predicted mHAs, which could be related to GVHD risk. Because different HLA molecules present variable numbers of peptide antigens, a predicted cumulative peptide-binding efficiency can be calculated for individual DRP based on the pair's HLA type. The purpose of this study was to test whether cumulative peptide-binding efficiency is associated with the risk of acute GVHD (aGVHD) or relapse. In this retrospective Center for International Blood and Marrow Transplant Research study, a total of 3242 HLA-matched DRPs were analyzed for predicted cumulative peptide-binding efficiency using their HLA types and were divided into tertiles based on their scores. Univariable and multivariable analyses was performed to test for associations between cumulative peptide-binding efficiency for DRPs, divided into the HLA-matched related donor (MRD) and HLA-matched unrelated donor (MUD) cohorts, and the primary outcomes of aGVHD and relapse. Secondary outcomes investigated included overall survival, disease-free survival, and transplantation-related mortality. Using a computationally generated peptidome as a test dataset, the tested series of HLA class I displayed peptide-binding frequencies ranging from 0.1% to 3.8% of the full peptidome, and HLA class II molecules had peptide-binding frequencies of 12% to 77% across the HLA-DRB1 allotypes. By increasing binding efficiency tertile, the cumulative incidence of aGVHD at 6 months for MUD patients was 41%, 41%, and 45% for HLA class I (P=.336) and 44%, 41%, and 42% for HLA class II (P=.452). The cumulative incidences of relapse at 3 years for MUD transplant recipients were 36%, 38%, and 38% for HLA class I (P=.533) and 37%, 37%, and 38% for HLA class II (P=.896). The findings were similar for MRD transplant recipients. Multivariable analysis did not identify any impact of peptide-binding efficiency on aGVHD or relapse in MUD or MRD transplant recipients. Whereas GVHD is mediated by minor antigen mismatches in the context of HLA-matched allo-HCT, peptide-binding efficiency, which was used as a surrogate measurement for predicted number of binding antigens, did not provide additional clinical information for GVHD risk assessment. The negative result may be due to the limitations of this surrogate marker, or it is possible that GVHD is driven by a subset of immunogenic mHAs. Further research should be directed at direct mHA epitope and immunogenicity prediction.

Step-by-Step Assembly of 2D Confined Chiral Space Endowing Achiral Clusters with Asymmetric Catalytic Activity for Epoxidation of Allylic Alcohols.

Endowing achiral polyoxometalates (POMs) with asymmetric catalytic properties is always an intriguing but challenging topic because of their high catalytic activities yet highly symmetrical molecular structures. In this work, a novel strategy was proposed to fabricate a series of two-dimensional chiral POM catalysts. Following the steps of exfoliation, covalent modification, and reassembly, the achiral POMs were orderly confined into the chiral interstitial domains of chiral ionic liquid (CIL)-modified layered double hydroxide materials with a decreased molecular symmetry. The chirality of POM molecules was induced by the l- or d-pyrrolidine-type CILs, and their asymmetric catalytic activity was enhanced by the confinement effect. Compared with the reported chiral POM-based catalysts [e.g., 8 turnover frequency (TOF) and 79% enantiomeric excess (ee) for chiral POM-based metal-organic frameworks], the constructed chiral POM catalysts showed a significantly higher TOF and enantioselectivity (up to 240 TOF and 93% ee) for the asymmetric epoxidation of allylic alcohols. The facilitated mass transfer in the IL channels and the increased binding efficiency between the chiral catalytic sites and reactants render this strategy highly promising for constructing efficient chiral catalysts from the catalytically active while achiral building blocks.

A bioconjugated MoS2 based nanoplatform with increased binding efficiency to cancer cells

We evaluate the application of surfactant-free liquid-phase exfoliated MoS2 nanosheets as a nanoplatform for a cancer detection and treatment system equipped with an antibody-antigen based recognition element. Employing antigen-antibody binding, we increased the probability of the endocytosis of MoS2 nanosheets into CAIX expressing cells by 30%. The nanosheets are functionalized with a specific antibody M75, which forms an antigen-antibody complex with CAIX. The bioconjugation of MoS2 nanosheets involves biocompatible components with low cytotoxicity, verified in the tested cell lines by fluorescence-based cell viability assay. The cellular internalization is quantified by flow cytometry, while the internalization is confirmed by label-free confocal Raman imaging. Raman measurements show increased lysosomal activity in the proximity of the internalized nanoplatforms.

A carbohydrate binding module-5 is essential for oxidative cleavage of chitin by a multi-modular lytic polysaccharide monooxygenase from Bacillus thuringiensis serovar kurstaki.

Conversion of crystalline chitin to soluble sugar molecules, using lytic polysaccharide mono-oxygenases (LPMOs) has emerged as a new avenue for the production of biofuels. The present study describes the role of accessory domains in a multi-modular LPMO from Bacillus thuringiensis serovar kurstaki (BtLPMO10A). The full length BtLPMO10A (BtLPMO10A-FL) possesses an N-terminal LPMO of AA10 family (BtAA10) and a C-terminal CBM5 (BtCBM5) connected via two fibronectin (Fn) III domains (aligned as AA10-FnIII-FnIII-CBM5 from N- to C-terminus). To determine the role of individual domains, we generated truncation mutants of BtLPMO10A-FL. Substrate binding and kinetic studies revealed that BtCBM5 was involved in increasing binding efficiency of BtAA10 which otherwise has feeble binding towards β-chitin and could not bind to α-chitin. Furthermore, binding assays also indicated that the presence of CBM5 increases the binding efficiency of BtLPMO10A-FL under extreme pH conditions. FnIII domains neither bind nor assist BtLPMO10A-FL in chitin binding and serve as linkers in BtLPMO10A-FL. BtLPMO10A-FL and BtAA10 generated oxidized chito-oligosaccharides from the insoluble β-chitin substrate. It is concluded that BtCBM5 is responsible for increasing binding efficiency of BtLPMO10A-FL, whereas; BtAA10 domain is accountable for oxidative cleavage of recalcitrant chitin.

Interactions of HLA-DR and Topoisomerase I Epitope Modulated Genetic Risk for Systemic Sclerosis

The association of systemic sclerosis with anti-Topoisomerase 1 antibody (ATASSc) with specific alleles of human leukocyte antigen (HLA)-DR has been observed among various ethnics. The anti-Topoisomerase 1 antibody is a common autoantibody in SSc with diffuse cutaneous scleroderma, which is one of the clinical subtypes of SSc. On the other hand, an immunodominant peptide of topoisomerase 1 (Top1) self-protein (residues 349–368) was reported to have strong association with ATASSc. In this study, molecular dynamics simulation was performed on the complexes of Top1 peptide with various HLA-DR subtypes divided into ATASSc-associated alleles (HLA-DRB1*08:02, HLA-DRB1*11:01 and HLA-DRB1*11:04), suspected allele (HLA-DRB5*01:02), and non-associated allele (HLA-DRB1*01:01). The unique interaction for each system was compared to the others in terms of dynamical behaviors, binding free energies and solvation effects. Our results showed that three HLA-DR/Top1 complexes of ATASSc association mostly exhibited high protein stability and increased binding efficiency without solvent interruption, in contrast to non-association. The suspected case (HLA-DRB5*01:02) binds Top1 as strongly as the ATASSc association case, which implied a highly possible risk for ATASSc development. This finding might support ATASSc development mechanism leading to a guideline for the treatment and avoidance of pathogens like Top1 self-peptide risk for ATASSc.

Molecular Docking Studies of Potential Inhibition of the Alcohol Dehydrogenase Enzyme by Phyllanthin, Hypophyllanthin and Gallic Acid.

A review of literature shows certain phytochemicals, phyllanthin, hypophyllanthin and gallic acid have beneficial effects in experimental animals for improving liver metabolism in alcoholic liver disease. We investigated the ability of these chemicals to exhibit the inhibitory effect on the enzyme, alcohol dehydrogenase active site. The software used were CASTp, AutoDock and Molinspiration in Windows platform. We observed the phytochemicals, phyllanthin (-2.37 kcal/mol), hypophyllanthin (-3.23 kcal/mol) and gallic acid (-5.85 kcal/mol) in the order of increasing binding efficiency, which was as good as 4-methyl pyrazole (-4.18 kcal/mol).

A new aptamer immobilization strategy for protein recognition

The interest towards aptamer-based surfaces in the field of bioaffinity assays is constantly growing. A crucial aspect is related to the ability of maintaining the high specificity of these molecules once immobilized on the surface. In this article we compare the immobilization of aptamers on a silanized silicon nitride surface as well as on a soft polymeric layer. An innovative immobilization approach, based on a copolymer, N-dimethylacrylamide-N-acryloyloxysuccinimide-3-(trimethoxysilyl)propylmethacrylate (DMA-NAS-MAPS) coating, able to crosslink itself to the substrate and to bind amino-modified biomolecules is proposed. Comparing this coating with a more classical functionalization process based on silane chemistry, we propose that the better results obtained on the polymeric layer are due to an increased binding efficiency of the aptamers bound to a soft material. The high specificity of immobilized DNA-aptamers is demonstrated using two sequences specific for thrombin detection and a non-sense sequence as negative control. The coating provides higher sensitivity compared to classical self-assembled silane coatings, probably due to a better mobility of bound aptamers. The aptamer immobilization on both surfaces was characterized and optimized using atomic force microscopy, X-ray photoelectron spectroscopy, contact angle and fluorescence microscopy. The comparison between the two different functionalizations highlights the better performances of the copolymer coating in terms of protein recognition, demonstrating thrombin detection down to 0.011nM in buffer solution and 4.9nM in complete human serum. Moreover, the localized immobilization of the aptameric sequences, utilized in this work, suggests the possibility of employing this platform also for multianalyte detection.

Increasing Binding Efficiency via Reporter Shape and Flux in a Viral Nanoparticle Lateral-Flow Assay.

To identify factors controlling the performance of reporter particles in a sensitive lateral-flow assay (LFA), we investigated the effect of the flux and shape of filamentous bacteriophage (phage) on the performance of phage LFAs. Phage of three different lengths and diameters were modified with biotin and AlexaFluor 555 as binding and read-out elements, respectively. The binding efficiencies of the functionalized phage were tested in a fibrous glass LFA membrane modified with avidin. The total binding rate, quantified using real-time particle counting and particle image velocimetry, decreased monotonically with the average bulk flux of phage through the membrane. At the pore scale, more phage bound in regions with faster local flow, confirming that both average and local flux increased binding. The number of bound phage increased with the aspect ratio of the phage and scaled with the phage surface area, consistent with a binding interaction controlled by the number of recognition elements on the surface. Together, these results indicate that increasing the likelihood that recognition elements on the surface of phage encounter the fibers enhances the assay binding efficiency and suggests one origin for the improved performance of nonspherical phage reporters.

Aluminum application to restore water quality in eutrophic lakes: maximizing binding efficiency between aluminum and phosphorus

ABSTRACTHuser BJ. 2017. Aluminum application to restore water quality in eutrophic lakes: maximizing binding efficiency between aluminum and phosphorus. Lake Reserv Manage. 33:143–151.Aluminum (Al), typically added to lakes to reduce internal cycling of legacy phosphorus (P) in sediment, was added to the littoral zone of Lake Harriet (Minnesota) to inhibit the uptake of sediment P by buoyancy regulating species of plankton. Analytical results from sediment collected over an 11-year period showed that the added Al (32 g/m2) started to move outside the treatment zone almost immediately after treatment, with <5% remaining within the treatment area after 6 months. Although the original treatment design failed, the application method was, unexpectedly, a success with respect to binding efficiency between Al and P in the sediment. As the Al drifted to deeper areas of the lake, internal P release in non-treated, deeper areas of the lake declined and the binding ratio between Al and Al-bound P decreased, reaching 2.1 (molar) in profundal sediments 10 years after treatment. The increased contact with available (mobile) sediment P increased binding efficiency, resulting in a 163–581% increase of P bound per unit Al compared to previous whole-lake aluminum treatments. The binding efficiency exceeded expectations showing that, in addition to the amount of Al added, treatment location and subsequent translocation of the Al floc can substantially affect binding efficiency and treatment effectiveness.

Murine anti-vaccinia virus D8 antibodies target different epitopes and differ in their ability to block D8 binding to CS-E.

The IMV envelope protein D8 is an adhesion molecule and a major immunodominant antigen of vaccinia virus (VACV). Here we identified the optimal D8 ligand to be chondroitin sulfate E (CS-E). CS-E is characterized by a disaccharide moiety with two sulfated hydroxyl groups at positions 4′ and 6′ of GalNAc. To study the role of antibodies in preventing D8 adhesion to CS-E, we have used a panel of murine monoclonal antibodies, and tested their ability to compete with CS-E for D8 binding. Among four antibody specificity groups, MAbs of one group (group IV) fully abrogated CS-E binding, while MAbs of a second group (group III) displayed widely varying levels of CS-E blocking. Using EM, we identified the binding site for each antibody specificity group on D8. Recombinant D8 forms a hexameric arrangement, mediated by self-association of a small C-terminal domain of D8. We propose a model in which D8 oligomerization on the IMV would allow VACV to adhere to heterogeneous population of CS, including CS-C and potentially CS-A, while overall increasing binding efficiency to CS-E.

Computational Design of a PDZ Domain Peptide Inhibitor that Rescues CFTR Activity

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel mutated in patients with cystic fibrosis (CF). The most prevalent CFTR mutation, ΔF508, blocks folding in the endoplasmic reticulum. Recent work has shown that some ΔF508-CFTR channel activity can be recovered by pharmaceutical modulators (“potentiators” and “correctors”), but ΔF508-CFTR can still be rapidly degraded via a lysosomal pathway involving the CFTR-associated ligand (CAL), which binds CFTR via a PDZ interaction domain. We present a study that goes from theory, to new structure-based computational design algorithms, to computational predictions, to biochemical testing and ultimately to epithelial-cell validation of novel, effective CAL PDZ inhibitors (called “stabilizers”) that rescue ΔF508-CFTR activity. To design the “stabilizers”, we extended our structural ensemble-based computational protein redesign algorithm to encompass protein-protein and protein-peptide interactions. The computational predictions achieved high accuracy: all of the top-predicted peptide inhibitors bound well to CAL. Furthermore, when compared to state-of-the-art CAL inhibitors, our design methodology achieved higher affinity and increased binding efficiency. The designed inhibitor with the highest affinity for CAL (kCAL01) binds six-fold more tightly than the previous best hexamer (iCAL35), and 170-fold more tightly than the CFTR C-terminus. We show that kCAL01 has physiological activity and can rescue chloride efflux in CF patient-derived airway epithelial cells. Since stabilizers address a different cellular CF defect from potentiators and correctors, our inhibitors provide an additional therapeutic pathway that can be used in conjunction with current methods.

Efficient detection of RNA-protein interactions using tethered RNAs

The diverse localization of transcripts in cells suggests that there are many specific RNA–protein interactions that have yet to be identified. Progress has been limited, however, by the lack of a robust method to detect and isolate the RNA-binding proteins. Here we describe the use of an RNA aptamer, scaffolded to a tRNA, to create an affinity matrix that efficiently pulls down transcript-specific RNA-binding proteins from cell lysates. The addition of the tRNA scaffold to a Streptavidin aptamer (tRSA) increased binding efficiency by ∼10-fold. The tRSA system with an attached G-quartet sequence also could efficiently and specifically capture endogenous Fragile X Mental Retardation Protein (FMRP), which recognizes this RNA sequence. An alternative method, using biotinylated RNA, captured FMRP less efficiently than did our tRSA method. Finally we demonstrate the identification of novel RNA-binding proteins that interact with intron2 or 3′-UTR of the polarity protein Crumbs3 transcript. Proteins captured by these RNA sequences attached to the tRNA scaffold were identified by mass spectrometry. GFP-tagged versions of these proteins also showed specific interaction with either the Crb3 intron2 or 3′-UTR. Our tRSA technique should find wide application in mapping the RNA–protein interactome.

Effect of Counterion Binding Efficiency on Structure and Dynamics of Wormlike Micelles

We have studied the effect of counterion binding efficiency on the linear viscoelastic properties of wormlike micelles formed from hexadecyltrimethylammonium bromide (CTAB) in the presence of different nonpenetrating inorganic salts: potassium bromide (KBr), sodium nitrate (NaNO(3)), and sodium chlorate (NaClO(3)). We have varied the salt/surfactant ratio R at fixed surfactant concentration of 350 mM. Results are compared to data for the system cetylpyridinium chloride (CPyCl) and the penetrating counterion sodium salicylate (NaSal) (Oelschlaeger, C.; Schopferer, M.; Scheffold, F.; Willenbacher, N. Langmuir 2009, 25, 716-723). Mechanical high-frequency rheology and diffusing wave spectroscopy (DWS) based tracer microrheology are used to determine the shear moduli G' and G'' in the frequency range from 0.1 Hz up to 1 MHz (Willenbacher, N.; Oelschlaeger, C.; Schopferer, M.; Fischer, P.; Cardinaux, F.; Scheffold, F. Phys. Rev. Lett. 2007, 99, 068302, 1-4). This enables us to determine the plateau modulus G(0), which is related to the cross-link density or mesh size of the entanglement network, the bending stiffness kappa (also expressed as persistence length l(p) = kappa/k(B)T) corresponding to the semiflexible nature of the micelles, and the scission energy E(sciss), which is related to their contour length. The viscosity maximum shifts to higher R values, and the variation of viscosity with R is less pronounced as the binding strength decreases. The plateau modulus increases with R at low ionic strength and is constant around the viscosity maximum; the increase in G(0) at high R, which is presumably due to branching, is weak compared to the system with penetrating counterion. The scission energy E(sciss) approximately = 20 k(B)T is independent of counterion binding efficiency irrespective of R and is slightly higher compared to the system CPyCl/NaSal, indicating that branching may be significant already at the viscosity maximum in this latter case. The micellar flexibility increases with increasing binding efficiency of counterions according to the Hofmeister series. The persistence length values for systems CTAB/KBr, CTAB/NaNO(3), and CTAB/NaClO(3) are 40, 34, and 29 nm, respectively, independent of R, and are significantly higher than in the case of CPyCl/NaSal.

Recognition of Achiral and Chiral Ammonium Salts by Neutral Ditopic Receptors Based on Chiral Salen-UO2 Macrocycles

A mononuclear (M20) and a dinuclear (M40) uranyl chiral macrocyclic complex, incorporating both a salen unit containing two phenyl rings linked to a chiral diimine bridge and the (R)-BINOL unit, behaves as an efficient ditopic receptor for achiral and chiral quaternary ammonium salts. Binding affinities in chloroform solution have been measured for 1:1 complexes of many quaternary salts encompassing tetramethylammonium (TMA), tetraethylammonium (TEA), tetrabutylammonium (TBA), and acetylcholine (ACh), as well as trimethylanilinium (TriMAn), benzyltrimethylammonium (BnTriMA), (alpha-methylbenzyl)trimethylammonium and pyrrolidinium cations. The anion of the salt is bound by the hard Lewis acidic uranyl site, with an increasing binding efficiency on increasing the anion hardness (I(-) < Br(-) < Cl(-)), whereas CH-pi or pi-pi attractions by binapthyl moiety, or the salicylaldehyde unit, or the phenyl rings of diimine bridge ensure the recognition of the cation partner. Optimized structures of receptor-anion-cation ternary complexes obtained by MM calculations are supported by 2D-ROESY NMR measurements.

Onconase and amphinase, the antitumor ribonucleases from Rana pipiens oocytes.

Rana pipiens oocytes contain two homologues of pancreatic ribonuclease A that are cytostatic and cytotoxic to human cancer cells. Extensively studied Onconase is in advanced Phase IIIb clinical trials against malignant mesothelioma, while Amphinase is a novel enzyme in pre-clinical development. Onconase is the smallest (104 amino acid residues) member of the ribonuclease A superfamily while Amphinase (114 residues) is the largest among amphibian ribonucleases. Both enzymes share the characteristic frog ribonucleases C-terminal disulfide bond but another signature of this group, the N-terminal pyroglutamate, an integral part of Onconase active site is not conserved in Amphinase. Although Onconase and Amphinase are weak catalysts their enzymatic activities are required for cytostatic and cytotoxic activity. While it was postulated that tRNA is the primary substrate of Onconase in vivo there is also extensive indirect evidence that suggests other RNA species, in particular micro RNAs, may actually be the critical target of these ribonucleases. The cytostatic effects of Onconase and Amphinase are manifested as cell arrest in the G(1) cell cycle phase. Apoptosis then follows involving activation of endonucleases(s), caspases, serine proteases and transglutaminase. Onconase was shown to be strongly synergistic when combined with numerous other antitumor modalities. Onconase and Amphinase are highly cationic molecules and their preferential toxicity towards cancer cells (having distinctly higher negative charge compared to normal cells) may depend on increased binding efficiency to the cell surface by electrostatic interactions.

The Proteasome Inhibitor PR-171 Inhibits Cell Growth, Induces Apoptosis, and Overcomes De Novo and Acquired Drug Resistance in Human Multiple Myeloma Cells.

Proteasome inhibitors are a new class of drugs that have shown to be cytotoxic and induce apoptosis in tumor cells, particularly, multiple myeloma (MM). This led to the clinical evaluation and subsequent FDA approval of the proteasome inhibitor, bortezomib, for the treatment of refractory and relapsed multiple myeloma. However, continued occurrence of refractoriness, development of resistance, and the toxicity of bortezomib in treated patients has led to the recent development of novel agents with increased binding efficiency and specificity to the proteasome. PR-171 is a member of the epoxomicin-class of compounds that act as potent and selective irreversible inhibitors of the chymotrypsin-like protease activity of the 20S proteasome resulting in accumulation of ubiquinated proteins and apoptosis of tumor cells (Ming et al., PNAS 96:10403). In this study, we evaluated the effects of PR-171 on human MM cells' survival, proteasome activity and drug resistance. PR-171 induced growth inhibition and apoptosis in the drug sensitive human MM cell lines 8226/S, MM1, H929, and U266, with IC50 values ranging from 9 to 21nM. PR-171 retains activity on cells resistant to melphalan, mitoxantrone, and zarnestra, but has reduced activity on cells resistant to doxorubicin. Pretreatment with verapamil restored sensitivity of the doxorubicin resistant cells (8226/Dox40) to PR-171 back to levels seen in the parental, drug sensitive cell line (8226/S). Proteasome activity was inhibited by PR-171 in the drug sensitive and drug resistant MM cell lines, although much less inhibition was observed in the doxorubicin resistant cells, consistent with the reduced effects of PR-171 on cell viability. In freshly isolated MM cells from patients, PR-171 at doses of 10–250nM induced apoptosis in 9/9 samples tested from both newly diagnosed and relapsed refractory patients. To test the effects of PR-171 on Cell Adhesion Mediated Drug Resistance (CAM-DR), MM cell lines were treated with drug after adhesion to fibronectin. PR-171 at 1–100nM overcomes CAM-DR in 8226/S and H929 cell lines, but not in MM1. The ability of PR-171 to overcome CAM-DR was also seen in primary MM cells from patients in 6 out of 9 samples tested. In summary, we have shown that PR-171 directly inhibits proteasome activity, proliferation, and induces apoptosis in human MM sensitive and resistant cell lines, induces apoptosis in isolated MM cells from newly diagnosed and relapsed refractory patients, and overcomes de novo drug resistance. These studies provide the foundation for clinical investigation of PR-171 to improve the outcome of patients with MM and other hematological malignancies.

Method for Selecting and Enriching Cells Expressing Low Affinity Ligands for Cell Surface Receptors

The affinity of protein-protein interaction in biological systems ranges from extremely strong, such as that between avidin and biotin (Ka = approximately 10 15 M-1) to the very weak, such as E-selectin and E-selectin ligand-1 (Ka = approximately 7.4 × 104 M-1S-1) (1). It is known that the interactions mediated by the surface receptors of immune cells are often of low-affinity and transient in nature (2). Despite weak affinities, cell surface interactions among immune cells have been shown to play an important role in many functions such as surveillance, immunosynapse formation, and activation (2). It is therefore desirable to devise a way to select and enrich cells expressing low-affinity ligands for specific immune cell surface receptors. Here we use the well-studied interaction between CD97 and CD55 as a model system to describe a simple method for this purpose. Both CD97 and CD55 are cell surface proteins expressed predominantly on leukocytes (3–5). The binding of CD97 to CD55 has been demonstrated to be both of low affinity (86 μM) and transient (off-rate; 0.6/s) (6). We and others have previously shown that the interaction is mediated exclusively by the epidermal growth factor (EGF)-like domains of CD97 and the short consensus repeats (SCR) of CD55 in a Ca2+-dependent fashion (6,7). Furthermore, a CD97-like molecule, EMR2, has been shown not to bind CD55 even though it contains highly homologous EGF-like domains (97.5% identity), therefore providing an ideal negative control (8). Our method is based on (i) the efficient isolation and coupling of Fc-fusion proteins to protein AGconjugated paramagnetic beads (Protein A/G Dynabeads®; Dynal A.S., Oslo, Norway) (Figure 1A) and (ii) the simple capture and separation of ligand-positive cells from the Fcfusion protein-coated paramagnetic beads (Figure 1B). The coupling of the Fc-fusion proteins to the protein AG-conjugated paramagnetic beads generates a multivalent protein probe, thereby increasing binding efficiency (Figure 1, A and B). As the major CD55-binding CD97 isoform contains three EGF-domains, we fused the three EGF-domains immediately upstream of the mouse Fc region (Figure 1A). CD97(EGF-1,2,5)-mFc and EMR2(EGF-1,2,5)-mFc fusion proteins were produced as described previously in transiently transfected HEK293T cells (Figure 1C). Protein AG-conjugated paramagnetic beads were washed three times in binding buffer [0.5% bovine serum albumin (BSA) in Hank’s Balanced Salt solution (HBSS)], reconstituted in the binding buffer to the original concentration (10 mg/mL), and incubated at 4°C for 30 min with or without 2 μg mFc-fusion protein/10 μL beads/reaction. Following the removal of unbound proteins by magnetic separation, the fusion protein-bead complexes were then resuspended in 20 μL binding buffer/reaction. K562 cells (CD55+) were washed, adjusted to a cell density of 3 × 106 cells/mL, and incubated at 4°C with the protein-bead

Amino Acid 36 in the Human Immunodeficiency Virus Type 1 gp41 Ectodomain Controls Fusogenic Activity: Implications for the Molecular Mechanism of Viral Escape from a Fusion Inhibitor

We have previously described a human immunodeficiency virus type 1 (HIV-1) proviral clone, pL2, derived from defective viral particles with higher fusogenicity than the prototypic NL4-3 virus. In this study, we attempted to determine the region that confers the enhanced fusion activity by creating envelope recombinants between pL2 and pNL4-3, as well as point mutants based on pNL4-3. The results indicate that amino acid 36 of gp41 is key for the fusogenic activity and infectivity enhancement and that glycine 36 (36G) of gp41 in pL2 is conserved in nearly all HIV-1 isolates except for pNL4-3. The mutation 36G-->D in a primary-isolate-derived Env decreased syncytium-forming activity and infectivity. The assays for cell-cell fusion and viral binding suggested that the enhanced fusion mediated by the 36D-->G mutation is not due to increased binding efficiency but is directly due to actual enhancement of viral fusion activity. Interestingly, this amino acid position is exactly equivalent to that at which the mutation of HIV-1 isolates that have escaped from a fusion inhibitor, enfuvirtide (T-20), has been frequently observed. The correlation between these previous findings and our findings was suggested by structural analysis. Our finding, therefore, has implications for a molecular basis of the viral escape from this drug.