Flap Region Research Articles

A molecular dynamics study comparing a wild‐type with a multiple drug resistant HIV protease: Differences in flap and aspartate 25 cavity dimensions

HIV proteases can develop resistance to therapeutic drugs by mutating specific residues, but still maintain activity with their natural substrates. To gain insight into why mutations confer such resistance, long ( approximately 70 ns) Molecular Dynamics simulations in explicit solvent were performed on a multiple drug resistant (MDR) mutant (with Asn25 in the crystal structure mutated in silico back to the catalytically active Asp25) and a wild type (WT) protease. HIV proteases are homodimers, with characteristic flap tips whose conformations and dynamics are known to be important influences of ligand binding to the aspartates that form the catalytic center. The WT protease undergoes a transition between 25 and 35 ns that is absent in the MDR protease. The origin of this distinction is investigated using principal component analysis, and is related to differences in motion mainly in the flap region of each monomer. Trajectory analysis suggests that the WT transition arises from a concerted motion of the flap tip distances to their catalytic aspartate residues, and the distance between the two flap tips. These distances form a triangle that in the WT expands the active site from an initial (semi-open) form to an open form, in a correlated manner. In contrast, the MDR protease remains in a more closed configuration, with uncorrelated fluctuations in the distances defining the triangle. This contrasting behavior suggests that the MDR mutant achieves its resistance to drugs by making its active site less accessible to inhibitors. The migration of water to the active site aspartates is monitored. Water molecules move in and out of the active site and individual waters hydrogen bond to both aspartate carboxylate oxygens, with residence times in the ns time regime.

Insight into analysis of interactions of saquinavir with HIV-1 protease in comparison between the wild-type and G48V and G48V/L90M mutants based on QM and QM/MM calculations

Saquinavir (SQV) was the first HIV-1 PR inhibitor licensed for clinical use and widely used for acquired immunodeficiency syndrome (AIDS) therapy. Its effectiveness, however, has been hindered by the emergence of resistant mutations. The two most important HIV-1 PR mutants are G48V and G48V/L90M. Inhibition studies of SQV on these mutants demonstrated 13.5- and 419-fold reductions of susceptibility, respectively. In this study, an analysis of energetic binding affinity between saquinavir and the HIV-1 PR wild-type and these two mutants has been performed in detail based on density functional theory and the hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. We have found that the interaction of SQV with flap residue 48 of the mutants is significantly perturbed, as shown by the reduced stability of binding between SQV and residue 48 for the G48V and G48V/L90M mutants over the wild-type. This was associated with conformational changes of the inhibitor and the enzyme, leading to the loss of hydrogen bonding between the binding subsite P2 and the backbone carbonyl of residue 48. Moreover, the G48V/L90M mutations cause the repositioning of the residues close to residues 48 and 90, at important locations as a part of the flap and catalytic regions, respectively. The repositioning of these residues consequently perturbed the binding affinity of SQV in the pocket as indicated by the decreasing interaction energies. In addition to the loss of inhibitor/enzyme binding, it is interesting to observe that the mutation leads significantly to an increase of the stability of the enzyme.

Structural analysis of lead fullerene-based inhibitor bound to human immunodeficiency virus type 1 protease in solution from molecular dynamics simulations

Molecular dynamics (MD) simulations of the HIV-1 protease (HIVP) complexed with lead fullerene-based inhibitor (diphenyl C 60 alcohol) in the three protonated states, unprotonated (Un-), monoprotonated (Mono-), and diprotonated (Di-) states at Asp25 and Asp25′ were performed. As the X-ray structure of the investigated complex is not available, it was built up starting with the X-ray crystallographic structure of the HIVP complexed with non-peptide inhibitor (PDB code: 1AID) and that of the diphenyl C 60 alcohol optimized using the integrated ONIOM molecular orbital calculations. The inhibitor was, then, introduced into the enzyme pocket using a molecular docking method. Change of the HIVP binding cavity for all three states were evaluated in terms of distance between the two catalytic residues, Asp25 and Asp25′ as well as those between the catalytic residues and the flap regions. The torsional angles formed by the O–C–C–O of the two carboxyl groups of the catalytic dyad show the non-planar configuration with the most frequency at about −45° for the Un-, 35° and −95° for the Mono- and 60° for the Di-systems. At equilibrium, different orientations of the fullerene-based inhibitor in the three protonation states were observed. For the Di-state, the OH group of the inhibitor stably forms hydrogen bonds with the two aspartic residues. It turns to the flap region to form hydrogen bonding to the backbone N of Ile50′ for the Un-state. In contrast, the OH group turns to locate between the catalytic and the flap region for the Mono-states. Beside the molecular orientation, the rotation of the OH group of the inhibitor in the Un-state was also detected. In terms of solvation, the carboxylate oxygens of the aspartic residues in the Un- and Mono-states were solvated by one to three water molecules while the OH group in these two states was coordinated by one water molecule. This is in contrast to the Di-state in which no water molecule is available in the radius of 5–6 Å around the oxygen atoms of the carboxylate groups of enzyme and of the OH group of the inhibitor. The simulated results lead to the conclusion that the active site of the HIVP complexed with the diphenyl C 60 alcohol is the diprotonation states on Asp25 and Asp25′.

Crystal structure of an FIV/HIV chimeric protease complexed with the broad-based inhibitor, TL-3

We have obtained the 1.7 Å crystal structure of FIV protease (PR) in which 12 critical residues around the active site have been substituted with the structurally equivalent residues of HIV PR (12X FIV PR). The chimeric PR was crystallized in complex with the broad-based inhibitor TL-3, which inhibits wild type FIV and HIV PRs, as well as 12X FIV PR and several drug-resistant HIV mutants [1-4]. Biochemical analyses have demonstrated that TL-3 inhibits these PRs in the order HIV PR > 12X FIV PR > FIV PR, with Ki values of 1.5 nM, 10 nM, and 41 nM, respectively [2-4]. Comparison of the crystal structures of the TL-3 complexes of 12X FIV and wild-typeFIV PR revealed theformation of additinal van der Waals interactions between the enzyme inhibitor in the mutant PR. The 12X FIV PR retained the hydrogen bonding interactions between residues in the flap regions and active site involving the enzyme and the TL-3 inhibitor in comparison to both FIV PR and HIV PR. However, the flap regions of the 12X FIV PR more closely resemble those of HIV PR, having gained several stabilizing intra-flap interactions not present in wild type FIV PR. These findings offer a structural explanation for the observed inhibitor/substrate binding properties of the chimeric PR.

The HIV-1 central DNA flap region contains a “flapping” third strand

Due to the discontinuous nature of HIV-1 plus-strand DNA synthesis, a 99-nt plus-strand overhang termed the “central DNA flap” is present near the center of the proviral DNA prior to integration. The flap appears to have stabilizing and/or protective effects on viral DNA, which has been hypothesized to be due to a specific conformation adopted by the three-stranded region. The 5′ end of the flap sequence is very purine rich and has the potential to adopt different secondary structures (e.g., duplex, triplex or quadruplex). In the present work, circular dichroism spectroscopy and thermal unfolding techniques were used to characterize an 89-nt long DNA sequence designed to mimic the three-stranded region at the 5′ end of HIV-1 proviral DNA. The effect of addition of the HIV-1 nucleocapsid protein (NC) on the nucleic acid structure was also examined. Although, guanine-rich short oligonucleotides derived from the DNA flap demonstrated CD spectra characteristic to parallel quadruplexes, this analysis reveals that the extended 89-nt construct folds into a canonical duplex with a “flapping” third strand both in the absence and presence of NC.

β-Secretase (BACE-1) inhibitors: Accounting for 10s loop flexibility using rigid active sites

BACE-1 is a flexible enzyme with experimentally determined motion in the flap region, the catalytic aspartates, and the 10s loop. Four in-house crystallographically determined complexes of tertiary carbinamine inhibitors revealed 10s loop motion in the S 3 pocket. These X-ray structures were used to correlate K i values, which span over five orders of magnitude, with the calculated interaction energy, using the Merck Molecular Force Field for a series of 19 tertiary carbinamine inhibitors.

N-Linked Oligosaccharides ofAspergillus awamoriFeruloyl Esterase Are Important for Thermostability and Catalysis

A unique N-linked glycosylation motif (Asn(79)-Tyr-Thr) was found in the sequence of type-A feruloyl esterases from Aspergillus spp. To clarify the function of the flap, the role of N-linked oligosaccharides located in the flap region on the biochemical properties of feruloyl esterase (AwFAEA) from Aspergillus awamori expressed in Pichia pastoris was analyzed by removing the N-linked glycosylation recognition site by site-directed mutagenesis. N79 was replaced with A or Q. N-glycosylation-free N79A and N79Q mutant enzymes had lower activity than that of the glycosylated recombinant AwFAEA wild-type enzyme toward alpha-naphthylbutyrate (C4), alpha-naphthylcaprylate (C8), and phenolic acid methyl esters. Kinetic analysis of the mutant enzymes indicated that the lower catalytic efficiency was due to a combination of increased Km and decreased k(cat) for N79A, and to a considerably decreased k(cat) for N79Q. N79A and N79Q mutant enzymes also exhibited considerably reduced thermostability relative to the wild-type.

Finite element computation of turbulent flow past a multi-element airfoil

Flow past multi-element airfoil is studied via two-dimensional numerical simulations. The incompressible Reynolds averaged Navier–Stokes equations, in primitive variables, are solved using a stabilized finite element formulation. The Spalart–Allmaras and Baldwin–Lomax models are employed for turbulence closure. The implementation of the Spalart–Allmaras model is verified by computing flow over a flat plate with a specified trip location. Good agreement is seen between the results obtained with the two models for flow past a NACA 0012 airfoil at 5° angle of attack. Results for the multi-element airfoil, with the two turbulence models, are compared with experiments for various angles of attack. In general, the pressure distribution, from both the models matches quite well with the experimental results. However, at larger angles of attack, the computational results overpredict the suction peak on the slat. The velocity profiles from the Baldwin–Lomax model are, in general, more diffused compared to those from the Spalart–Allmaras model. The agreement between the computed and experimental results is not too good in the flap region for large angles of attack. Both the models are unable to predict the stall; the flow remains attached even for relatively large angles of attack. Consequently, the lift coefficient is over predicted at large α by the computations. Overall, compared to the Baldwin–Lomax model, the predictions from the Spalart–Allmaras model are closer to experimental measurements.

Role of Invariant Thr80 in Human Immunodeficiency Virus Type 1 Protease Structure, Function, and Viral Infectivity

Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. While the importance of conserved active-site residues is easily understood, the role of other invariant residues is not. This work focuses on invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. In a previous study, we postulated, on the basis of a molecular dynamics simulation of the unliganded protease, that Thr80 may play a role in the mobility of the flaps of protease. In the present study, both experimental and computational methods were used to study the role of Thr80 in HIV protease. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. Both T80V and T80N had decreased the affinity for saquinavir. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain.

Computational Simulations of HIV-1 ProteasesMulti-drug Resistance Due to Nonactive Site Mutation L90M

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the proteins that currently available anti-HIV-1 drugs target. Inhibitors of HIV-1 PR have become available, and they have lowered the rate of mortality from acquired immune deficiency syndrome (AIDS) in advanced countries. However, the rate of emergence of drug-resistant HIV-1 variants is quite high because of their short retroviral life cycle and their high mutation rate. Serious drug-resistant mutations against HIV-1 PR inhibitors (PIs) frequently appear at the active site of PR. Exceptionally, some other mutations such as L90M cause drug resistance, although these appear at nonactive sites. The mechanism of resistance due to nonactive site mutations is difficult to explain. In this study, we carried out computational simulations of L90M PR in complex with each of three kinds of inhibitors and one typical substrate, and we clarified the mechanism of resistance. The L90M mutation causes changes in interaction between the side chain atoms of the 90th residue and the main chain atoms of the 25th residue, and a slight dislocation of the 25th residue causes rotation of the side chain at the 84th residue. The rotation of the 84th residue leads to displacement of the inhibitor from the appropriate binding location, resulting in a collision with the flap or loop region. The difference in levels of resistance to the three inhibitors has been explained from energetic and structural viewpoints, which provides the suggestion for promising drugs keeping its efficacy even for the L90M mutant.

Mechanism of Drug Resistance Revealed by the Crystal Structure of the Unliganded HIV-1 Protease with F53L Mutation

Mutations in HIV-1 protease (PR) that produce resistance to antiviral PR inhibitors are a major problem in AIDS therapy. The mutation F53L arising from antiretroviral therapy was introduced into the flexible flap region of the wild-type PR to study its effect and potential role in developing drug resistance. Compared to wild-type PR, PR(F53L) showed lower (15%) catalytic efficiency, 20-fold weaker inhibition by the clinical drug indinavir, and reduced dimer stability, while the inhibition constants of two peptide analog inhibitors were slightly lower than those for PR. The crystal structure of PR(F53L) was determined in the unliganded form at 1.35 Angstrom resolution in space group P4(1)2(1)2. The tips of the flaps in PR(F53L) had a wider separation than in unliganded wild-type PR, probably due to the absence of hydrophobic interactions of the side-chains of Phe53 and Ile50'. The changes in interactions between the flaps agreed with the reduced stability of PR(F53L) relative to wild-type PR. The altered flap interactions in the unliganded form of PR(F53L) suggest a distinct mechanism for drug resistance, which has not been observed in other common drug-resistant mutants.

Dichloroacetate Reduces Tissue Necrosis in a Rat Transverse Rectus Abdominis Musculocutaneous Flap Model

Ischemia-related complications may occur during postmastectomy transverse rectus abdominis musculocutaneous (TRAM) flap reconstruction. The aim of our study was to investigate whether necrosis of susceptible flap regions could be reduced by dichloroacetate (DCA)-induced stimulation of oxidative metabolism in hypoxic tissue. The study was a randomized control trial using male Sprague-Dawley rats. A pedicled TRAM flap based upon the right inferior epigastric artery was elevated and reapproximated. Animals were randomly assigned to 1 of 5 treatment groups (n = 6). Group I received no DCA; groups II through V were administered 75 mg/kg DCA orally 24 hours preoperative; in addition, groups II through IV received 75 mg/kg/d DCA orally postoperative for 4 days; group III also received 75 mg/kg DCA (IP) intraoperatively; groups IV and V were given 15 mg/kg/d DCA orally for 6 days before the 24-hour preoperative treatment. Four days postsurgery, skin paddles were photographed and assessed for viability. Underlying TRAM muscle was biopsied for histologic analysis. Blood lactate levels were measured at pre- and postoperative time points. The mean percentages of viable skin paddle were as follows: 32.0%+/- 4.0% (group I), 68.1% +/- 6.2% (group II), 84.3% +/- 5.9% (group III), 92.8% +/- 2.0% (group IV), 82.6% +/- 5.8% (group V). Statistically significant differences were found in all experimental (DCA) groups relative to the controls (P < 0.01). Group IV (6-day DCA preconditioning, plus 24-hour preoperative and 4-day postoperative treatment) displayed the greatest improvement in flap viability, significantly better than other DCA groups (P < 0.01). Group IV also had significantly lower serum lactate levels than controls (P < 0.05). Histologic examination of muscle biopsies revealed reductions in inflammation and necrosis correlating with DCA treatment and skin paddle survival. This study indicates that DCA may provide a useful pharmacologic tool for reducing ischemia-related necrosis in TRAM flaps.

Drug Resistance of HIV-1 Protease Against JE-2147: I47V Mutation Investigated by Molecular Dynamics Simulation

Anti-retroviral therapies for acquired immunodeficiency syndrome (AIDS) patients are at risk because of drug resistance that has been identified with a number of currently marketed drugs. HIV-1 protease (HIV-pr), a well-validated AIDS therapeutic target, undergoes various mutations leading to resistance such existing drugs. However, the molecular basis of drug resistance of HIV-pr is still not fully understood. JE-2147, an experimental inhibitor of HIV-pr, shows a resistance profile different from that of known drugs. Noteworthy, it is less susceptible to several common mutations, but it is still susceptible to a few mutations, including I47V which appears to be specific for JE-2147. In this work, the molecular details of the effect of I47V mutation is investigated using molecular dynamics simulation. Four simulations of apo and complexed proteins in their wild type (WT) and mutant forms have been performed. It is found that the mobility of the side chain of mutant Val47 in chain B of HIV-pr about the inhibitor increases significantly relative to WT Ile47 in chain B. This is due to loss of optimized packing of the inhibitor to the residue 47 in chain B of the mutant when compared with WT enzyme. There also are subtle differences in motion involving residues in the flap region, which are more prominent in the apo form.

Unexpected Novel Binding Mode of Pyrrolidine‐Based Aspartyl Protease Inhibitors: Design, Synthesis and Crystal Structure in Complex with HIV Protease

At present nine FDA-approved HIV protease inhibitors have been launched to market, however rapid drug resistance arising under antiviral therapy calls upon novel concepts. Possible strategies are the development of ligands with less peptide-like character or the stabilization of a new and unexpected binding-competent conformation of the protein through a novel ligand-binding mode. Our rational design of pyrrolidinedimethylene diamines was inspired by the idea to incorporate key structural elements from classical peptidomimetics with a non-peptidic heterocyclic core comprising an endocyclic amino function to address the catalytic aspartic acid side chains of Asp 25 and 25'. The basic scaffolds were decorated by side chains already optimized for the recognition pockets of HIV protease or cathepsin D. A multistep synthesis has been established to produce the central heterocycle and to give flexible access to side chain decorations. Depending on the substitution pattern of the pyrrolidine moiety, single-digit micromolar inhibition of HIV-1 protease and cathepsin D has been achieved. Successful design is suggested in agreement with our modelling concepts. The subsequently determined crystal structure with HIV protease shows that the pyrrolidine moiety binds as expected to the pivotal position between both aspartic acid side chains. However, even though the inhibitors have been equipped symmetrically by polar acceptor groups to address the flap water molecule, it is repelled from the complex, and only one direct hydrogen bond is formed to the flap. A strong distortion of the flap region is detected, leading to a novel hydrogen bond which cross-links the flap loops. Furthermore, the inhibitor addresses only three of the four available recognition pockets. It achieves only an incomplete desolvation compared with the similarly decorated amprenavir. Taking these considerations into account it is surprising that the produced pyrrolidine derivatives achieve micromolar inhibition and it suggests extraordinary potency of the new compound class. Most likely, the protonated pyrrolidine moiety experiences strong enthalpic interactions with the enzyme through the formation of two salt bridges to the aspartic acid side chains. This might provide challenging opportunities to combat resistance of the rapidly mutating virus.

Review of monitoring free muscle flap transfers in reconstructive surgery: role of 99mTc sestamibi scintigraphy

Free tissue transfer is a method of moving any tissue from a donor area of the body to a recipient site and re-attaching the arteries and veins to the blood vessels at the recipient site by microvascular surgery. Improved microsurgical techniques have resulted in a high percentage of successful free tissue transfers. Post-operative monitoring of tissue viability can detect early problems in free tissue transfer which may allow early intervention and salvage. Although many flap monitoring methods have been described, there is still no consensus on which of these techniques will become the standard accepted method for monitoring free muscle flaps. In present study, we investigated the use of 99mTc sestamibi scintigraphy in determining free muscle flap viability and complications, and also in directing treatment. Thirteen patients were examined prospectively during the post-operative period after free tissue transfer for foot defects. The cause of the defect was diabetic foot ulcer in 10 patients, dermatofibrosarcoma in one patient, squamous cell carcinoma in one patient and gunshot wound in one patient. Foot defect covering was carried out with a free latissimus dorsi muscle flap and skin graft (n=12) and a free gracilis muscle flap (n=1). All patients were examined with a monitoring system that consisted of visual inspection, hand-held Doppler ultrasonography and scintigraphic examinations. Scintigraphic imaging of all cases was performed routinely within the first 48 h post-operatively, and also on days 10 and 91 in two patients. There were four flap failures during the study. One of these patients had viable findings upon visual inspection and no evidence of vascular compromise on Doppler at the first examination. In the other patient, visual inspection of the flap showed that it was ischaemic in one region, but there was no vascular compromise on Doppler examination. Scintigraphic images of each of these patients showed a partial hypoperfused area in the flap region. Later, these two flaps showed positive clinical indications of hypoperfusion (colour of muscle and appearance of skin graft) and Doppler abnormalities. The remaining two patients had non-viable scintigraphic images as well as positive clinical indicators of hypoperfusion and evidence of vascular compromise on Doppler. Nine patients each had a viable flap. In these patients, all three examination tools demonstrated that the flaps were totally viable and there were no vascular complications. According to the results of this study, 99mTc sestamibi scintigraphy appears to be a feasible and promising method in the evaluation of free muscle flap viability and complications. On the other hand, to demonstrate any impact on management or patient outcome, further evaluation of 99mTc sestamibi imaging, including comparative studies with different established methods in a larger patient population, is highly recommended.

Specific Sequences in the N and C Termini of Apolipoprotein A-IV Modulate Its Conformation and Lipid Association

Apolipoprotein (apoA-IV) is a 376-residue exchangeable apolipoprotein that may play a number of important roles in lipid metabolism, including chylomicron assembly, reverse cholesterol transport, and appetite regulation. In vivo, apoA-IV exists in both lipid-poor and lipid-associated forms, and the balance between these states may determine its function. We examined the structural elements that modulate apoA-IV lipid binding by producing a series of deletion mutants and determining their ability to interact with phospholipid liposomes. We found that the deletion of residues 333-343 strongly increased the lipid association rate versus native apoA-IV. Additional mutagenesis revealed that two phenylalanine residues at positions 334 and 335 mediated this lipid binding inhibitory effect. We also observed that residues 11-20 in the N terminus were required for the enhanced lipid affinity induced by deletion of the C-terminal sequence. We propose a structural model in which these sequences can modulate the conformation and lipid affinity of apoA-IV.

Cockroach allergen Bla g 2: An unusual aspartic proteinase

Enzymatic activity of mite, fungal, and bee venom allergens is thought to potentiate their allergenicity. Bla g 2 is a potent cockroach allergen, but despite sharing sequence homology with aspartic proteinases, it contains critical amino acid substitutions that impair proteolytic activity. The biologic function of Bla g 2 remains unclear. We sought to investigate the effects of specific amino acid substitutions on enzymatic activity, and the peptide-binding capability of Bla g 2. Site-directed mutagenesis was used to produce a recombinant Bla g 2 mutant (Mut) with corrected canonical triads and a flap region. Another mutant (MutF - ) was expressed after additional mutations in the flap region of Mut. Bla g 2 wild-type (Wt), Mut, and MutF - were assayed for aspartic proteinase activity, and Bla g 2 Wt was tested for pepstatin binding. Recombinant Bla g 2 Wt and Mut did not show enzymatic activity in a milk-clotting and hemoglobin assay. By using a modified hemoglobin assay, residual activity inhibited by pepstatin was detected for MutF - and Wt at 20 microg/mL, whereas pepsin was active at a 1000-fold lower concentration. Most of Bla g 2 binding to pepstatin-agarose was nonspecific. Residual proteolytic activity was found for Bla g 2 at concentrations of approximately 4 mM. This weak activity suggests that proteolysis is not the primary function of this allergen and that it is unlikely to contribute to the allergenicity of Bla g 2. Bla g 2 has a cleft that might specifically bind ligands other than pepstatin.

1131. Generation of Whole Cell Vaccines for Acute Myeloid Leukaemia by Lentivirus Mediated IL-2/CD80 Transduction

Despite recent treatment advances, the prognosis for the majority of AML patients remains bleak, particularly in the elderly, those with poor prognostic cytogenetics, and/or progression from myelodysplasia. We and others have previously shown that tumour cells engineered to express CD80 and IL-2 can stimulate T and NK cell responses against the unmodified tumour cells, and that vaccination with irradiated CD80/IL-2 expressing leukaemic cells can prolong survival in mouse models of leukaemia. In order to test the therapeutic potential of this approach, we have devised an efficient gene transfer protocol for primary human acute myeloid leukaemia blasts. Based on a previously established methodology for retroviral vectors, we show that disabled SIN-lentiviral vectors can be conjugated to streptavidin paramagnetic microparticles (PMP) after covalent linkage of biotin to 293T packaging cells. Biotin coated lentivirus is then captured and magnetically concentrated, resulting in up to 2600 fold titre enhancements after 100 fold volume reductions to achieve titres in excess of 109 cfu/ml for amphotropic or VSV-G pseudotypes. A self-inactivating lentiviral backbone, incorporating the HIV flap region and a myeloid efficient internal promoter, derived from spleen focus forming virus (SFFV), is used for the efficient co-expression of CD80 and IL-2 in primary AML blasts. These vectors express CD80 and IL-2 as a single fusion protein that incorporates the recognition sequence for a golgi/ER located ubiquitous endoprotease |[ndash]| furin between the CD80 and IL-2 moieties. This fusion protein is post-synthetically cleaved by furin to generate biologically active membrane anchored CD80 and secreted IL-2. Following a single round of infection of primary or established AML cells at an MOI of 10, between 40%-99% of AML blasts express CD80 and secrete 2-15 ng of IL-2/106/24hrs. The combined expression of IL-2 and CD80 enables the increased in vitro stimulation of allogeneic and autologous T cells obtained from AML patients, either before or following the induction of remission, with or without preceding bone marrow transplantation. The stimulated lymphocytes have a TH1 cytokine secretion profile and show evidence of specificity, as indicated by their increased proliferation in the presence of unmodified AML compared to remission bone marrow cells. The stimulated T cells also demonstrate cytotoxic activity against AML blasts. This data supports the notion that IL-2/CD80 whole cell vaccination could stimulate therapeutically useful immune responses in AML patients, particularly following chemotherapy induced remission and allogeneic bone marrow transplantation. We have recently been granted permission for the vaccination of poor prognosis AML patients with lentivirus mediated ex vivo transduced AML blasts expressing CD80 and IL-2.

Mutational analysis of a feruloyl esterase from involved in substrate discrimination and pH dependence

We cloned the feruloyl esterase A gene from Aspergillus awamori (AwfaeA) and engineered it to study substrate specificity and pH dependence of catalysis. Based on the crystal structures of two type-A feruloyl esterases (FAE-III and AnFAEA) from Aspergillus niger, residues located in the flap region of AwFAEA (Asp71, Thr72, Asp77, and Tyr80) were replaced with corresponding amino acid residues (Ile, Arg, Asn, and Phe), respectively, found in the lid of lipases from Rhizomucor miehei (RmLIP) and Humicola lanuginose (HlLIP). Furthermore, Asp77 of AwFAEA, which is conserved in Aspergillus FAEs and lipases, was replaced with a hydrophobic residue (Ile). Kinetic analysis of the mutant enzymes showed that the higher catalytic efficiency of the D77I and Y80F mutants toward alpha-naphthylbutyrate (C4) and alpha-naphthylcaprylate (C8), respectively, was due to a lower K(m) value. The higher catalytic efficiency of D77N toward C4 substrate was due to a combination of decreased K(m) and considerably increased k(cat). The D71I and Y80F mutants showed some activity toward long-acyl chain esters. On the other hand, the D77I mutant had no detectable activity toward phenolic acid methyl esters and feruloylated arabinoxylan. Moreover, the pH optima of the D77I, D77N, and Y80F mutants increased from 5.0 to 7.0-8.0, 7.0, and 6.0, respectively.

Resistant Mechanism against Nelfinavir of Human Immunodeficiency Virus Type 1 Proteases

Inhibitors against human immunodeficiency virus type-1 (HIV-1) proteases are finely effective for anti-HIV-1 treatments. However, the therapeutic efficacy is reduced by the rapid emergence of inhibitor-resistant variants of the protease. Among patients who failed in the inhibitor nelfinavir (NFV) treatment, D30N, N88D, and L90M mutations of HIV-1 protease are often observed. Despite the serious clinical problem, it is not clear how these mutations, especially nonactive site mutations N88D and L90M, affect the affinity of NFV or why they cause the resistance to NFV. In this study, we executed molecular dynamics simulations of the NFV-bound proteases in the wild-type and D30N, N88D, D30N/N88D, and L90M mutants. Our simulations clarified the conformational change at the active site of the protease and the change of the affinity with NFV for all of these mutations, even though the 88th and 90th residues are not located in the NFV-bound cavity and not able to directly interact with NFV. D30N mutation causes the disappearance of the hydrogen bond between the m-phenol group of NFV and the 30th residue. N88D mutation alters the active site conformation slightly and induces a favorable hydrophobic contact. L90M mutation dramatically changes the conformation at the flap region and leads to an unfavorable distortion of the binding pocket of the protease, although 90M is largely far apart from the flap region. Furthermore, the changes of binding energies of the mutants from the wild-type protease are shown to be correlated with the mutant resistivity previously reported by the phenotypic experiments.