Biomedical subjects

Richard S Larson

Publications and source records attributed to Richard S Larson.

13 recordsLinked to original sources

PubMed2006

Transcriptional regulatory networks downstream of TAL1/SCL in T-cell acute lymphoblastic leukemia.

Aberrant expression of 1 or more transcription factor oncogenes is a critical component of the molecular pathogenesis of human T-cell acute lymphoblastic leukemia (T-ALL); however, oncogenic transcriptional programs downstream of T-ALL oncogenes are mostly unknown. TAL1/SCL is a basic helix-loop-helix (bHLH) transcription factor oncogene aberrantly expressed in 60% of human T-ALLs. We used chromatin immunoprecipitation (ChIP) on chip to identify 71 direct transcriptional targets of TAL1/SCL. Promoters occupied by TAL1 were also frequently bound by the class I bHLH proteins E2A and HEB, suggesting that TAL1/E2A as well as TAL1/HEB heterodimers play a role in transformation of T-cell precursors. Using RNA interference, we demonstrated that TAL1 is required for the maintenance of the leukemic phenotype in Jurkat cells and showed that TAL1 binding can be associated with either repression or activation of genes whose promoters occupied by TAL1, E2A, and HEB. In addition, oligonucleotide microarray analysis of RNA from 47 primary T-ALL samples showed specific expression signatures involving TAL1 targets in TAL1-expressing compared with -nonexpressing human T-ALLs. Our results indicate that TAL1 may act as a bifunctional transcriptional regulator (activator and repressor) at the top of a complex regulatory network that disrupts normal T-cell homeostasis and contributes to leukemogenesis.

Basic Helix-Loop-Helix Proteins↗

PubMed2006

Characterization of a radiolabeled small molecule targeting leukocyte function-associated antigen-1 expression in lymphoma and leukemia.

OBJECTIVE: Leukocyte function-associated antigen-1 (LFA-1) is constitutively expressed on leukocytes, including overexpression on lymphomas and leukemias. We have developed a derivative of BIRT 377, an allosteric inhibitor of LFA-1, which may be chemically tagged without affecting binding. In this study, we modified this derivative, (R)-1-(4-aminobutyl)-5-(4-bromobenzyl)-3-(3,5-dichlorophenyl)-5-methylimidazolidine- 2,4-dione (butylamino-NorBIRT), and demonstrated its potential as a noninvasive imaging agent. METHODS: Specific binding of fluorescein-labeled butylamino-NorBIRT to both human and murine cells was demonstrated using equilibrium binding and dissociation techniques. A radiometal, lutetium-177 (Lu-177), was incorporated into the butylamino-NorBIRT through 1,4,7,10-tetraazacyclododecane-N,N',N",N'''- tetraacetic acid (DOTA) as a chelator. RESULTS: Equilibrium-binding experiments demonstrated that fluorescein- labeled butylamino-NorBIRT specifically binds human and murine LFA-1 with affinity constants of 135 and 186 nM, respectively. Dissociation kinetic experiments demonstrated an off-rate of 0.168/second(1) on murine cells, consistent with the observed affinity constant. Lutetium-177 was used for labeling, with > or =99.99% radiochemical purity and incorporation yield. This radiolabeled derivative exhibited high stability in fetal bovine serum (FBS) at 37 degrees C over 72 hours. (177)Lu-DOTA-butylamino-NorBIRT showed a binding affinity of 235 nM to human LFA-1 for equilibrium binding and competitive binding experiments. CONCLUSIONS: The radiolabeled DOTA-butylamino-NorBIRT may have potential as a noninvasive imaging or therapeutic agent in both human and mouse models.

Animals↗

PubMed2006

Nuclear magnetic resonance-based screening methods for drug discovery.

Nuclear magnetic resonance (NMR) techniques are widely used in the drug discovery process. The primary feature exploited in these investigations is the large difference in mass between drugs and receptors (usually proteins) and the effect that this has on the rotational or translational correlation times for drugs bound to their targets. Many NMR parameters, such as the diffusion coefficient, spin diffusion, nuclear Overhauser enhancement, and transverse and longitudinal relaxation times, are strong functions of either the overall tumbling or translation of molecules in solution. This has led to the development of a wide variety of NMR techniques applicable to the elucidation of protein and nucleic acid structure in solution, the screening of drug candidates for binding to a target of choice, and the study of the conformational changes that occur in a target on drug binding. High-throughput screening by NMR methods has recently received a boost from the introduction of sophisticated computational techniques for reducing the time needed for the acquistion of the primary NMR data for multidimensional studies.

Binding Sites↗

PubMed2005

Dissociation of I domain and global conformational changes in LFA-1: refinement of small molecule-I domain structure-activity relationships.

LFA-1 (alphalbeta2) is constitutively expressed on leukocytes, but its activity is rapidly regulated. This rapid activation has been proposed to be associated with conformation changes in the inserted ("I") domain within the headpiece of LFA-1 as well as conversion of the molecules from bent to extended forms. To study these molecular changes as they relate to affinity regulation of LFA-1, we developed and synthesized a fluorescent derivative of BIRT-377 [Kelly et al. (2001) J. Immunol.] to examine changes in LFA-1 affinity in a flow cytometer with live cells. BIRT-377 binds to the ligand-binding or "I" domain of LFA-1. Structure-activity relationships studies indicated that an aminoalkyl group could be added to the central hydantoin group without significantly affecting binding. Using this modified derivative [1-(N-fluoresceinylthioureidobutyl)-[5R]-(4-bromobenzyl)-3-(3,5-dichlorophenyl)-5-methyl-imidazolidine-2,4-dione (FBABIRT)], we analyzed the affinity of FBABIRT binding to LFA-1 on live cells. The binding affinity increases, and the dissociation rate decreases with divalent cation (Mn(2+)) stimulation. We then used FBABIRT with fluorescent resonance energy transfer (FRET) to show that LFA-1 changes its height relative to the cell surface when cells were treated with dithiothreitol (DTT) but not Mn(2+). Competition assays among FBABIRT and BIRT derivatives defined structure-affinity relationships that refine the current model of BIRT-377 binding to the I domain. Our data supports the model in which BIRT-377 binds to the I domain and stabilizes the bent structure of LFA-1, while divalent cation activation results in a small conformational change in the I domain without significant extension of LFA-1. DTT, in contrast, induces a conversion to the extended form of LFA-1 in the presence of BIRT-377 on live cells. The structure-activity studies suggest that BIRT-377 is a fully optimized inhibitor.

Antibodies, Blocking↗

PubMed2005

CD4(+) CD56(+) lineage-negative malignancies are rare tumors of plasmacytoid dendritic cells.

CD4(+) CD56(+) lineage-negative malignancies are difficult to diagnose and classify. Recent studies have suggested that these malignancies may derive from plasmacytoid dendritic cells (pDC). In this report, we examine 10 cases of CD4+, CD56+ lineage-negative malignancies that presented in various tissue sites. The goal was to identify the morphologic, immunophenotypic, and genotypic findings to devise a diagnostic approach to tissue biopsies of these lesions and to confirm the proposed cell of origin. The mean age was 66 years (range, 45-80 years) with a male predominance (8 males/2 females). Frequent sites of disease included skin (60%) and peripheral blood/bone marrow (70%). Tumor cells were positive for CD45, CD43, CD4, and CD56 (9 of 10). The pDC markers, CD123 (9 of 10) and CD45RA (10 of 10), were detected by immunoperoxidase staining. Also noted was CD2 positivity (1 case), weak CD7 positivity (4 of 8 cases), weak CD33 (4 of 9 cases), TdT (2 cases), and CD68 (2 cases). All cases were otherwise negative for EBV (EBER), B-cell, T-cell, myeloid, and NK cell markers. T-cell receptor-gamma gene rearrangement was negative in all cases. Complex structural chromosomal abnormalities were seen in 3 of 5 cases, a subset of which may be recurrent in pDC malignancy. Overall prognosis was poor despite multiagent chemotherapy and/or radiation. Our study confirms that CD4+/CD56+ lineage-negative tumors are derived from pDC and have characteristic clinical, histopathologic, and immunophenotypic features. Furthermore, these rare neoplasms can be readily diagnosed using recently developed immunoperoxidase techniques.

Aged↗

PubMed2005

Peptide antagonists that inhibit Sin Nombre virus and hantaan virus entry through the beta3-integrin receptor.

Specific therapy is not available for the treatment of hantavirus cardiopulmonary syndrome caused by Sin Nombre virus (SNV). The entry of pathogenic hantaviruses into susceptible human cells is dependent upon expression of the alpha(v)beta(3) integrin, and transfection of human beta(3) integrin is sufficient to confer infectibility onto CHO (Chinese hamster ovary) cells. Furthermore, pretreatment of susceptible cells with anti-beta(3) antibodies such as c7E3 or its Fab fragment ReoPro prevents hantavirus entry. By using repeated selection of a cyclic nonamer peptide phage display library on purified alpha(v)beta(3), we identified 70 peptides that were competitively eluted with ReoPro. Each of these peptides was examined for its ability to reduce the number of foci of SNV strain SN77734 in a fluorescence-based focus reduction assay according to the method of Gavrilovskaya et al. (I. N. Gavrilovskaya, M. Shepley, R. Shaw, M. H. Ginsberg, and E. R. Mackow, Proc. Natl. Acad. Sci. USA 95:7074-7079, 1998). We found that 11 peptides reduced the number of foci to a greater extent than did 80 mug/ml ReoPro when preincubated with Vero E6 cells. In addition, 8 of the 70 peptides had sequence similarity to SNV glycoproteins. We compared all 18 peptide sequences (10 most potent, 7 peptides with sequence similarity to hantavirus glycoproteins, and 1 peptide that was in the group that displayed the greatest potency and had significant sequence similarity) for their abilities to inhibit SNV, Hantaan virus (HTNV), and Prospect Hill virus (PHV) infection. There was a marked trend for the peptides to inhibit SNV and HTNV to a greater extent than they inhibited PHV, a finding that supports the contention that SNV and HTNV use beta(3) integrins and PHV uses a different receptor, beta1 integrin. We then chemically synthesized the four peptides that showed the greatest ability to neutralize SNV. These peptides inhibited viral entry in vitro as free peptides outside of the context of a phage. Some combinations of peptides proved more inhibitory than did individual peptides. In all, we have identified novel peptides that inhibit entry by SNV and HTNV via beta(3) integrins and that can be used as lead compounds for further structural optimization and consequent enhancement of activity.

Amino Acid Sequence↗

PubMed2005

Design and structure of peptide and peptidomimetic antagonists of protein-protein interaction.

Peptides based on the amino acid sequences found at protein-protein interaction sites make excellent leads for antagonist development. A statistical picture of amino acids involved in protein-protein interactions indicates that proteins recognize and interact with one another through the restricted set of specialized interface amino acid residues, Pro, Ile, Tyr, Trp, Asp and Arg. These amino acids represent residues from each of the three classes of amino acids, hydrophobic, aromatic and charged, with one anionic and one cationic residue at neutral pH. The use of peptides as drug leads has been successfully used to search for antagonists of cell-surface receptors. Peptide, peptidomimetic, and non-peptide organic inhibitors of a class of cell surface receptors, the integrins, currently serve as therapeutic and diagnostic imaging agents. In this review, we discuss the structural features of protein-protein interactions as well as the design of peptides, peptidomimetics, and small organic molecules for the inhibition of protein-protein interactions. Information gained from studying inhibitors of integrin functions is now being applied to the design and testing of inhibitors of other protein-protein interactions. Most drug development progress in the past several decades has been made using the enzyme binding-pocket model of drug targets. Small molecules are designed to fit into the substrate-binding pockets of proteins based on a lock-and-key, induced-fit, or conformational ensemble model of the protein binding site. Traditionally, enzymes have been used as therapeutic drug targets because it was easier to develop rapid, sensitive screening assays, and to find low molecular weight inhibitors that blocked the active site. However, for proteins which interact with other proteins, rather than with small substrate molecules, the lack of binding pockets means that this approach will not generally succeed. There exist many diseases in which the inhibition of protein-protein interactions would provide therapeutic benefit, but there are no general methods available to address such problems. The focus of the first part of this review is to discuss the features of protein-protein interactions which may serve as general guidelines for the development and design of inhibitors for protein-protein interactions. In the second part we focus on the design of peptides (lead compounds) and their conversion into peptidomimetics or small organic molecules for the inhibition of protein-protein interactions. We draw examples from the important and emerging area of integrin-based cell adhesion and show how the principles of protein-protein interactions are followed in the discovery, optimization and usage of specific protein interface peptides as drug leads.

Amino Acids↗

PubMed2004

The signature molecular descriptor. 3. Inverse-quantitative structure-activity relationship of ICAM-1 inhibitory peptides.

We present a methodology for solving the inverse-quantitative structure-activity relationship (QSAR) problem using the molecular descriptor called signature. This methodology is detailed in four parts. First, we create a QSAR equation that correlates the occurrence of a signature to the activity values using a stepwise multilinear regression technique. Second, we construct constraint equations, specifically the graphicality and consistency equations, which facilitate the reconstruction of the solution compounds directly from the signatures. Third, we solve the set of constraint equations, which are both linear and Diophantine in nature. Last, we reconstruct and enumerate the solution molecules and calculate their activity values from the QSAR equation. We apply this inverse-QSAR method to a small set of LFA-1/ICAM-1 peptide inhibitors to assist in the search and design of more-potent inhibitory compounds. Many novel inhibitors were predicted, a number of which are predicted to be more potent than the strongest inhibitor in the training set. Two of the more potent inhibitors were synthesized and tested in-vivo, confirming them to be the strongest inhibiting peptides to date. Some of these compounds can be recycled to train a new QSAR and develop a more focused library of lead compounds.

Drug Design↗

PubMed2004

Relationship between molecular and cellular dissociation rates for VLA-4/VCAM-1 interaction in the absence of shear stress.

The rate of leukocyte recruitment to and detachment from the vasculature contributes to cellular tethering, rolling, firm adherence, and migration across an endothelium layer. The molecular rates depend on the type and number of bound integrin or selectin adhesion molecules, shear force acting on the bound adhesion molecules, and affinity state of integrins. Although little is known of the effect that the number of adhesion molecules has on leukocyte recruitment, it has been shown that firm adhesion for cells in suspension may be mediated by small numbers of bound adhesion molecules. We studied the disaggregation of aggregates composed of B78H1 cells transfected with human vascular cell adhesion molecule-1 (VCAM-1) and human monoblastoid U937 cells expressing Very Late Antigen-4 (VLA-4). Aggregate disaggregation rates were obtained and compared to dissociation rates for soluble rhVCAM-1 ligand and monoblastoid U937 cells. Under conditions without shear stress, it was found that average cellular disaggregation rates were a factor of 1.3 +/- 0.4 times slower than molecular dissociation rates for the 1 mM Mn(2+) and 1 mM Mn(2+) + 1 mM Ca(2+) conditions. A simple mathematical model was used to predict how much smaller the dissociation constant would be if the number of bonds holding an aggregate varied from one bond to N bonds under conditions without shear stress. The average number of adhesion bonds holding the cell aggregates together was found to be 1.5 +/- 0.7. This suggests that a few bonds were needed to form cellular aggregates and that increased aggregation was related to integrin affinity changes and not due to clustering or increased bond numbers.

Animals↗

PubMed2003

Alpha4beta1 integrin affinity changes govern cell adhesion.

Integrin alpha4beta1 is a receptor for vascular cell adhesion molecule-1 and fibronectin. It is important in lymphopoiesis, inflammatory recruitment of leukocytes, and other situations that require cell adhesion to the vascular endothelium. The avidity of the cells expressing alpha4beta1 integrin can be rapidly changed by chemokines and chemoattractants. Different mechanisms, including changes in the number of interacting molecules due to the alteration of the receptor topology or changes in the affinity of the individual bonds, have been proposed to explain the nature of these fast changes in avidity. Recently, we described a fluorescent LDV-containing small molecule, which we used to monitor the affinity changes on live cells in real time (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S. et al. (2001) J. Biol. Chem. 276, 48670-48678). Here we show that the affinity of the small molecule probe as well as the native ligand vascular cell adhesion molecule-1 varies in parallel when the integrin is modulated with divalent cations and that the affinity modulation leads to the changes in cell avidity. Using formyl peptide receptor-transfected U937 cells, we further show that the time course of avidity changes in response to the receptor activation coincides with the time course of the affinity changes. Taken together, these data are consistent with the idea that affinity regulation is a major factor that governs the avidity of cell adhesion mediated by the alpha4 integrin.

Animals↗

PubMed2003

Retinoic acid syndrome: manifestations, pathogenesis, and treatment.

All-trans retinoic acid (ATRA) is a potent differentiation agent that is effective therapy in acute promyelocytic leukaemia. Although ATRA is generally well tolerated, some patients develop retinoic acid syndrome. This syndrome is manifested by unexplained fever, weight gain, respiratory distress, interstitial pulmonary infiltrates, pleural and pericardial effusion, episodic hypotension, and acute renal failure. However, if identified early enough, effective therapy can be administered. This chapter discusses the clinical aspects and pathogenesis of retinoic acid syndrome.

Chemotaxis, Leukocyte↗

PubMed2002

Amelioration of ischemia-reperfusion injury with cyclic peptide blockade of ICAM-1.

Neutrophils are pivotal in the pathogenesis of ischemia-reperfusion (I/R) injury leading to muscle damage. Firm adhesion of neutrophils to the endothelium is initiated by an interaction between intercellular adhesion molecular-1 (ICAM-1) on the endothelium and beta(2)-integrins on neutrophils. Inhibition of ICAM-1-dependent binding using monoclonal antibodies has been shown to be efficacious in ameliorating I/R injury by preventing the influx of neutrophils into the ischemic tissue. We recently described a cyclic peptide that is a potent and selective inhibitor of ICAM-1 (IP25) in vitro. In this study, we tested the hypothesis that IP25-mediated blockade of ICAM-1 would inhibit neutrophil influx during reperfusion of ischemic tissue and consequently attenuate muscle injury in a tourniquet hindlimb murine model of I/R injury. Varying amounts of peptide drug were injected at the beginning of the reperfusion period. The neutrophil influx and size of infarction at the end of 2 h of reperfusion were compared with those in untreated control mice and contralateral nonischemic limbs. Mice receiving IP25 immediately before reperfusion showed a 56% reduction in neutrophil infiltration in the ischemic muscle, accompanied by a 40% reduction in the infarct size. No effect on I/R injury was seen if IP25 administration was delayed for 60 min after reperfusion. We conclude that IP25 effectively inhibits ICAM-1-mediated adhesion of neutrophils to the endothelium in mice leading to a protective effect and suggests that synthetic peptide antagonists have a potential role as therapeutic tools.

Amino Acid Sequence↗

PubMed2002

Regulation of human basophil adhesion to endothelium under flow conditions: Different very late antigen 4 regulation on umbilical cord blood-derived and peripheral blood basophils.

BACKGROUND: Although soluble mediators released by basophils in tissue sites contribute to the chronic injury that occurs in hypersensitivity diseases, only limited information is available about how circulating basophils are recruited to tissues. In particular, the interaction of basophils with endothelium under conditions that mimic physiologic flow has not been explored. OBJECTIVE: We sought to identify adhesion molecules regulating the attachment of human basophils to IL-4-activated human umbilical vein endothelial cells (HUVECs) under flow conditions. METHODS: A parallel-plate flow chamber and blocking mAbs were used to define the adhesion molecules involved in the interactions of peripheral blood basophils (PBBs) and cord blood-derived basophils (CBDBs) with IL-4-activated HUVECs and with Chinese hamster ovary (CHO) cell transfectants expressing specific adhesion molecules. A fluorescent ligand specific for very late antigen 4 (VLA-4) was used to directly examine the VLA-4 affinity state of basophils. RESULTS: Flowing PBBs and CBDBs attached to activated HUVECs and to CHO cells expressing P- or E-selectin. However, only CBDBs attached to vascular cell adhesion molecule 1 (VCAM-1)-transfected CHO cells under flow conditions. The attachment of CBDBs to CHO cells was blocked by mAbs directed against E-selectin, P-selectin, and VCAM-1, whereas attachment of PBBs was blocked by E-selectin and P-selectin mAbs. Activating VLA-4 with Mn(2+) on PBBs resulted in adhesion to the VCAM-1-transfected CHO cells, indicating that VLA-4 activity on PBBs can be regulated, at least in part, through affinity changes. The Mn(2+)-induced upregulation of basophil VLA-4 affinity was demonstrated directly by using a fluorescent ligand for VLA-4 and flow cytometry. CONCLUSIONS: The interaction of human CBDBs and PBBs with endothelium under flow conditions is mediated in part by both P- and E-selectin. VLA-4 additionally contributes to the adhesion of flowing CBDBs. However, the affinity of VLA-4 is too low to support the adhesion under flow conditions of unstimulated PBBs.

Animals↗