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1.
ACS Pharmacol Transl Sci ; 5(5): 344-361, 2022 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-35592439

RESUMO

During the development of a melanocortin (MC) peptide drug to treat the condition of cachexia (a hypermetabolic state producing lean body mass wasting), we were confronted with the need for peptide transport across the blood-brain barrier (BBB): the MC-4 receptors (MC4Rs) for metabolic rate control are located in the hypothalamus, i.e., behind the BBB. Using the term "peptides with BBB transport", we screened the medical literature like a peptide library. This revealed numerous "hits"-peptides with BBB transport and/or oral activity. We noted several features common to most peptides in this class, including a dipeptide sequence of nonpolar residues, primary structure cyclization (whole or partial), and a Pro-aromatic motif usually within the cyclized region. Based on this, we designed an MC4R antagonist peptide, TCMCB07, that successfully treated many forms of cachexia. As part of our pharmacokinetic characterization of TCMCB07, we discovered that hepatobiliary extraction from blood accounted for a majority of the circulating peptide's excretion. Further screening of the literature revealed that TCMCB07 is a member of a long-forgotten peptide class, showing active transport by a multi-specific bile salt carrier. Bile salt transport peptides have predictable pharmacokinetics, including BBB transport, but rapid hepatic clearance inhibited their development as drugs. TCMCB07 shares the general characteristics of the bile salt peptide class but with a much longer half-life of hours, not minutes. A change in its C-terminal amino acid sequence slows hepatic clearance. This modification is transferable to other peptides in this class, suggesting a platform approach for producing drug-like peptides.

2.
J Magn Reson ; 201(2): 218-21, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19819173

RESUMO

The envelope glycoproteins S1 and S2 of severe acute respiratory syndrome coronavirus (SARS-CoV) mediate viral entry by conformational change from a prefusion state to a postfusion state that enables fusion of the viral and target membranes. In this work we present the characterization of the dynamic properties of the SARS-CoV S2-HR2 domain (residues 1141-1193 of S) in the prefusion and newly discovered transition states by NMR (15)N relaxation studies. The dynamic properties of the different states, which are stabilized under different experimental conditions, extend the current model of viral membrane fusion and give insight into the design of structure-based antagonists of SARS-CoV in particular, as well as other enveloped viruses such as HIV.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/ultraestrutura , Cinética , Transição de Fase , Conformação Proteica , Estrutura Terciária de Proteína
3.
Biochemistry ; 47(26): 6802-8, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18540634

RESUMO

The envelope glycoproteins of the class I family, which include human immunodeficiency virus (HIV), influenza, and severe acute respiratory syndrome coronavirus (SARS-CoV), mediate viral entry by first binding to their cellular receptors and subsequently inducing fusion of the viral and cellular membranes. In the case of SARS-CoV, heptad repeat domains of the envelope glycoprotein, termed S2-HR1 and S2-HR2, are thought to undergo structural changes from a prefusion state, in which S2-HR1 and S2-HR2 do not interact, to a postfusion state in which S2-HR1 and S2-HR2 associate to form a six-helix bundle. In the present work, the structural and dynamic properties of S2-HR2 have been characterized. Evidence is presented for an equilibrium between a structured trimer thought to represent a prefusion state and an ensemble of unstructured monomers thought to represent a novel transition state. A model for viral entry is presented in which S2-HR2 is in a dynamic equilibrium between an ensemble of unstructured monomers in the transition state and a structured trimer in the prefusion state. Conversion from the prefusion state to the postfusion state requires passage through the transition state, a state that may give insight into the design of structure-based antagonists of SARS-CoV in particular, as well as other enveloped viruses in general.


Assuntos
Glicoproteínas/química , Glicoproteínas/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Internalização do Vírus , Sequência de Aminoácidos , Dicroísmo Circular , Glicoproteínas/genética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Quaternária de Proteína , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Proteínas Virais/genética
4.
Protein Sci ; 16(11): 2519-30, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17905835

RESUMO

We previously determined the solution structures of the first 156 residues of human erythroid alpha-spectrin (SpalphaI-1-156, or simply Spalpha). Spalpha consists of the tetramerization site of alpha-spectrin and associates with a model beta-spectrin protein (Spbeta) with an affinity similar to that of native alpha- and beta-spectrin. Upon alphabeta-complex formation, our previous results indicate that there is an increase in helicity in the complex, suggesting conformational change in either Spalpha or Spbeta or in both. We have now used isothermal titration calorimetry, circular dichroism, static and dynamic light scattering, and solution NMR methods to investigate properties of the complex as well as the conformation of Spalpha in the complex. The results reveal a highly asymmetric complex, with a Perrin shape parameter of 1.23, which could correspond to a prolate ellipsoid with a major axis of about five and a minor axis of about one. We identified 12 residues, five prior to and seven following the partial domain helix in Spalpha that moved freely relative to the structural domain in the absence of Spbeta but when in the complex moved with a mobility similar to that of the structural domain. Thus, it appears that the association with Spbeta induced an unstructured-to-helical conformational transition in these residues to produce a rigid and asymmetric complex. Our findings may provide insight toward understanding different association affinities of alphabeta-spectrin at the tetramerization site for erythroid and non-erythroid spectrin and a possible mechanism to understand some of the clinical mutations, such as L49F of alpha-spectrin, which occur outside the functional partial domain region.


Assuntos
Eritrócitos/metabolismo , Espectrina/química , Calorimetria/métodos , Cromatografia/métodos , Dicroísmo Circular , Humanos , Espectroscopia de Ressonância Magnética/métodos , Modelos Estatísticos , Peso Molecular , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Espalhamento de Radiação
5.
Protein Sci ; 16(3): 539-42, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17322536

RESUMO

The coxsackievirus and adenovirus receptor (CAR) mediates entry of coxsackievirus and adenovirus. CAR possesses an extracellular region that is comprised of 2 immunoglobulin domains termed CAR-D1 and CAR-D2. In the present work, the solution structure of CAR-D2, consisting of residues 142-235 of human CAR, has been determined by NMR spectroscopy. CAR-D2 is shown to be a beta-sandwich motif comprised of two beta-sheets, which are stabilized by two disulfide bonds. The first beta-sheet is comprised of beta-strands A, B, and E, and the second beta-sheet is comprised of beta-strands C, F, and G. A relatively hydrophobic helix is found between beta-strands C and E, which replaces beta-strand D of the classical c-type immunoglobulin fold.


Assuntos
Adenoviridae , Enterovirus , Imunoglobulinas/química , Receptores Virais/química , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Soluções
6.
J Biol Chem ; 281(17): 11965-71, 2006 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-16507566

RESUMO

The envelope glycoprotein, termed the spike protein, of severe acute respiratory syndrome coronavirus (SARS-CoV) is known to mediate viral entry. Similar to other class 1 viral fusion proteins, the heptad repeat regions of SARS-CoV spike are thought to undergo conformational changes from a prefusion form to a subsequent post-fusion form that enables fusion of the viral and host membranes. Recently, the structure of a post-fusion form of SARS-CoV spike, which consists of isolated domains of heptad repeats 1 and 2 (HR1 and HR2), has been determined by x-ray crystallography. To date there is no structural information for the prefusion conformations of SARS-CoV HR1 and HR2. In this work we present the NMR structure of the HR2 domain (residues 1141-1193) from SARS-CoV (termed S2-HR2) in the presence of the co-solvent trifluoroethanol. We find that in the absence of HR1, S2-HR2 forms a coiled coil symmetric trimer with a complex molecular mass of 18 kDa. The S2-HR2 structure, which is the first example of the prefusion form of coronavirus envelope, supports the current model of viral membrane fusion and gives insight into the design of structure-based antagonists of SARS.


Assuntos
Glicoproteínas de Membrana/síntese química , Modelos Moleculares , Sequências Repetitivas de Aminoácidos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Proteínas do Envelope Viral/química , Proteínas Virais de Fusão/síntese química , Cristalografia por Raios X , Hemaglutininas Virais , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Conformação Proteica , Sequências Repetitivas de Aminoácidos/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Solubilidade , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo
7.
Protein Pept Lett ; 12(6): 537-9, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16101391

RESUMO

The Coxsackievirus and Adenovirus receptor (CAR) mediates entry of coxsackievirus and adenovirus. CAR possesses an extracellular region that is comprised of 2 Ig domains termed CAR-D1 and CAR-D2. The 1H, 13C and 15N resonances of CAR-D2 have been assigned and the secondary structure has been deduced.


Assuntos
Receptores Virais/química , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Humanos , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
8.
Biochemistry ; 43(7): 1847-53, 2004 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-14967025

RESUMO

The coxsackievirus and adenovirus receptor (CAR) mediates entry of coxsackievirus B (CVB) and adenovirus (Ad). The normal cellular function of CAR, which is expressed in a wide variety of tissue types, is thought to involve homophilic cell adhesion in the developing brain. The extracellular domain of CAR consists of two immunoglobulin (Ig) domains termed CAR-D1 and CAR-D2. CAR-D1 is shown by sedimentation velocity to be monomeric at pH 3.0. The solution structure and the dynamic properties of monomeric CAR-D1 have been determined by NMR spectroscopy at pH 3.0. The determinants of the CAR-D1 monomer-dimer equilibrium, as well as the binding site of CVB and Ad on CAR, are discussed in light of the monomer structure.


Assuntos
Adenoviridae/química , Enterovirus Humano B/química , Fragmentos de Peptídeos/química , Receptores Virais/química , Adenoviridae/metabolismo , Cristalografia por Raios X , Dimerização , Enterovirus Humano B/metabolismo , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Virais/metabolismo , Soluções , Termodinâmica , Ultracentrifugação
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