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1.
Proc Natl Acad Sci U S A ; 119(50): e2214396119, 2022 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-36472957

RESUMO

Osteoporosis is a major public health problem. Currently, there are no orally available therapies that increase bone formation. Intermittent parathyroid hormone (PTH) stimulates bone formation through a signal transduction pathway that involves inhibition of salt-inducible kinase isoforms 2 and 3 (SIK2 and SIK3). Here, we further validate SIK2/SIK3 as osteoporosis drug targets by demonstrating that ubiquitous deletion of these genes in adult mice increases bone formation without extraskeletal toxicities. Previous efforts to target these kinases to stimulate bone formation have been limited by lack of pharmacologically acceptable, specific, orally available SIK2/SIK3 inhibitors. Here, we used structure-based drug design followed by iterative medicinal chemistry to identify SK-124 as a lead compound that potently inhibits SIK2 and SIK3. SK-124 inhibits SIK2 and SIK3 with single-digit nanomolar potency in vitro and in cell-based target engagement assays and shows acceptable kinome selectivity and oral bioavailability. SK-124 reduces SIK2/SIK3 substrate phosphorylation levels in human and mouse cultured bone cells and regulates gene expression patterns in a PTH-like manner. Once-daily oral SK-124 treatment for 3 wk in mice led to PTH-like effects on mineral metabolism including increased blood levels of calcium and 1,25-vitamin D and suppressed endogenous PTH levels. Furthermore, SK-124 treatment increased bone formation by osteoblasts and boosted trabecular bone mass without evidence of short-term toxicity. Taken together, these findings demonstrate PTH-like effects in bone and mineral metabolism upon in vivo treatment with orally available SIK2/SIK3 inhibitor SK-124.


Assuntos
Inibição Psicológica , Osteogênese , Humanos , Camundongos , Animais , Chumbo , Proteínas Serina-Treonina Quinases/genética
2.
J Clin Invest ; 133(6)2023 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-36719747

RESUMO

Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2/STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2/STAT5 pathway and is overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2-mutated MPNs. The nonessential roles of Plek2 under physiologic conditions make it an ideal target for MPN therapy. Here, we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening approach and cell-based assays, followed by the synthesis of analogs. Plek2-specific small-molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2-signaling complex also includes Hsp72, which protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to the Plek2 dishevelled, Egl-10 and pleckstrin (DEP) domain. The role of Plek2 in activating Akt signaling was further confirmed in vivo using a hematopoietic-specific Pten-knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to that seen with the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34-positive cells from MPN patients. Our studies reveal a Plek2/Akt complex that drives cell proliferation and can be targeted by a class of antiproliferative compounds for MPN therapy.


Assuntos
Transtornos Mieloproliferativos , Neoplasias , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Proliferação de Células , Janus Quinase 2/metabolismo
3.
Nature ; 439(7078): 879-84, 2006 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-16482161

RESUMO

Nucleic acid damage by environmental and endogenous alkylation reagents creates lesions that are both mutagenic and cytotoxic, with the latter effect accounting for their widespread use in clinical cancer chemotherapy. Escherichia coli AlkB and the homologous human proteins ABH2 and ABH3 (refs 5, 7) promiscuously repair DNA and RNA bases damaged by S(N)2 alkylation reagents, which attach hydrocarbons to endocyclic ring nitrogen atoms (N1 of adenine and guanine and N3 of thymine and cytosine). Although the role of AlkB in DNA repair has long been established based on phenotypic studies, its exact biochemical activity was only elucidated recently after sequence profile analysis revealed it to be a member of the Fe-oxoglutarate-dependent dioxygenase superfamily. These enzymes use an Fe(II) cofactor and 2-oxoglutarate co-substrate to oxidize organic substrates. AlkB hydroxylates an alkylated nucleotide base to produce an unstable product that releases an aldehyde to regenerate the unmodified base. Here we have determined crystal structures of substrate and product complexes of E. coli AlkB at resolutions from 1.8 to 2.3 A. Whereas the Fe-2-oxoglutarate dioxygenase core matches that in other superfamily members, a unique subdomain holds a methylated trinucleotide substrate into the active site through contacts to the polynucleotide backbone. Amide hydrogen exchange studies and crystallographic analyses suggest that this substrate-binding 'lid' is conformationally flexible, which may enable docking of diverse alkylated nucleotide substrates in optimal catalytic geometry. Different crystal structures show open and closed states of a tunnel putatively gating O2 diffusion into the active site. Exposing crystals of the anaerobic Michaelis complex to air yields slow but substantial oxidation of 2-oxoglutarate that is inefficiently coupled to nucleotide oxidation. These observations suggest that protein dynamics modulate redox chemistry and that a hypothesized migration of the reactive oxy-ferryl ligand on the catalytic Fe ion may be impeded when the protein is constrained in the crystal lattice.


Assuntos
Reparo do DNA , DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , RNA/metabolismo , Alquilação , Anaerobiose , Sítios de Ligação , Catálise , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Oxirredução , Maleabilidade , Conformação Proteica
4.
J Med Chem ; 50(10): 2310-8, 2007 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-17444623

RESUMO

The structures of both the native holo-HCV NS3/4A protease domain and the protease domain with a serine 139 to alanine (S139A) mutation were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contribution to the binding energy arises from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets [the numbering of the subsites is as defined in Berger, A.; Schechter, I. Philos. Trans. R. Soc. London, Ser. B 1970, 257, 249-264]. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease that is currently in clinical trials.


Assuntos
Antivirais/síntese química , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/química , Hepacivirus/enzimologia , Prolina/análogos & derivados , Inibidores de Serina Proteinase/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Antivirais/química , Sítios de Ligação , Cristalografia por Raios X , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Prolina/síntese química , Prolina/química , Conformação Proteica , Estereoisomerismo , Relação Estrutura-Atividade
5.
Curr Opin Struct Biol ; 13(1): 115-21, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12581668

RESUMO

Recent studies report the application of isothermal titration calorimetry and differential scanning calorimetry to the study of protein-ligand interactions, allosteric cooperativity and aspects of protein folding. New methods of data analysis compare alternative methods for determining ligand binding enthalpy and analyze potential sources of error in the experimental measurement of other thermodynamic parameters. Several reports examine issues concerning drug design and the correlation of thermodynamic and X-ray structural data. New instruments allow volumetric effects in biochemical systems to be evaluated calorimetrically and to substantially expand the throughput of differential scanning calorimetry measurements in drug discovery and other high-throughput applications.


Assuntos
Calorimetria/instrumentação , Calorimetria/métodos , Proteínas/química , Varredura Diferencial de Calorimetria/instrumentação , Varredura Diferencial de Calorimetria/métodos , Desenho de Fármacos , Transferência de Energia , Entropia , Ligantes , Substâncias Macromoleculares , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Estrutura Terciária de Proteína , Temperatura
6.
J Med Chem ; 48(11): 3704-13, 2005 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-15916422

RESUMO

New therapeutics to combat malaria are desperately needed. Here we show that the enzyme protein farnesyltransferase (PFT) from the malaria parasite Plasmodium falciparum (P. falciparum) is an ideal drug target. PFT inhibitors (PFTIs) are well tolerated in man, but are highly cytotoxic to P. falciparum. Because of their anticancer properties, PFTIs comprise a highly developed class of compounds. PFTIs are ideal for the rapid development of antimalarials, allowing "piggy-backing" on previously garnered information. Low nanomolar concentrations of tetrahydroquinoline (THQ)-based PFTIs inhibit P. falciparum PFT and are cytotoxic to cultured parasites. Biochemical studies suggest inhibition of parasite PFT as the mode of THQ cytotoxicity. Studies with malaria-infected mice show that THQ PFTIs dramatically reduce parasitemia and lead to parasite eradication in the majority of animals. These studies validate P. falciparum PFT as a target for the development of antimalarials and describe a potent new class of THQ PFTIs with antimalaria activity.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antimaláricos/síntese química , Plasmodium falciparum/efeitos dos fármacos , Quinolonas/síntese química , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Farnesiltranstransferase , Feminino , Humanos , Malária/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Plasmodium berghei , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Prenilação de Proteína , Quinolonas/química , Quinolonas/farmacologia , Ratos , Relação Estrutura-Atividade
7.
Protein Sci ; 11(11): 2676-87, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12381849

RESUMO

Accelerated proteolytic cleavage of proteins under controlled microwave irradiation has been achieved. Selective peptide fragmentation by endoproteases trypsin or lysine C led to smaller peptides that were analyzed by matrix-assisted laser desorption ionization (MALDI) or liquid chromatography-electrospray ionization (LC-ESI) techniques. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analyses of the peptide fragmentation of several biologically active proteins, including cytochrome c, ubiquitin, lysozyme, myoglobin, and interferon alpha-2b. Most important, using this novel approach digestion of proteins occurs in minutes, in contrast to the hours required by conventional methods.


Assuntos
Micro-Ondas , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos/métodos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Bovinos , Grupo dos Citocromos c/química , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Interferon alfa-2 , Interferon-alfa/química , Interferon-alfa/genética , Interferon-alfa/metabolismo , Peso Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes , Fatores de Tempo , Tripsina/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo
9.
Protein Eng Des Sel ; 23(5): 375-84, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20150177

RESUMO

Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646(Delta405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-A resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The DeltaF508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína/genética , Sítios de Ligação/genética , Clonagem Molecular , Cristalização , Regulador de Condutância Transmembrana em Fibrose Cística/isolamento & purificação , Primers do DNA/genética , Dimerização , Humanos , Mutação/genética
10.
J Synchrotron Radiat ; 15(Pt 3): 204-7, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18421139

RESUMO

The structures of both native and S139A holo-HCV NS3/4A protease domain were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contributions to the binding energy arise from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease, which is currently in clinical trials.


Assuntos
Hepacivirus/enzimologia , Prolina/análogos & derivados , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Modelos Moleculares , Estrutura Molecular , Prolina/química
12.
J Virol ; 78(2): 700-9, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14694101

RESUMO

Inspection of over 250 hepatitis C virus (HCV) genome sequences shows that a threonine is strictly conserved at the P1 position in the NS3-NS4A (NS3-4A) autoproteolysis junction, while a cysteine is maintained as the P1 residue in all of the putative trans cleavage sites (NS4A-4B, NS4B-5A, and NS5A-5B). To understand why T631 is conserved at the NS3-4A junction of HCV, a series of in vitro transcription-translation studies were carried out using wild-type and mutant (T631C) NS3-4A constructs bearing native, truncated, and mutant NS4A segments. The autocleavage of the wild-type junction was found to be dependent on the presence of the central cofactor domain of NS4A (residues 21 to 34). In contrast, all NS3-4A T631C mutant proteins underwent self-cleavage even in the absence of the cofactor. Subgenomic replicons derived from the Con1 strain of HCV and bearing the T631C mutation showed reduced levels of colony formation in transfection studies. Similarly, replicons derived from a second genotype 1b virus, HCV-N, demonstrated a comparable reduction in replication efficiency in transient-transfection assays. These data suggest that the threonine is conserved at position 631 because it serves two functions: (i) to slow processing at the NS3-4A cleavage site, ensuring proper intercalation of the NS4A cofactor with NS3 prior to polyprotein scission, and (ii) to prevent subsequent product inhibition by the NS3 C terminus.


Assuntos
Regulação Viral da Expressão Gênica , Hepacivirus/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Hepacivirus/genética , Humanos , Mutação , Poliproteínas/metabolismo , Biossíntese de Proteínas , Replicon , Treonina/química , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Replicação Viral
13.
Methods Enzymol ; 276: 13-22, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-27799088
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