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1.
Nature ; 466(7309): 935-40, 2010 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-20686482

RESUMO

Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , DNA Girase/química , Quinolinas/química , Quinolinas/farmacologia , Staphylococcus aureus/enzimologia , Inibidores da Topoisomerase II , Antibacterianos/metabolismo , Apoenzimas/química , Apoenzimas/metabolismo , Arginina/metabolismo , Ácido Aspártico/metabolismo , Sítios de Ligação , Domínio Catalítico , Ciprofloxacina/química , Ciprofloxacina/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Clivagem do DNA , DNA Girase/metabolismo , DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , Desenho de Fármacos , Resistência a Medicamentos , Escherichia coli/enzimologia , Manganês/metabolismo , Modelos Moleculares , Conformação Proteica , Quinolinas/metabolismo , Quinolonas/química , Quinolonas/metabolismo , Relação Estrutura-Atividade
2.
Biochem J ; 456(2): 263-73, 2013 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24015710

RESUMO

DNA gyrase, a type II topoisomerase, regulates DNA topology by creating a double-stranded break in one DNA duplex and transporting another DNA duplex [T-DNA (transported DNA)] through this break. The ATPase domains dimerize, in the presence of ATP, to trap the T-DNA segment. Hydrolysis of only one of the two ATPs, and release of the resulting Pi, is rate-limiting in DNA strand passage. A long unresolved puzzle is how the non-hydrolysable ATP analogue AMP-PNP (adenosine 5'-[ß,γ-imido]triphosphate) can catalyse one round of DNA strand passage without Pi release. In the present paper we discuss two crystal structures of the Mycobacterium tuberculosis DNA gyrase ATPase domain: one complexed with AMP-PCP (adenosine 5'-[ß,γ-methylene]triphosphate) was unexpectedly monomeric, the other, an AMP-PNP complex, crystallized as a dimer. In the AMP-PNP structure, the unprotonated nitrogen (P-N=P imino) accepts hydrogen bonds from a well-ordered 'ATP lid', which is known to be required for dimerization. The equivalent CH2 group, in AMP-PCP, cannot accept hydrogen bonds, leaving the 'ATP lid' region disordered. Further analysis suggested that AMP-PNP can be converted from the imino (P-N=P) form into the imido form (P-NH-P) during the catalytic cycle. A main-chain NH is proposed to move to either protonate AMP-P-N=P to AMP-P-NH-P, or to protonate ATP to initiate ATP hydrolysis. This suggests a novel dissociative mechanism for ATP hydrolysis that could be applicable not only to GHKL phosphotransferases, but also to unrelated ATPases and GTPases such as Ras. On the basis of the domain orientation in our AMP-PCP structure we propose a mechanochemical scheme to explain how ATP hydrolysis is coupled to domain motion.


Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/análogos & derivados , Proteínas de Bactérias/química , DNA Girase/química , Mycobacterium tuberculosis/enzimologia , Trifosfato de Adenosina/química , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , DNA Topoisomerases Tipo II/química , Ligação de Hidrogênio , Hidrólise , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína
3.
Bioorg Med Chem Lett ; 23(19): 5437-41, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23968823

RESUMO

During the course of our research to find novel mode of action antibacterials, we discovered a series of hydroxyl tricyclic compounds that showed good potency against Gram-positive and Gram-negative pathogens. These compounds inhibit bacterial type IIA topoisomerases. Herein we will discuss structure-activity relationships in this series and report advanced studies on compound 1 (GSK966587) which demonstrates good PK and in vivo efficacy properties. X-ray crystallographic studies were used to provide insight into the structural basis for the difference in antibacterial potency between enantiomers.


Assuntos
Bactérias/enzimologia , Naftiridinas/química , Naftiridinas/farmacologia , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Animais , Cristalografia por Raios X , Cães , Ativação Enzimática/efeitos dos fármacos , Haplorrinos , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ratos
4.
Front Immunol ; 13: 968206, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36148231

RESUMO

Myasthenia Gravis (MG) is mediated by autoantibodies against acetylcholine receptors that cause loss of the receptors in the neuromuscular junction. Eculizumab, a C5-inhibitor, is the only approved treatment for MG that mechanistically addresses complement-mediated loss of nicotinic acetylcholine receptors. It is an expensive drug and was approved despite missing the primary efficacy endpoint in the Phase 3 REGAIN study. There are two observations to highlight. Firstly, further C5 inhibitors are in clinical development, but other terminal pathway proteins, such as C7, have been relatively understudied as therapeutic targets, despite the potential for lower and less frequent dosing. Secondly, given the known heterogenous mechanisms of action of autoantibodies in MG, effective patient stratification in the REGAIN trial may have provided more favorable efficacy readouts. We investigated C7 as a target and assessed the in vitro function, binding epitopes and mechanism of action of three mAbs against C7. We found the mAbs were human, cynomolgus monkey and/or rat cross-reactive and each had a distinct, novel mechanism of C7 inhibition. TPP1820 was effective in preventing experimental MG in rats in both prophylactic and therapeutic dosing regimens. To enable identification of MG patients that are likely to respond to C7 inhibition, we developed a patient stratification assay and showed in a small cohort of MG patients (n=19) that 63% had significant complement activation and C7-dependent loss of AChRs in this in vitro set up. This study provides validation of C7 as a target for treatment of MG and provides a means of identifying patients likely to respond to anti-C7 therapy based on complement-activating properties of patient autoantibodies.


Assuntos
Antineoplásicos Imunológicos , Miastenia Gravis Autoimune Experimental , Receptores Nicotínicos , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Autoanticorpos/metabolismo , Proteínas do Sistema Complemento/metabolismo , Epitopos , Humanos , Macaca fascicularis , Nicotina , Ratos , Receptores Colinérgicos
5.
J Med Chem ; 59(6): 2452-67, 2016 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-26938474

RESUMO

Inhibitors of mitochondrial branched chain aminotransferase (BCATm), identified using fragment screening, are described. This was carried out using a combination of STD-NMR, thermal melt (Tm), and biochemical assays to identify compounds that bound to BCATm, which were subsequently progressed to X-ray crystallography, where a number of exemplars showed significant diversity in their binding modes. The hits identified were supplemented by searching and screening of additional analogues, which enabled the gathering of further X-ray data where the original hits had not produced liganded structures. The fragment hits were optimized using structure-based design, with some transfer of information between series, which enabled the identification of ligand efficient lead molecules with micromolar levels of inhibition, cellular activity, and good solubility.


Assuntos
Mitocôndrias/enzimologia , Transaminases/antagonistas & inibidores , Adipócitos/efeitos dos fármacos , Adipócitos/enzimologia , Cristalografia por Raios X , Ensaios de Triagem em Larga Escala , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Relação Estrutura-Atividade
6.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 10): 1242-6, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26457513

RESUMO

Fluoroquinolone drugs such as moxifloxacin kill bacteria by stabilizing the normally transient double-stranded DNA breaks created by bacterial type IIA topoisomerases. Previous crystal structures of Staphylococcus aureus DNA gyrase with asymmetric DNAs have had static disorder (with the DNA duplex observed in two orientations related by the pseudo-twofold axis of the complex). Here, 20-base-pair DNA homoduplexes were used to obtain crystals of covalent DNA-cleavage complexes of S. aureus DNA gyrase. Crystals with QPT-1, moxifloxacin or etoposide diffracted to between 2.45 and 3.15 Šresolution. A G/T mismatch introduced at the ends of the DNA duplexes facilitated the crystallization of slightly asymmetric complexes of the inherently flexible DNA-cleavage complexes.


Assuntos
Clivagem do DNA , DNA Girase/química , Etoposídeo/química , Fluoroquinolonas/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos de Espiro/química , Staphylococcus aureus/enzimologia , Sequência de Bases , Cristalização , Cristalografia por Raios X , Dados de Sequência Molecular , Moxifloxacina
7.
Nat Commun ; 6: 10048, 2015 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-26640131

RESUMO

New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a 'pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested.


Assuntos
Antibacterianos/química , Antineoplásicos/química , DNA Girase/química , Etoposídeo/química , Fluoroquinolonas/química , Staphylococcus aureus/enzimologia , Inibidores da Topoisomerase II/química , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , DNA Girase/genética , DNA Girase/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana , Etoposídeo/farmacologia , Fluoroquinolonas/farmacologia , Modelos Moleculares , Estrutura Molecular , Moxifloxacina , Staphylococcus aureus/química , Staphylococcus aureus/efeitos dos fármacos , Inibidores da Topoisomerase II/farmacologia
8.
J Med Chem ; 58(18): 7140-63, 2015 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-26090771

RESUMO

The hybridization of hits, identified by complementary fragment and high throughput screens, enabled the discovery of the first series of potent inhibitors of mitochondrial branched-chain aminotransferase (BCATm) based on a 2-benzylamino-pyrazolo[1,5-a]pyrimidinone-3-carbonitrile template. Structure-guided growth enabled rapid optimization of potency with maintenance of ligand efficiency, while the focus on physicochemical properties delivered compounds with excellent pharmacokinetic exposure that enabled a proof of concept experiment in mice. Oral administration of 2-((4-chloro-2,6-difluorobenzyl)amino)-7-oxo-5-propyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile 61 significantly raised the circulating levels of the branched-chain amino acids leucine, isoleucine, and valine in this acute study.


Assuntos
Proteínas Mitocondriais/antagonistas & inibidores , Pirazóis/química , Pirimidinonas/química , Transaminases/antagonistas & inibidores , Adipócitos/efeitos dos fármacos , Adipócitos/enzimologia , Animais , Cristalografia por Raios X , Humanos , Isoleucina/sangue , Leucina/sangue , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Moleculares , Pirazóis/síntese química , Pirazóis/farmacologia , Pirimidinonas/síntese química , Pirimidinonas/farmacologia , Relação Estrutura-Atividade , Transaminases/química , Valina/sangue
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