RESUMEN
Evolving antimicrobial resistance has motivated the search for novel targets and alternative therapies. Caseinolytic protease (ClpP) has emerged as an enticing new target since its function is conserved and essential for bacterial fitness, and because its inhibition or dysregulation leads to bacterial cell death. ClpP protease function controls global protein homeostasis and is, therefore, crucial for the maintenance of the bacterial proteome during growth and infection. Previously, acyldepsipeptides (ADEPs) were discovered to dysregulate ClpP, leading to bactericidal activity against both actively growing and dormant Gram-positive pathogens. Unfortunately, these compounds had very low efficacy against Gram-negative bacteria. Hence, we sought to develop non-ADEP ClpP-targeting compounds with activity against Gram-negative species and called these activators of self-compartmentalizing proteases (ACPs). These ACPs bind and dysregulate ClpP in a manner similar to ADEPs, effectively digesting bacteria from the inside out. Here, we performed further ACP derivatization and testing to improve the efficacy and breadth of coverage of selected ACPs against Gram-negative bacteria. We observed that a diverse collection of Neisseria meningitidis and Neisseria gonorrhoeae clinical isolates were exquisitely sensitive to these ACP analogues. Furthermore, based on the ACP-ClpP cocrystal structure solved here, we demonstrate that ACPs could be designed to be species specific. This validates the feasibility of drug-based targeting of ClpP in Gram-negative bacteria.
Asunto(s)
Antibacterianos , Depsipéptidos , Péptido Hidrolasas , Antibacterianos/farmacología , Bacterias , Depsipéptidos/farmacología , Bacterias GramnegativasRESUMEN
Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia coli ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation.
Asunto(s)
Endopeptidasa Clp/química , Modelos Moleculares , Electricidad Estática , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Endopeptidasa Clp/metabolismo , Activación Enzimática , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Proteínas Tirosina Fosfatasas/químicaRESUMEN
Acyldepsipeptides (ADEPs) are potential antibiotics that dysregulate the activity of the highly conserved tetradecameric bacterial ClpP protease, leading to bacterial cell death. Here, we identified ADEP analogs that are potent dysregulators of the human mitochondrial ClpP (HsClpP). These ADEPs interact tightly with HsClpP, causing the protease to non-specifically degrade model substrates. Dysregulation of HsClpP activity by ADEP was found to induce cytotoxic effects via activation of the intrinsic, caspase-dependent apoptosis. ADEP-HsClpP co-crystal structure was solved for one of the analogs revealing a highly complementary binding interface formed by two HsClpP neighboring subunits but, unexpectedly, with HsClpP in the compact conformation. Given that HsClpP is highly expressed in multiple cancers and has important roles in cell metastasis, our findings suggest a therapeutic potential for ADEPs in cancer treatment.
Asunto(s)
Antibacterianos/efectos adversos , Antibacterianos/química , Apoptosis/efectos de los fármacos , Depsipéptidos/efectos adversos , Depsipéptidos/química , Endopeptidasa Clp/metabolismo , Mitocondrias/efectos de los fármacos , Acilación , Antibacterianos/farmacología , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Línea Celular Tumoral , Depsipéptidos/farmacología , Endopeptidasa Clp/química , Células HEK293 , Humanos , Mitocondrias/enzimología , Simulación del Acoplamiento Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/enzimologíaRESUMEN
The problem of antibiotic resistance has prompted the search for new antibiotics with novel mechanisms of action. Analogues of the A54556 cyclic acyldepsipeptides (ADEPs) represent an attractive class of antimicrobial agents that act through dysregulation of caseinolytic protease (ClpP). Previous studies have shown that ADEPs are active against Gram-positive bacteria (e.g., MRSA, VRE, PRSP (penicillin-resistant Streptococcus pneumoniae)); however, there are currently few studies examining Gram-negative bacteria. In this study, the synthesis and biological evaluation of 14 novel ADEPs against a variety of pathogenic Gram-negative and Gram-positive organisms is outlined. Optimization of the macrocyclic core residues and N-acyl side chain culminated in the development of 26, which shows potent activity against the Gram-negative species Neisseria meningitidis and Neisseria gonorrheae and improved activity against the Gram-positive organisms Staphylococcus aureus and Enterococcus faecalis in comparison with known analogues. In addition, the co-crystal structure of an ADEP-ClpP complex derived from N. meningitidis was solved.
Asunto(s)
Antibacterianos/síntesis química , Antibacterianos/farmacología , Caseínas/metabolismo , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Péptido Hidrolasas/metabolismo , Relación Estructura-ActividadRESUMEN
The effect of dysprosium(III) triflate on macrolactonization reactions to form depsipeptides using MNBA (Shiina's reagent) is reported. Improved yields were obtained for the formation of 16-membered depsipeptides using lanthanide triflate additives. The use of a macrocyclization strategy permits the use of a semiautomated solid-phase synthesis approach for the rapid synthesis of analogues of the antibacterial A54556 acyldepsipeptides in only two physical operations, requiring only final product purification after cyclization.
Asunto(s)
Depsipéptidos/síntesis química , Elementos de la Serie de los Lantanoides/química , Antibacterianos , Ciclización , Depsipéptidos/química , Lactonas/síntesis química , Estructura Molecular , Técnicas de Síntesis en Fase SólidaRESUMEN
Ruthenium-catalyzed asymmetric [2 + 2] cycloadditions between chiral acyl camphorsultam-functionalized alkynes and bicyclic alkenes were examined, providing adducts with complete exo stereoselectivity in good overall yield and enantioselectivity (up to 99% and 166:1, respectively), as well as appreciable diastereoselectivity (up to 163:1). The diastereoselectivity showed dependence on the solvent and temperature, as well as on the substitution pattern of the reacting alkyne and bicyclic alkene components. In general, higher diastereoselectivities were observed for reactions conducted in ethereal solvents and at lower temperatures between N-propynoyl camphorsultams and bicyclic alkenes.
Asunto(s)
Alquenos/química , Alquinos/química , Compuestos Bicíclicos con Puentes/química , Compuestos Heterocíclicos con 3 Anillos/química , Rutenio/química , Catálisis , Reacción de Cicloadición , Estructura Molecular , EstereoisomerismoRESUMEN
The first total synthesis of all six known A54556 acyldepsipeptide (ADEP) antibiotics from Streptomyces hawaiiensis is reported. This family of compounds has a unique mechanism of antibacterial action, acting as activators of caseinolytic protease (ClpP). Assembly of the 16-membered depsipeptide core was accomplished via a pentafluorophenyl ester-based macrolactamization strategy. Late stage amine deprotection was carried out under neutral conditions by employing a mild hydrogenolysis strategy, which avoids the formation of undesired ring-opened depsipeptide side products encountered during deprotection of acid-labile protecting groups. The free amines were found to be significantly more reactive toward late stage amide bond formation as compared to the corresponding ammonium salts, giving final products in excellent yields. A thorough NMR spectroscopic analysis of these compounds was carried out to formally assign the structures and to aid with the spectroscopic assignment of ADEP analogues. The identity of two of the structures was confirmed by comparison with biologically produced samples from S. hawaiiensis. An X-ray crystallographic analysis of an ADEP analogue reveals a conformation similar to that found in cocrystal structures of ADEPs with ClpP protease. The degree of antibacterial activity of the different compounds was evaluated in vitro using MIC assays employing both Gram-positive and Gram-negative strains and a fluorescence-based biochemical assay.
Asunto(s)
Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Depsipéptidos/síntesis química , Depsipéptidos/farmacología , Streptomyces/química , Antibacterianos/química , Cristalografía por Rayos X , Depsipéptidos/química , Endopeptidasa Clp , Proteínas de Escherichia coli/agonistas , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Neisseria meningitidis/efectos de los fármacos , Resonancia Magnética Nuclear Biomolecular , Staphylococcus aureus/efectos de los fármacos , Streptococcus pneumoniae/efectos de los fármacosRESUMEN
A general method for the synthesis of amides involving the direct coupling of alkali metal carboxylate salts with amines is described. Amidation of a wide variety of carboxylate salts with either free amines or their ammonium hydrochloride salts can be achieved using HBTU as a coupling agent in combination with Hünig's base. The reaction is highly efficient and is generally complete in as little as 1-2 h, giving the products in good to excellent yields. The protocol is valuable for the coupling of carboxylates for which the corresponding carboxylic acids or acyl chlorides are unstable, less conveniently manipulated/isolated, or are not commercially available. For example, the coupling of amines and α-amino acids with lithium 5-bromo-1H-pyrrole-2-carboxylate, whose corresponding acid that is prone to decarboxylation, allowed for the synthesis of 5-bromo-1H-pyrrole-2-carboxamides, which are analogues of the pyrrole-2-aminoimidazole marine alkaloids. The protocol can be combined with other reactions in a sequenced fashion, as exemplified by the synthesis of acetylenic amides, in a one-pot procedure, via the coupling of a lithium carboxylate salt formed initially by the addition of carbon dioxide to a lithiated terminal alkyne.
Asunto(s)
Alquinos/química , Amidas/síntesis química , Aminas/química , Dióxido de Carbono/química , Ácidos Carboxílicos/química , Litio/química , Metales/química , Sales (Química)/química , Amidas/química , Catálisis , Estructura MolecularRESUMEN
ClpP is a cylindrical serine protease whose ability to degrade proteins is regulated by the unfoldase ATP-dependent chaperones. ClpP on its own can only degrade small peptides. Here, we used ClpP as a target in a high-throughput screen for compounds, which activate the protease and allow it to degrade larger proteins, hence, abolishing the specificity arising from the ATP-dependent chaperones. Our screen resulted in five distinct compounds, which we designate as Activators of Self-Compartmentalizing Proteases 1 to 5 (ACP1 to 5). The compounds are found to stabilize the ClpP double-ring structure. The ACP1 chemical structure was considered to have drug-like characteristics and was further optimized to give analogs with bactericidal activity. Hence, the ACPs represent classes of compounds that can activate ClpP and that can be developed as potential novel antibiotics.
Asunto(s)
Antibacterianos/química , Endopeptidasa Clp/química , Proteínas de Escherichia coli/química , Antibacterianos/farmacología , Sitios de Unión , Simulación por Computador , Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , Activación Enzimática/efectos de los fármacos , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Pruebas de Sensibilidad Microbiana , Chaperonas Moleculares/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The Mn(hfac)(2) complex of the paramagnetic 4-(benzoxazol-2'-yl)-1,2,3,5-dithiadiazolyl ligand is reported (hfac = 1,1,1,5,5,5-hexafluoroacetylacetonato-). The Mn(ii) and radical ligand spins are coupled antiferromagnetically (AF) in the coordination complex. Short sulfur-oxygen contacts between molecules provide an efficient pathway for AF coupling between the radical ligand of one molecule and the Mn(ii) of a neighbouring molecule, resulting in a large total spin ground state (S(T) = 4) for a pair of molecules.
RESUMEN
The crystal structure of a third polymorphic form of the known 4-(2,6-difluorophenyl)-1,2,3,5-dithiadiazolyl radical, C(7)H(3)F(2)N(2)S(2), is reported. This new polymorph represents a unique crystal-packing motif never before observed for 1,2,3,5-dithiadiazolyl (DTDA) radicals. In the two known polymorphic forms of the title compound, all of the molecules form cis-cofacial dimers, such that two molecules are pi-stacked with like atoms one on top of the other, a common arrangement for DTDA species. By contrast, the third polymorph, reported herein, contains two crystallographically unique molecules organized such that only 50% are dimerized, while the other 50% remain monomeric radicals. The dimerized molecules are arranged in the trans-antarafacial mode. This less common dimer motif for DTDA species is characterized by pi-pi interactions between the S atoms [S...S = 3.208 (1) A at 110 K], such that the two molecules of the dimer are related by a centre of inversion. The most remarkable aspect of this third polymorph is that the DTDA dimers are co-packed with monomers. The monomeric radicals are arranged in one-dimensional chains directed by close lateral intermolecular contacts between the two S atoms of one DTDA heterocycle and an N atom of a neighbouring coplanar DTDA heterocycle [S...N = 2.857 (2) and 3.147 (2) A at 110 K].
RESUMEN
The regio- and absolute stereochemistry of (7S)-N-[4-(3-thienyl)tricyclo[4.2.1.0(2,5)]non-3-en-3-ylcarbonyl]-2,10-camphorsultam tetrahydrofuran hemisolvate, C(24)H(29)NO(3)S(2).0.5C(4)H(8)O, and (7S)-N-[4-(4-tolyl)tricyclo[4.2.1.0(2,5)]non-3-en-3-ylcarbonyl]-2,10-camphorsultam, C(27)H(33)NO(3)S, have been established. One contains a half-occupancy tetrahydrofuran solvent molecule located on a twofold axis and the other contains two crystallographically unique molecules which are nearly identical. The extended structures of both complexes can be explained via weak C-H...O interactions, which link the molecules together into two-dimensional sheets in the ab plane for the thienyl complex and ultimately into a three-dimensional structure for the tolyl derivative. The stereochemistry of both structures confirms that [2+2] cycloadditions of bicyclic alkenes and alkynes catalysed by ruthenium are exclusively exo.