Fluorescein analogues inhibit SecA ATPase: the first sub-micromolar inhibitor of bacterial protein translocation

SecA is a central component of the general secretion system that is essential for bacterial growth and thus an ideal target for antimicrobial agents. A series of fluorescein analogues were first screened against the ATPase activity using the truncated unregulated SecA catalytic domain. Rose bengal (RB) and erythrosin B (EB) were found to be potent inhibitors SecA with IC(50) values of 0.5 μM and 2 μM, respectively. RB and EB inhibit the catalytic SecA ATPase more effectively than the F(1) F(0) -proton ATPase. We used three assays to test the effect of these compounds on full-length SecA ATPase: in solution (intrinsic ATPase), in membrane preparation, and translocation ATPase. RB and EB show the following trend in terms of IC(50) values: translocation ATPase相似文献   

Design,Synthesis and Biological Evaluation of Rose Bengal Analogues as SecA Inhibitors

SecA, a key component of bacterial Sec‐dependent secretion pathway, is an attractive target for exploring novel antimicrobials. Rose bengal (RB), a polyhalogenated fluorescein derivative, was found from our previous study as a potent SecA inhibitor. Here we describe the synthesis and structure–activity relationships (SAR) of 23 RB analogues that were designed by systematical dissection of RB. Evaluation of these analogues allowed us to establish an initial SAR in SecA inhibition. The antimicrobial effects of these SecA inhibitors are confirmed in experiments using E. coli and B. subtilis.     

Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

With the widespread emergence of drug resistance, there is an urgent need to search for new antimicrobials, especially those against Gram‐negative bacteria. Along this line, the identification of viable targets is a critical first step. The protein translocase SecA is commonly believed to be an excellent target for the development of broad‐spectrum antimicrobials. In recent years, we developed three structural classes of SecA inhibitors that have proven to be very effective against Gram‐positive bacteria. However, we have not achieved the same level of success against Gram‐negative bacteria, despite the potent inhibition of SecA in enzyme assays by the same inhibitors. In this study, we use representative inhibitors as chemical probes to gain an understanding as to why these inhibitors were not effective against Gram‐negative bacteria. The results validate our initial postulation that the major difference in effectiveness against Gram‐positive and Gram‐negative bacteria is in the additional permeability barrier posed by the outer membrane of Gram‐negative bacteria. We also found that the expression of efflux pumps, which are responsible for multidrug resistance (MDR), have no effect on the effectiveness of these SecA inhibitors. Identification of an inhibitor‐resistant mutant and complementation tests of the plasmids containing secA in a secAts mutant showed that a single secA‐azi‐9 mutation increased the resistance, providing genetic evidence that SecA is indeed the target of these inhibitors in bacteria. Such results strongly suggest SecA as an excellent target for developing effective antimicrobials against Gram‐negative bacteria with the intrinsic ability to overcome MDR. A key future research direction should be the optimization of membrane permeability.     

Time‐Dependent Diaryl Ether Inhibitors of InhA: Structure–Activity Relationship Studies of Enzyme Inhibition,Antibacterial Activity,and in vivo Efficacy

The diaryl ethers are a novel class of antituberculosis drug candidates that inhibit InhA, the enoyl‐ACP reductase involved in the fatty acid biosynthesis (FASII) pathway, and have antibacterial activity against both drug‐sensitive and drug‐resistant strains of Mycobacterium tuberculosis. In the present work, we demonstrate that two time‐dependent B‐ring modified diaryl ether InhA inhibitors have antibacterial activity in a mouse model of TB infection when delivered by intraperitoneal injection. We propose that the efficacy of these compounds is related to their residence time on the enzyme, and to identify structural features that modulate drug–target residence time in this system, we have explored the inhibition of InhA by a series of B‐ring modified analogues. Seven ortho‐substituted compounds were found to be time‐dependent inhibitors of InhA, where the slow step leading to the final enzyme–inhibitor complex (EI*) is thought to correlate with closure and ordering of the InhA substrate binding loop. A detailed mechanistic understanding of the molecular basis for residence time in this system will facilitate the development of InhA inhibitors with improved in vivo activity.     

N‐Benzyl‐4‐((heteroaryl)methyl)benzamides: A New Class of Direct NADH‐Dependent 2‐trans Enoyl–Acyl Carrier Protein Reductase (InhA) Inhibitors with Antitubercular Activity

Isoniazid (INH) remains one of the cornerstones of antitubercular chemotherapy for drug‐sensitive strains of M. tuberculosis bacteria. However, the increasing prevalence of multidrug‐resistant (MDR) and extensively drug‐resistant (XDR) strains containing mutations in the KatG enzyme, which is responsible for the activation of INH into its antitubercular form, have rendered this drug of little or no use in many cases of drug‐resistant tuberculosis. Presented herein is a novel family of antitubercular direct NADH‐dependent 2‐trans enoyl–acyl carrier protein reductase (InhA) inhibitors based on an N‐benzyl‐4‐((heteroaryl)methyl)benzamide template; unlike INH, these do not require prior activation by KatG. Given their direct InhA target engagement, these compounds should be able to circumvent KatG‐related resistance in the clinic. The lead molecules were shown to be potent inhibitors of InhA and showed activity against M. tuberculosis bacteria. This new family of inhibitors was found to be chemically tractable, as exemplified by the facile synthesis of analogues and the establishment of structure–activity relationships. Furthermore, a co‐crystal structure of the initial hit with the enzyme is disclosed, providing valuable information toward the design of new InhA inhibitors for the treatment of MDR/XDR tuberculosis.     

Design and Structural Analysis of Aromatic Inhibitors of Type II Dehydroquinase from Mycobacterium tuberculosis

3‐Dehydroquinase, the third enzyme in the shikimate pathway, is a potential target for drugs against tuberculosis. Whilst a number of potent inhibitors of the Mycobacterium tuberculosis enzyme based on a 3‐dehydroquinate core have been identified, they generally show little or no in vivo activity, and were synthetically complex to prepare. This report describes studies to develop tractable and drug‐like aromatic analogues of the most potent inhibitors. A range of carbon–carbon linked biaryl analogues were prepared to investigate the effect of hydrogen bond acceptor and donor patterns on inhibition. These exhibited inhibitory activity in the high‐micromolar range. The addition of flexible linkers in the compounds led to the identification of more potent 3‐nitrobenzylgallate‐ and 5‐aminoisophthalate‐based analogues.     

Design,Synthesis, and SAR of Naphthyl‐Substituted Diarylpyrimidines as Non‐Nucleoside Inhibitors of HIV‐1 Reverse Transcriptase

A series of 38 2‐naphthyl‐substituted diarylpyrimidine (DAPY) analogues, characterized by various substitution patterns on the pyrimidine and naphthalene rings, was synthesized in a straightforward fashion by means of parallel synthesis and evaluated as inhibitors of the HIV‐1 wild‐type and double mutant (K103N+Y181C) strains. Most of the compounds displayed strong activity against wild‐type HIV‐1. The most active compound, with a cyano group at position C6 on the naphthalene ring, exhibited activity against wild‐type HIV‐1 with an EC₅₀ value of 0.002 μM and against the double mutant strain with an EC₅₀ value of 0.24 μM ; the selectivity index (SI) against wild‐type is >180 000, the highest SI value among DAPY analogues. The structure–activity relationship (SAR) of the newly synthesized DAPYs is presented herein.     

Chemical Interrogation of the Malaria Kinome

Malaria, an infectious disease caused by eukaryotic parasites of the genus Plasmodium, afflicts hundreds of millions of people every year. Both the parasite and its host utilize protein kinases to regulate essential cellular processes. Bioinformatic analyses of parasite genomes predict at least 65 protein kinases, but their biological functions and therapeutic potential are largely unknown. We profiled 1358 small‐molecule kinase inhibitors to evaluate the role of both the human and the malaria kinomes in Plasmodium infection of liver cells, the parasites' obligatory but transient developmental stage that precedes the symptomatic blood stage. The screen identified several small molecules that inhibit parasite load in liver cells, some with nanomolar efficacy, and each compound was subsequently assessed for activity against blood‐stage malaria. Most of the screening hits inhibited both liver‐ and blood‐stage malaria parasites, which have dissimilar gene expression profiles and infect different host cells. Evaluation of existing kinase activity profiling data for the library members suggests that several kinases are essential to malaria parasites, including cyclin‐dependent kinases (CDKs), glycogen synthase kinases, and phosphoinositide‐3‐kinases. CDK inhibitors were found to bind to Plasmodium protein kinase 5, but it is likely that these compounds target multiple parasite kinases. The dual‐stage inhibition of the identified kinase inhibitors makes them useful chemical probes and promising starting points for antimalarial development.     

Cyclobutanone Analogues of β‐Lactam Antibiotics: β‐Lactamase Inhibitors with Untapped Potential?

β‐Lactam antibiotics have been used for many years to treat bacterial infections. However the effective treatment of an increasing range of microbial infections is threatened by bacterial resistance to β‐lactams: the prolonged, widespread (and at times reckless) use of these drugs has spawned widespread resistance, which renders them ineffective against many bacterial strains. The cyclobutanone ring system is isosteric with β‐lactam: in cyclobutanone analogues, the eponymous cyclic amide is replaced with an all‐carbon ring, the amide N is substituted by a tertiary C?H α to a ketone. Cyclobutanone analogues of various β‐lactam antibiotics have been investigated over the last 35 years, initially as prospective antibiotics in their own right and inhibitors of the β‐lactamase enzymes that impart resistance to β‐lactams. More recently they have been tested as inhibitors of other serine proteases and as mechanistic probes of β‐lactam biosynthesis. Cyclobutanone analogues of the penam ring system are the first reversible inhibitors with moderate activity against all classes of β‐lactamase; other compounds from this family inhibit Streptomyces R61 dd ‐carboxypeptidase/transpeptidase, human neutrophil elastase and porcine pancreatic elastase. But has their potential as enzyme inhibitors been fully exploited? Challenges in synthesising diversely functionalised cyclobutanone derivatives mean that only a limited number have been made (with limited structural diversity) and evaluated. This review surveys the different synthetic approaches that have been taken to these compounds, the investigations made to evaluate their biological activity and prospects for future developments in this area.     

Discovery of 3‐Hydroxy‐3‐phenacyloxindole Analogues of Isatin as Potential Monoamine Oxidase Inhibitors

A series of 3‐hydroxy‐3‐phenacyloxindole analogues of isatin were designed, synthesized, and evaluated in vitro for their inhibitory activity toward monoamine oxidase (MAO) A and B. Most of the synthesized compounds proved to be potent and selective inhibitors of MAO‐A rather than MAO‐B. 1‐Benzyl‐3‐hydroxy‐3‐(4′‐hydroxyphenacyl)oxindole (compound 18 ) showed the highest MAO‐A inhibitory activity (IC₅₀: 0.009±0.001 μm , K_i: 3.69±0.003 nm ) and good selectivity (selectivity index: 60.44). Kinetic studies revealed that compounds 18 and 16 (1‐benzyl‐3‐hydroxy‐3‐(4′‐bromophenacyl)oxindole) exhibit competitive inhibition against MAO‐A and MAO‐B, respectively. Structure–activity relationship studies suggested that the 3‐hydroxy group is an essential feature for these analogues to exhibit potent MAO‐A inhibitory activity. Computational studies revealed the possible molecular interactions between the inhibitors and MAO isozymes. The computational data obtained are congruent with experimental results. Further studies on the lead inhibitors, including co‐crystallization of inhibitor–MAO complexes and in vivo evaluations, are essential for their development as potential therapeutic agents for the treatment of MAO‐associated neurological disorders.     

Benzyl and naphthalene methylphosphonic acid inhibitors of autotaxin with anti-invasive and anti-metastatic activity

Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) by lysophospholipase D activity, which leads to generation of the growth‐factor‐like lipid mediator lysophosphatidic acid (LPA). ATX is highly upregulated in metastatic and chemotherapy‐resistant carcinomas and represents a potential target to mediate cancer invasion and metastasis. Herein we report the synthesis and pharmacological characterization of ATX inhibitors based on the 4‐tetradecanoylaminobenzylphosphonic acid scaffold, which was previously found to lack sufficient stability in cellular systems. The new 4‐substituted benzylphosphonic acid and 6‐substituted naphthalen‐2‐ylmethylphosphonic acid analogues block ATX activity with K_i values in the low micromolar to nanomolar range against FS3, LPC, and nucleotide substrates through a mixed‐mode inhibition mechanism. None of the compounds tested inhibit the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor (LPAR) subtypes. Analogues 22 and 30 b , the two most potent ATX inhibitors, inhibit the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers in vitro in a dose‐dependent manner. The average terminal half‐life for compound 22 is 10±5.4 h and it causes a long‐lasting decrease in plasma LPA levels. Compounds 22 and 30 b significantly decrease lung metastasis of B16‐F10 syngeneic mouse melanoma in a post‐inoculation treatment paradigm. The 4‐substituted benzylphosphonic acids and 6‐substituted naphthalen‐2‐ylmethylphosphonic acids described herein represent new lead compounds that effectively inhibit the ATX–LPA–LPAR axis both in vitro and in vivo.     

Biological Evaluation of Potent Triclosan‐Derived Inhibitors of the Enoyl–Acyl Carrier Protein Reductase InhA in Drug‐Sensitive and Drug‐Resistant Strains of Mycobacterium tuberculosis

New triclosan (TRC) analogues were evaluated for their activity against the enoyl–acyl carrier protein reductase InhA in Mycobacterium tuberculosis (Mtb). TRC is a well‐known inhibitor of InhA, and specific modifications to its positions 5 and 4′ afforded 27 derivatives; of these compounds, seven derivatives showed improved potency over that of TRC. These analogues were active against both drug‐susceptible and drug‐resistant Mtb strains. The most active compound in this series, 4‐(n‐butyl)‐1,2,3‐triazolyl TRC derivative 3 , had an MIC value of 0.6 μg mL^?1 (1.5 μM ) against wild‐type Mtb. At a concentration equal to its MIC, this compound inhibited purified InhA by 98 %, and showed an IC₅₀ value of 90 nM . Compound 3 and the 5‐methylisoxazole‐modified TRC 14 were able to inhibit the biosynthesis of mycolic acids. Furthermore, mc²4914, an Mtb strain overexpressing inhA, was found to be less susceptible to compounds 3 and 14 , supporting the notion that InhA is the likely molecular target of the TRC derivatives presented herein.     

Optimization of Marine Triterpene Sipholenols as Inhibitors of Breast Cancer Migration and Invasion

Sipholenol A, a sipholane triterpene isolated from the Red Sea sponge Callyspongia siphonella, has the ability to reverse multidrug resistance in cancer cells that overexpress P‐glycoprotein (P‐gp). Here, the antimigratory activity of sipholenol A and analogues are reported against the highly metastatic human breast cancer cell line MDA‐MB‐231 in a wound‐healing assay. Sipholenol A and sipholenone A were semisynthetically optimized using ligand‐based strategies to generate structurally diverse analogues in an attempt to maximize their antimigratory activity. A total of 22 semisynthetic ester, ether, oxime, and carbamate analogues were generated and identified by extensive one‐ and two‐dimensional NMR spectroscopy and high‐resolution mass spectrometry analyses. Sipholenol A 4β‐4‐chlorobenzoate and 19,20‐anhydrosipholenol A 4β‐4‐chlorobenzoate esters were the most potent of all tested analogues in the wound‐healing assay, with IC₅₀ values of 5.3 and 5.9 μM , respectively. Generally, ester derivatives showed better antimigratory activities than the carbamate analogues. A KINOMEscan of 19,20‐anhydrosipholenol A 4β‐benzoate ester against 451 human protein kinases identified protein tyrosine kinase 6 (PTK6) as a potential target. In breast tumor cells, PTK6 promotes growth factor signaling and migration, and as such the semisynthetic sipholanes were evaluated for their ability to inhibit PTK6 phosphorylation in vitro. The two analogues with the highest antimigratory activities, sipholenol A 4β‐4‐chlorobenzoate and 19,20‐anhydrosipholenol A 4β‐4‐chlorobenzoate esters, also exhibited the most potent inhibition of PTK6 phosphorylation inhibition. None of the compounds exhibited cytotoxicity in a normal epithelial breast cell line. These derivatives were evaluated in an in vitro invasion assay, where sipholenol A succinate potently inhibited MDA‐MB‐231 cell invasion at 10 μM . These results highlight sipholane triterpenoids as novel antimigratory marine natural products with potential for further development as agents for the control of metastatic breast malignancies.     

Gold(I) and Gold(III) N-Heterocyclic Carbene Complexes as Antibacterial Agents and Inhibitors of Bacterial Thioredoxin Reductase

A series of (NHC)Au(I)Cl monocarbene complexes and their gold(III) analogues (NHC)Au(III)Cl₃ were prepared and investigated as antibacterial agents and inhibitors of bacterial TrxR. The complexes showed stronger antibacterial effects against the Gram-positive MRSA and E. faecium strains than against several Gram-negative bacteria. All complexes were efficient inhibitors of bacterial thioredoxin reductase, indicating that inhibition of this enzyme might be involved in their mechanism of action. The efficacy of gold(I) and gold(III) analogues was comparable in most of the assays. The cytotoxicity of the gold NHC compounds against cancer and human cells was overall weaker than the activity against the Gram-positive bacteria, suggesting that their optimization as antibacterials warrants further investigation.     

Discovery of Pyridone‐Based Histone Deacetylase Inhibitors: Approaches for Metabolic Stability

Histone deacetylases (HDACs) are important enzymes in epigenetic regulation and are therapeutic targets for cancer. Most zinc‐dependent HDACs induce proliferation, dedifferentiation, and anti‐apoptotic effects in cancer cells. We designed and synthesized a new series of pyridone‐based HDAC inhibitors that have a pyridone ring in the core structure and a conjugated system with an olefin connecting the hydroxamic acid moiety. Consequently, most of the selected pyridone‐based HDAC inhibitors showed similar or higher inhibition profiles in addition to remarkable metabolic stability against hydrolysis relative to the corresponding lactam‐based HDAC inhibitors. Furthermore, the selectivity of the novel pyridine‐based compounds was evaluated across all of the HDAC isoforms. One of these compounds, (E)‐N‐hydroxy‐3‐{1‐[3‐(naphthalen‐2‐yl)propyl]‐2‐oxo‐1,2‐dihydropyridin‐3‐yl}acrylamide, exhibited the highest level of HDAC inhibition (IC₅₀=0.07 μM ), highly selective inhibition of class I HDAC1 and class II HDAC6 enzymes, metabolic stability in mouse liver microsomal studies, and effective growth inhibition of various cancer cell lines. Docking studies indicated that a long alkyl linker and bulky hydrophobic cap groups affect in vitro activities. Overall, the findings reported herein regarding pyridone‐based HDAC inhibitors can be used to guide future research efforts to develop new and effective anticancer therapeutics.     

Novel 2-substituted quinolin-4-yl-benzenesulfonate derivatives: synthesis, antiproliferative activity, and inhibition of cellular tubulin polymerization

A series of 2‐substituted quinolin‐4‐yl‐benzenesulfonate derivatives were synthesized for the purpose of evaluating antiproliferative activity. Structure–activity relationships of the newly synthesized compounds against human lymphoblastic leukemia and various solid tumor cell growths in culture are discussed. Of these derivatives, 2‐phenyl‐6‐pyrrolidinyl‐4‐quinoline sulfonate analogues 10 f , 10 g , and 10 k , and 4′‐nitrophenyl sulfonate 10 m exhibit superior cytotoxicity over other sulfonates. The antiproliferative activities of these compounds correlate well with their abilities to induce mitotic arrest and apoptosis. Mechanistic studies indicate that they target the vinblastine binding site of tubulin and inhibit cellular tubulin polymerization. Hence, these compounds induce the formation of aberrant mitotic spindles and mitotic arrest, resulting in intensive apoptosis. The tested compounds were shown to be poor substrates for membrane multidrug resistance transporters. The present studies suggest that these newly synthesized compounds are promising tubulin polymerization inhibitors and are worthy of further investigation as antitumor agents.     

Development of Novel Selective Peptidomimetics Containing a Boronic Acid Moiety,Targeting the 20S Proteasome as Anticancer Agents

This paper describes the design, synthesis, and biological evaluation of peptidomimetic boronates as inhibitors of the 20S proteasome, a validated target in the treatment of multiple myeloma. The synthesized compounds showed a good inhibitory profile against the ChT‐L activity of 20S proteasome. Compounds bearing a β‐alanine residue at the P2 position were the most active, that is, 3‐ethylphenylamino and 4‐methoxyphenylamino (R)‐1‐{3‐[4‐(substituted)‐2‐oxopyridin‐1(2H)‐yl]propanamido}‐3‐methylbutylboronic acids ( 3 c and 3 d , respectively), and these derivatives showed inhibition constants (K_i) of 17 and 20 nM , respectively. In addition, they co‐inhibited post glutamyl peptide hydrolase activity ( 3 c , K_i=2.57 μM ; 3 d , K_i=3.81 μM ). No inhibition was recorded against the bovine pancreatic α‐chymotrypsin, which thus confirms the selectivity towards the target enzyme. Docking studies of 3 c and related inhibitors into the yeast proteasome revealed the structural basis for specificity. The evaluation of growth inhibitory effects against 60 human tumor cell lines was performed at the US National Cancer Institute. Among the selected compounds, 3 c showed 50 % growth inhibition (GI₅₀) values at the sub‐micromolar level on all cell lines.