ABSTRACT
The flavin mononucleotide (FMN) riboswitch is an emerging target for the development of novel RNA-targeting antibiotics. We previously discovered an FMN derivative, 5FDQD, that protects mice against diarrhea-causing Clostridium difficile bacteria. Here, we present the structure-based drug design strategy that led to the discovery of this fluoro-phenyl derivative with antibacterial properties. This approach involved the following stages: (1) structural analysis of all available free and bound FMN riboswitch structures; (2) design, synthesis, and purification of derivatives; (3) in vitro testing for productive binding using two chemical probing methods; (4) in vitro transcription termination assays; and (5) resolution of the crystal structures of the FMN riboswitch in complex with the most mature candidates. In the process, we delineated principles for productive binding to this riboswitch, thereby demonstrating the effectiveness of a coordinated structure-guided approach to designing drugs against RNA.
Subject(s)
Anti-Bacterial Agents/pharmacology , Flavin Mononucleotide/pharmacology , Quinoxalines/pharmacology , RNA, Bacterial/antagonists & inhibitors , Riboswitch , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Bacteria/drug effects , Base Sequence , Binding Sites , Drug Design , Flavin Mononucleotide/chemical synthesis , Flavin Mononucleotide/chemistry , Ligands , Molecular Structure , Quinoxalines/chemical synthesis , Quinoxalines/chemistry , RNA, Bacterial/genetics , Structure-Activity RelationshipABSTRACT
AFN-1252 is a novel inhibitor of FabI, an essential enzyme in fatty acid biosynthesis in Staphylococcus spp. AFN-1252 exhibits typical MIC(90) values of ≤0·015 µg/ml against diverse clinical isolates of S. aureus, oral absorption, long elimination half-live and efficacy in animal models. We now report high binding (â¼95%) to serum proteins of mouse, rat, dog and humans, associated with an eight-fold increase in minimal inhibitory concentration (MIC) and which may be responsible for the long elimination half-lives on pharmacokinetic studies. Unlike daptomycin, AFN-1252 activity is not reduced in the presence of lung surfactant. AFN-1252 exhibits a short post-antibiotic effect of 1·1 hours against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) following a 4-hour exposure period. The AFN-1252 unique spectrum of activity is not compromised by interactions with major antibiotic classes, but demonstrates synergy with low concentrations of gentamicin against MSSA and MRSA. These studies support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.
Subject(s)
Anti-Bacterial Agents/blood , Anti-Bacterial Agents/pharmacology , Benzofurans/blood , Benzofurans/pharmacology , Blood Proteins/metabolism , Pyrones/blood , Pyrones/pharmacology , Animals , Daptomycin/blood , Daptomycin/pharmacology , Dogs , Gentamicins/blood , Gentamicins/pharmacology , Half-Life , Humans , Male , Methicillin/blood , Methicillin/pharmacology , Methicillin Resistance/drug effects , Methicillin-Resistant Staphylococcus aureus/drug effects , Mice , Microbial Sensitivity Tests , Pulmonary Surfactants/metabolism , Rats , Staphylococcal Infections/metabolism , Staphylococcus aureus/drug effectsABSTRACT
The mechanism of action of AFN-1252, a selective inhibitor of Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI), which is involved in fatty acid biosynthesis, was confirmed by using biochemistry, macromolecular synthesis, genetics, and cocrystallization of an AFN-1252-FabI complex. AFN-1252 demonstrated a low propensity for spontaneous resistance development and a time-dependent reduction of the viability of both methicillin-susceptible and methicillin-resistant S. aureus, achieving a ≥2-log(10) reduction in S. aureus counts over 24 h, and was extremely potent against clinical isolates of S. aureus (MIC(90), 0.015 µg/ml) and coagulase-negative staphylococci (MIC(90), 0.12 µg/ml), regardless of their drug resistance, hospital- or community-associated origin, or other clinical subgroup. AFN-1252 was orally available in mouse pharmacokinetic studies, and a single oral dose of 1 mg/kg AFN-1252 was efficacious in a mouse model of septicemia, providing 100% protection from an otherwise lethal peritoneal infection of S. aureus Smith. A median effective dose of 0.15 mg/kg indicated that AFN-1252 was 12 to 24 times more potent than linezolid in the model. These studies, demonstrating a selective mode of action, potent in vitro activity, and in vivo efficacy, support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Bacterial Proteins/antagonists & inhibitors , Benzofurans/therapeutic use , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/antagonists & inhibitors , Pyrones/therapeutic use , Sepsis/drug therapy , Staphylococcal Infections/drug therapy , Staphylococcus aureus/drug effects , Administration, Oral , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Benzofurans/pharmacology , Community-Acquired Infections/drug therapy , Community-Acquired Infections/microbiology , Cross Infection/drug therapy , Cross Infection/microbiology , Drug Administration Schedule , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/metabolism , Female , Humans , Kinetics , Mice , Microbial Sensitivity Tests , Pyrones/pharmacology , Sepsis/microbiology , Sepsis/mortality , Staphylococcal Infections/microbiology , Staphylococcus aureus/enzymology , Staphylococcus aureus/growth & development , Survival RateABSTRACT
MLN4924 is a selective inhibitor of the NEDD8-activating enzyme (NAE) and has advanced into clinical trials for the treatment of both solid and hematological malignancies. In contrast, the structurally similar compound 1 (developed by Millennium: The Takeda Oncology Company) is a pan inhibitor of the E1 enzymes NAE, ubiquitin activating enzyme (UAE), and SUMO-activating enzyme (SAE) and is currently viewed as unsuitable for clinical use given its broad spectrum of E1 inhibition. Here, we sought to understand the determinants of NAE selectivity. A series of compound 1 analogues were synthesized through iterative functionalization of the purine C6 position and evaluated for NAE specificity. Optimal NAE specificity was achieved through substitution with primary N-alkyl groups, while bulky or secondary N-alkyl substituents were poorly tolerated. When assessed in vitro, inhibitors reduced the growth and viability of malignant K562 leukemia cells. Through this study, we have successfully identified a series of sub-10 nM NAE-specific inhibitors and thereby highlighted the functionalities that promote NAE selectivity.
ABSTRACT
An optimized synthetic route to prepare ring-locked retinoid 1a has been developed. We fully describe a purification protocol that provides isomerically pure 1a in support of on-going proof of concept studies for the development of therapeutic agents to treat human ADRP. Additionally, we have found that isomerically pure 1a can be stored in amber vials under argon at -20°C for use over time (up to six months) without degradation. Thus, enabling 1a to be an accessible and valuable biological tool.
ABSTRACT
In the search for new antibacterial agents, the enzyme FabI has been identified as an attractive target. Employing a structure guided approach, the previously reported ene-amide series of FabI inhibitors were expanded to include 2,3,4,5-tetrahydro-1H-pyrido[2,3-b and e][1,4]diazepines. These novel series incorporate additional H-bonding functions and can be more water soluble than their naphthyridinone progenitors; diazepine 16c is shown to be efficacious in a mouse infection model.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Azepines/chemistry , Azepines/pharmacology , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/antagonists & inhibitors , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/metabolism , Animals , Anti-Bacterial Agents/therapeutic use , Azepines/therapeutic use , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli Infections/drug therapy , Mice , Models, Molecular , Protein Binding , Staphylococcal Infections/drug therapy , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymologyABSTRACT
Spiropiperidine naphthyridinone inhibitors of Staphylococcus aureus and Escherichia coli FabI have been prepared. Compounds 14a and 14c were identified as having sub-nanomolar E. coli FabI activity and are among the most potent FabI inhibitors yet described. The structural model of 14a bound to E. coli FabI is shown.
Subject(s)
Anti-Bacterial Agents/pharmacology , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/antagonists & inhibitors , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/metabolism , Escherichia coli/enzymology , Naphthyridines/pharmacology , Piperidines/pharmacology , Spiro Compounds/pharmacology , Staphylococcus aureus/enzymology , Anti-Bacterial Agents/chemistry , Catalytic Domain , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/chemistry , Models, Molecular , Naphthyridines/chemistry , Piperidines/chemistry , Protein Binding , Spiro Compounds/chemistry , Structure-Activity RelationshipABSTRACT
The high-throughput manual solid-phase parallel synthesis of libraries comprising thousands of discrete samples using pellicular supports (i.e. SynPhase crowns and lanterns) and a suite of novel tools and techniques is described. Key aspects of this approach include the combination of a split-split-split synthesis strategy with spatial encoding to differentiate thousands of crowns, the rapid washing and filtration of up to 48 reaction vessels in parallel, the application of an inexpensive and environmentally friendly technique to remove trifluoroacetic acid from sixteen 96-well plates in parallel, and a high-throughput method for removing cleaved crowns from reusable pin racks. Tens of thousands of discrete samples have been produced in-house using this conceptually and operationally straightforward strategy.
ABSTRACT
Novel 2'-heteroaryl-2-(phenoxymethyl)imidazolines have been identified as potent agonists of the cloned human alpha(1)-adrenoceptors in vitro. The nature of the 2'-heteroaryl group can have significant effects on the potency, efficacy, and subtype selectivity in this series. alpha(1A) Subtype selective agonists have been identified.