Genomic Discovery and Structure-Activity Exploration of a Novel Family of Enzyme-Activated Covalent Cyclin-Dependent Kinase Inhibitors.

Fungi have historically been the source of numerous important medicinal compounds, but full exploitation of their genetic potential for drug development has been hampered in traditional discovery paradigms. Here we describe a radically different approach, top-down drug discovery (TD3), starting with a massive digital search through a database of over 100,000 fully genomicized fungi to identify loci encoding molecules with a predetermined human target. We exemplify TD3 by the selection of cyclin-dependent kinases (CDKs) as targets and the discovery of two molecules, 1 and 2, which inhibit therapeutically important human CDKs. 1 and 2 exhibit a remarkable mechanism, forming a site-selective covalent bond to the CDK active site Lys. We explored the structure-activity relationship via semi- and total synthesis, generating an analog, 43, with improved kinase selectivity, bioavailability, and efficacy. This work highlights the power of TD3 to identify mechanistically and structurally novel molecules for the development of new medicines.

Correction to "Structure Based Design of Non-Natural Peptidic Macrocyclic Mcl-1 Inhibitors".

[This corrects the article DOI: 10.1021/acsmedchemlett.6b00464.].

Structure Based Design of Non-Natural Peptidic Macrocyclic Mcl-1 Inhibitors.

Mcl-1 is a pro-apoptotic BH3 protein family member similar to Bcl-2 and Bcl-xL. Overexpression of Mcl-1 is often seen in various tumors and allows cancer cells to evade apoptosis. Here we report the discovery and optimization of a series of non-natural peptide Mcl-1 inhibitors. Screening of DNA-encoded libraries resulted in hit compound 1, a 1.5 µM Mcl-1 inhibitor. A subsequent crystal structure demonstrated that compound 1 bound to Mcl-1 in a ß-turn conformation, such that the two ends of the peptide were close together. This proximity allowed for the linking of the two ends of the peptide to form a macrocycle. Macrocyclization resulted in an approximately 10-fold improvement in binding potency. Further exploration of a key hydrophobic interaction with Mcl-1 protein and also with the moiety that engages Arg256 led to additional potency improvements. The use of protein-ligand crystal structures and binding kinetics contributed to the design and understanding of the potency gains. Optimized compound 26 is a <3 nM Mcl-1 inhibitor, while inhibiting Bcl-2 at only 5 µM and Bcl-xL at >99 µM, and induces cleaved caspase-3 in MV4-11 cells with an IC50 of 3 µM after 6 h.

Absorption, distribution, metabolism and elimination of 14C-ETX0914, a novel inhibitor of bacterial type-II topoisomerases in rodents.

1. ETX0914 is a novel bacterial topoisomerase inhibitor that has a novel mode-of-inhibition and is in clinical development for the treatment of infections caused by Neisseria gonorrhoeae. 2. The in vitro biotransformation studies of ETX0914 using mouse, rat, dog and human hepatocytes showed moderate intrinsic clearance in mouse and rat and low intrinsic clearance in dog and human. 3. Following intravenous administration of [14C]-ETX0914 in rats, the mean recovery of administered dose in urine, bile and feces was approximately 15%, 55% and 24%, respectively. Unchanged ETX0914 recovered in urine and bile was less than 5% of the dose, indicating that ETX0914 underwent extensive metabolism in rats. Metabolites M1, M2, M4, M6 and M12 detected in both rat and mouse urine samples were not detected in mouse urine when predosed with 1-aminobenzotriazole, indicating that these metabolites were cytochrome P450 mediated products. The major fecal metabolites observed in rats were not formed when ETX0914 was incubated with fresh feces from germ free rats under sterile condition or in incubations with rat intestinal microsome and cytosol, suggesting that most likely ETX0914 was directly excreted into gut lumen where metabolites were formed as intestinal microflora-mediated products. The major sites of metabolism by CYP enzymes were in the morpholine and oxazolidinone rings while it was benzisoxazole reduction with the gut microflora.

Discovery and Characterization of New Hydroxamate Siderophores, Baumannoferrin A and B, produced by Acinetobacter baumannii.

Acinetobacter baumannii AYE does not produce acinetobactin but grows under iron limitation. Accordingly, analyses of AYE iron-restricted culture supernatants resulted in the isolation of two fractions, which contained only hydroxamates and showed siderophore activity. Structural analyses identified baumannoferrin A and baumannoferrin B, which differ only by a double bond. These siderophores are composed of citrate, 1,3-diaminopropane, 2,4-diaminobutyrate, decenoic acid, and α-ketoglutarate. Analysis of the AYE genome showed the presence of a 12-gene cluster coding for proteins similar to those involved in the production and utilization of the hydroxamate siderophores acinetoferrin and achromobactin. As A. baumannii AYE does not produce acinetobactin and harbors only one gene cluster encoding the production and utilization of a siderophore, this strain's growth under iron limitation depends on baumannoferrin, a novel hydroxamate that could play a role in its virulence.

Thinking outside the "bug": a unique assay to measure intracellular drug penetration in gram-negative bacteria.

Significant challenges are present in antibiotic drug discovery and development. One of these is the number of efficient approaches Gram-negative bacteria have developed to avoid intracellular accumulation of drugs and other cell-toxic species. In order to better understand these processes and correlate in vitro enzyme inhibition to whole cell activity, a better assay to evaluate a key factor, intracellular accumulation of the drug, is urgently needed. Here, we describe a unique liquid chromatography (LC)-mass spectrometry (MS) approach to measure the amount of cellular uptake of antibiotics by Gram-negative bacteria. This method, which measures the change of extracellular drug concentration, was evaluated by comparing the relative uptake of linezolid by Escherichia coli wild-type versus an efflux pump deficient strain. A higher dosage of the drug showed a higher accumulation in these bacteria in a dosing range of 5-50 ng/mL. The Escherichia coli efflux pump deficient strain had a higher accumulation of the drug than the wild-type strain as predicted. The approach was further validated by determining the relative meropenem uptake by Pseudomonas aeruginosa wild-type versus a mutant strain lacking multiple porins. These studies show great promise of being applied within antibiotic drug discovery, as a universal tool to aid in the search for compounds that can easily penetrate bacterial cells.

In vitro and in vivo metabolism of 14C-AZ11, a novel inhibitor of bacterial DNA gyrase/type II topoisomerase.

1. (2R,4S,4aS)-11-Fluoro-2,4-dimethyl-8-((S)-4-methyl-2-oxooxazolidin-3-yl)-2,4,4a,6-tetrahydro-1H,1'H-spiro [isoxazolo[4,5-g][1,4]oxazino[4,3-a]quinoline-5,5'-pyrimidine]-2',4',6'(3'H)-trione (AZ11) is a novel mode-of-inhibition bacterial topoisomerase inhibitor that entered preclinical development for the treatment of Gram-positive bacteria infection. 2. The in vitro biotransformation studies of AZ11 using mouse, rat, dog and human hepatocytes showed low-intrinsic clearance in all species attributed to microsomal metabolism. 3. After a single intravenous administration of [14C]AZ11 in bile duct cannulated rats, the mean percentage of dose recovered in rat urine, bile and feces was approximately 18, 36 and 42%, respectively. Unchanged AZ11 recovered in rat urine and bile was less than 9% of the dose, indicating that AZ11 underwent extensive metabolism in rats. 4. The most abundant in vivo metabolite detected in urine and bile was M1 formed via ring opening on the piperidine and morpholine rings accounting for 20% of the administered dose. The major fecal metabolite was M5, which accounted for approximately 32% of administered dose. M5 was not formed when AZ11 incubated with rat intestinal microsomes and cytosol but was formed when incubated with fresh rat feces, suggesting that unchanged AZ11 was directly excreted into gut lumen where M5 formed as an intestinal microflora-mediated product. This process could have significant impact on bioavailability or exposure of AZ11 in rat.

Synthesis of a tetrahydronaphthyridine spiropyrimidinetrione DNA gyrase inhibiting antibacterial agent--differential substitution at all five carbon atoms of pyridine.

The synthesis of (-)-1, a potent antibacterial agent, was achieved stereoselectively in nine steps from readily available starting materials. Directed metalations were developed to assemble a pentasubstituted pyridine with appropriately positioned aldehyde and dimethylmorpholine substituents for a key tertiary amino effect reaction (T-reaction) that led to the spirocylic architecture. Ultimately, (-)-1 was isolated as the thermodynamically most favored stereoisomer.

Enantioselective Synthesis and Profiling of Two Novel Diazabicyclooctanone ß-Lactamase Inhibitors.

The enantioselective synthesis of two novel cyclopropane-fused diazabicyclooctanones is reported here. Starting from butadiene monoxide, the key enone intermediate 7 was prepared in six steps. Subsequent stereoselective introduction of the cyclopropane group and further transformation led to compounds 1a and 1b as their corresponding sodium salt. The great disparity regarding their hydrolytic stability was rationalized by the steric interaction between the cyclopropyl methylene and urea carbonyl. These two novel ß-lactamase inhibitors were active against class A, C, and D enzymes.

An aminoquinazoline inhibitor of the essential bacterial cell wall synthetic enzyme GlmU has a unique non-protein-kinase-like binding mode.

GlmU is a bifunctional enzyme with acetyltransferase and uridyltransferase activities, and is essential for the biosynthesis of the bacterial cell wall. Inhibition results in a loss of cell viability. GlmU is therefore considered a potential target for novel antibacterial agents. A HTS (high-throughput screen) identified a series of aminoquinazolines with submicromolar potency against the uridyltransferase reaction. Biochemical and biophysical characterization showed competition with UTP binding. We determined the crystal structure of a representative aminoquinazoline bound to the Haemophilus influenzae isoenzyme at a resolution of 2.0 Å. The inhibitor occupies part of the UTP site, skirts the outer perimeter of the GlcNAc1-P (N-acetylglucosamine-1-phosphate) pocket and anchors a hydrophobic moiety into a lipophilic pocket. Our SAR (structure-activity relationship) analysis shows that all of these interactions are essential for inhibitory activity in this series. The crystal structure suggests that the compound would block binding of UTP and lock GlmU in an apo-enzyme-like conformation, thus interfering with its enzymatic activity. Our lead generation effort provides ample scope for further optimization of these compounds for antibacterial drug discovery.

Novel structures derived from 2-[[(2-pyridyl)methyl]thio]-1H-benzimidazole as anti-Helicobacter pylori agents, Part 2.

A parallel chemistry expansion of the 2-([3-[(1H-benzimidazol-2-ylsulfanyl)methyl]-phenyl]sulfanyl)-1-ethanol scaffold (2) successfully provided a set of 2-([3-[(1H-benzimidazol-2-ylsulfanyl)methyl]-2-methylphenyl]sulfanyl)ethyl carbamates with the generic structure 12, which displayed potent and selective activities against the gastric pathogen Helicobacter pylori. A prototype carbamate 12a was studied further and found to meet several significant in vitro microbiological criteria required for a novel anti-H. pylori agent. The compound displayed low minimal inhibition concentration (MIC) values against a panel of 27 different clinically relevant H. pylori strains (MIC(90) = 0.25 microg/mL), including strains resistant to either metronidazole or clarithromycin or both. Additionally, 12a was almost inactive against a wide range of commensal or pathogenic microorganisms comprising panels of 25 aerobic bacterial strains including two strains of methicillin resistant Staphylococcus aureus (MIC(90) = >64 microg/mL) and 18 anaerobic bacterial strains (MIC(90) = >64 microg/mL). The measured rate of resistance development against 12a was found to be less than 10(-9), a clinically acceptable level, and pharmacokinetic studies revealed in vivo exposure levels comparable with those established for antimicrobials currently used in H. pylori triple regimen.