Jörg Haupenthal

Discovery of Novel Bacterial RNA Polymerase Inhibitors: Pharmacophore-Based Virtual Screening and Hit Optimization

The bacterial RNA polymerase (RNAP) is a validated target for broad spectrum antibiotics. However, the efficiency of drugs is reduced by resistance. To discover novel RNAP inhibitors, a pharmacophore based on the alignment of described inhibitors was used for virtual screening. In an optimization process of hit compounds, novel derivatives with improved in vitro potency were discovered. Investigations concerning the molecular mechanism of RNAP inhibition reveal that they prevent the protein-protein interaction (PPI) between σ(70) and the RNAP core enzyme. Besides of reducing RNA formation, the inhibitors were shown to interfere with bacterial lipid biosynthesis. The compounds were active against Gram-positive pathogens and revealed significantly lower resistance frequencies compared to clinically used rifampicin.

Synthesis of some novel androstanes as potential aromatase inhibitors

Novel pyrazole and isoxazole derivatives (6-9) were synthesized as a aromatase inhibitors. Pyrazole was synthesized from hydrazine hydrate and isoxazoles from hydroxylamine hydrochloride under different conditions. Molecular docking studies were carried out for the synthesized compounds. The best score was obtained for the compound (9) followed by compound (6) while compound (8) afforded poorest of the score. Aromatase inhibitory activity for compound (6) having pyrazole ring at 2,3 position showed highest activity followed by nitrile derivative (9). Isomeric forms of isoxazole (7 and 8) showed very poor activity compared to fadrozole and aminoglutethimide. Preliminary kinetic studies have shown that both of the active compounds (6 and 9) are reversible inhibitors of the enzyme.

Benzamidobenzoic acids as potent PqsD inhibitors for the treatment of Pseudomonas aeruginosa infections.

Targeting PqsD is a promising novel approach to disrupt bacterial cell-to-cell-communication in Pseudomonas aeruginosa. In search of selective PqsD inhibitors, two series of benzamidobenzoic acids - one published as RNAP inhibitors and the other as PqsD inhibitors - were investigated for inhibitory activity toward the respective other enzyme. Additionally, novel derivatives were synthesized and biologically evaluated. By this means, the structural features needed for benzamidobenzoic acids to be potent and, most notably, selective PqsD inhibitors were identified. The most interesting compound of this study was the 3-Cl substituted compound 5 which strongly inhibits PqsD (IC₅₀ 6.2 μM) while exhibiting no inhibition of RNAP.

Expanding the scaffold for bacterial RNA polymerase inhibitors: design, synthesis and structure–activity relationships of ureido-heterocyclic-carboxylic acids

The emergence of bacterial resistance requires the development of new antibiotics with an alternative mode of action. Based on class I, developed in our previous study, a new series of RNA polymerase (RNAP) inhibitors targeting the switch region was designed. Feasible synthetic procedures for the aryl-ureido-heterocyclic-carboxylic acids were developed including three regioisomeric thiophene classes (II–IV), as well as three isosteric furan (V, VI) and thiazole (VII) classes. Biological evaluation using a RNAP transcription inhibition assay revealed that class II compounds possess the same activity as the parent class I, whereas classes III, V–VII were active, however with lower potency. Structure–activity relationship (SAR) studies, supported by molecular modeling, elucidated the structural requirements necessary for interaction with the binding site. Beside the RNAP inhibitory effects, the new compounds displayed good antibacterial activities against Gram positive bacteria and the Gram negative E. coli TolC strain. Moreover, they showed no cross resistance with the clinically used RNAP inhibitor rifampicin (Rif) and a lower rate of resistance compared to Rif.

A detective story in drug discovery: elucidation of a screening artifact reveals polymeric carboxylic acids as potent inhibitors of RNA polymerase.

Chasing the active impurity: In the validation of a screening hit it was discovered that a polymeric trace impurity was responsible for the biological activity. Such a side product can be formed with similar compounds. During the investigations it was discovered that the negatively charged macromolecule interacts very efficiently with the protein surface of E. coli RNAP via electrostatic interactions.

Synthesis and biological evaluation of novel unsaturated carboxysteroids as human 5α-reductase inhibitors: a legitimate approach.

In the present study, novel steroidal 17a-substituted 3-cyano-17a-aza-D-homo-3,5-androstadien-17-ones (12-19) and 17a-substituted 17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic acids (20-26) were synthesized from dehydroepiandrosterone acetate (6) along with 17-oxo-19-nor-3,5-androstadien-3-oic acid (30) through a multistep synthesis. Compounds were evaluated for their in vitro 5α-reductase inhibitory activity by measuring the conversion of [(3)H] androstenedione in human embryonic kidney (HEK) cells. In vivo 5α-reductase inhibitory activity was also determined using rat prostate weighing method. Compounds 21-23 and 25 showed potent inhibition of 5α-reductase II enzyme with IC(50) values of 54.1 ± 9.5, 22.1 ± 2.4, 72.8 ± 2.3 and 26.5 ± 4.4 nM respectively as compared to Finasteride (30.3 nM) along with a significant (p < 0.05) reduction in rat prostate weight.

Binding mode characterization of novel RNA polymerase inhibitors using a combined biochemical and NMR approach.

Bacterial RNA polymerase (RNAP) represents a validated target for the development of broad-spectrum antibiotics. However, the medical value of RNAP inhibitors in clinical use is limited by the prevalence of resistant strains. To overcome this problem, we focused on the exploration of alternative target sites within the RNAP. Previously, we described the discovery of a novel RNAP inhibitor class containing an ureidothiophene-2-carboxylic acid core structure. Herein, we demonstrate that these compounds are potent against a set of methicillin-resistant Staphylococcus aureus (MRSA) strains (MIC 2-16 μg mL(-1)) and rifampicin-resistant Escherichia coli TolC strains (MIC 12.5-50 μg mL(-1)). Additionally, an abortive transcription assay revealed that these compounds inhibit the bacterial transcription process during the initiation phase. Furthermore, the binding mode of the ureidothiophene-2-carboxylic acids was characterized by mutagenesis studies and ligand-based NMR spectroscopy. Competition saturation transfer difference (STD) NMR experiments with the described RNAP inhibitor myxopyronin A (Myx) suggest that the ureidothiophene-2-carboxylic acids compete with Myx for the same binding site in the RNAP switch region. INPHARMA (interligand NOE for pharmacophore mapping) experiments and molecular docking simulations provided a binding model in which the ureidothiophene-2-carboxylic acids occupy the region of the Myx western chain binding site and slightly occlude that of the eastern chain. These results demonstrate that the ureidothiophene-2-carboxylic acids are a highly attractive new class of RNAP inhibitors that can avoid the problem of resistance.

New Insights into the Bacterial RNA Polymerase Inhibitor CBR703 as a Starting Point for Optimization as an Anti-Infective Agent

ABSTRACT CBR703 was reported to inhibit bacterial RNA polymerase (RNAP) and biofilm formation, considering it to be a good candidate for further optimization. While synthesized derivatives of CBR703 did not result in more-active RNAP inhibitors, we observed promising antibacterial activities. These again correlated with a significant cytotoxicity toward mammalian cells. Furthermore, we suspect the promising effects on biofilm formation to be artifacts. Consequently, this class of compounds can be considered unattractive as antibacterial agents.

Quorum Sensing Inhibitors as Pathoblockers for Pseudomonas aeruginosa Infections: A New Concept in Anti-Infective Drug Discovery

Decades after the Golden Age of antibiotics, it is evident that antibiotic resistance is rapidly evolving and spreading among clinically relevant pathogens. The medical need for alternative anti-infective treatments is tremendously high today. A novel concept has emerged focusing on “pathoblockers” disarming a pathogen of its virulence weaponries instead of directly killing it. As such compounds should not instigate selection pressure – in contrast to conventional antibiotics – treated bacteria ought to be less prone to resistance development. The discovery of anti-pathogenic compounds necessitates a profound understanding of the respective pathogenic mechanisms and modern medicinal chemistry strategies in order to develop highly effective therapies. Interference with the bacterial cell-to-cell communication that globally regulates the production of various virulence factors and biofilm formation is a very promising approach. As an example, we herein review recent advances and forward-looking concepts in the development of quorum sensing inhibitors selectively targeting the ESKAPE pathogen Pseudomonas aeruginosa.

Synthesis and biological evaluation of 3-tetrazolo steroidal analogs: Novel class of 5α-reductase inhibitors

In the present study, a series of steroidal tetrazole derivatives of androstane and pregnane have been prepared in which the tetrazole moiety was appended at C-3 and 17a-aza locations. 3-Tetrazolo-3,5-androstadien-17-one (6), 3-tetrazolo-19-nor-3,5-androstadien-17-one (10), 3-tetrazolo-3,5-pregnadien-20-one (14), 17a-substituted 3-tetrazolo-17a-aza-D-homo-3,5-androstadien-17-one (26-31) and 3-(2-acetyltetrazolo)-17a-aza-d-homo-3,5-androstadien-17-one (32) were synthesized from dehydroepiandrosterone acetate (1) through multiple synthetic steps. Some of the synthesized compounds were evaluated for their in vitro 5α-reductase (5AR) inhibitory activity by measuring the conversion of [(3)H] androstenedione in human embryonic kidney (HEK) cells. In vivo 5α-reductase inhibitory activity also showed a significant reduction (p <0.05) in rat prostate weight. The most potent compound 14 showed 5AR-2 inhibition with IC50 being 15.6nM as compared to clinically used drug finasteride (40nM). There was also a significant inhibition of 5AR-1 with IC50 547nM compared to finasteride (453nM).