Optimized Pyridazinone Nutrient Channel Inhibitors Are Potent and Specific Antimalarial Leads.

Human and animal malaria parasites increase their host erythrocyte permeability to a broad range of solutes as mediated by parasite-associated ion channels. Molecular and pharmacological studies have implicated an essential role in parasite nutrient acquisition, but inhibitors suitable for development of antimalarial drugs are missing. Here, we generated a potent and specific drug lead using Plasmodium falciparum, a virulent human pathogen, and derivatives of MBX-2366, a nanomolar affinity pyridazinone inhibitor from a high-throughput screen. As this screening hit lacks the bioavailability and stability needed for in vivo efficacy, we synthesized 315 derivatives to optimize drug-like properties, establish target specificity, and retain potent activity against the parasite-induced permeability. Using a robust, iterative pipeline, we generated MBX-4055, a derivative active against divergent human parasite strains. MBX-4055 has improved oral absorption with acceptable in vivo tolerability and pharmacokinetics. It also has no activity against a battery of 35 human channels and receptors and is refractory to acquired resistance during extended in vitro selection. Single-molecule and single-cell patch-clamp indicate direct action on the plasmodial surface anion channel, a channel linked to parasite-encoded RhopH proteins. These studies identify pyridazinones as novel and tractable antimalarial scaffolds with a defined mechanism of action. SIGNIFICANCE STATEMENT: Because antimalarial drugs are prone to evolving resistance in the virulent human P. falciparum pathogen, new therapies are needed. This study has now developed a novel drug-like series of pyridazinones that target an unexploited parasite anion channel on the host cell surface, display excellent in vitro and in vivo ADME properties, are refractory to acquired resistance, and demonstrate a well defined mechanism of action.

Optimization of a small-molecule Lipid II binder.

Lipid II is an essential precursor of bacterial cell wall biosynthesis and an attractive target for antibiotics. Lipid II is comprised of specialized lipid (bactoprenol) linked to a hydrophilic head group consisting of a peptidoglycan subunit (N-acetylglucosamine (GlcNAc)-N-acetylmuramic acid (MurNAc) disaccharide coupled to a short pentapeptide moiety) via a pyrophosphate. We previously identified a (E)-2,4-bis(4-bromophenyl)-6-(4-(dimethylamino)styryl)pyrylium boron tetrafluoride salt, termed 6jc48-1, that interacts with the MurNAc moiety, the phosphate cage and the isoprenyl tail of Lipid II. Here, we report on the structure-activity relationship of 6jc48-1 derivatives obtained by de novo chemical synthesis. Our results indicate that bacterial killing is positively driven by bi-phenyl stacking with peptidoglycan units. Replacement of bromides by fluorides resulted in activity against S. aureus without affecting Lipid II binding and cytotoxicity. Antibacterial activity was affected negatively by extended interaction of the scaffold with Lipid II isoprenyl units.

Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives.

The Escherichia coli AcrAB-TolC efflux pump is the archetype of the resistance nodulation cell division (RND) exporters from Gram-negative bacteria. Overexpression of RND-type efflux pumps is a major factor in multidrug resistance (MDR), which makes these pumps important antibacterial drug discovery targets. We have recently developed novel pyranopyridine-based inhibitors of AcrB, which are orders of magnitude more powerful than the previously known inhibitors. However, further development of such inhibitors has been hindered by the lack of structural information for rational drug design. Although only the soluble, periplasmic part of AcrB binds and exports the ligands, the presence of the membrane-embedded domain in AcrB and its polyspecific binding behavior have made cocrystallization with drugs challenging. To overcome this obstacle, we have engineered and produced a soluble version of AcrB [AcrB periplasmic domain (AcrBper)], which is highly congruent in structure with the periplasmic part of the full-length protein, and is capable of binding substrates and potent inhibitors. Here, we describe the molecular basis for pyranopyridine-based inhibition of AcrB using a combination of cellular, X-ray crystallographic, and molecular dynamics (MD) simulations studies. The pyranopyridines bind within a phenylalanine-rich cage that branches from the deep binding pocket of AcrB, where they form extensive hydrophobic interactions. Moreover, the increasing potency of improved inhibitors correlates with the formation of a delicate protein- and water-mediated hydrogen bond network. These detailed insights provide a molecular platform for the development of novel combinational therapies using efflux pump inhibitors for combating multidrug resistant Gram-negative pathogens.

DNA Targeting as a Likely Mechanism Underlying the Antibacterial Activity of Synthetic Bis-Indole Antibiotics.

We previously reported the synthesis and biological activity of a series of cationic bis-indoles with potent, broad-spectrum antibacterial properties. Here, we describe mechanism of action studies to test the hypothesis that these compounds bind to DNA and that this target plays an important role in their antibacterial outcome. The results reported here indicate that the bis-indoles bind selectively to DNA at A/T-rich sites, which is correlated with the inhibition of DNA and RNA synthesis in representative Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) organisms. Further, exposure of E. coli and S. aureus to representative bis-indoles resulted in induction of the DNA damage-inducible SOS response. In addition, the bis-indoles were found to be potent inhibitors of cell wall biosynthesis; however, they do not induce the cell wall stress stimulon in S. aureus, suggesting that this pathway is inhibited by an indirect mechanism. In light of these findings, the most likely basis for the observed activities of these compounds is their ability to bind to the minor groove of DNA, resulting in the inhibition of DNA and RNA synthesis and other secondary effects.

Discovery of bacterial fatty acid synthase type II inhibitors using a novel cellular bioluminescent reporter assay.

Novel, cellular, gain-of-signal, bioluminescent reporter assays for fatty acid synthesis type II (FASII) inhibitors were constructed in an efflux-deficient strain of Pseudomonas aeruginosa and based on the discovery that FASII genes in P. aeruginosa are coordinately upregulated in response to pathway disruption. A screen of 115,000 compounds identified a series of sulfonamidobenzamide (SABA) analogs, which generated strong luminescent signals in two FASII reporter strains but not in four control reporter strains designed to respond to inhibitors of pathways other than FASII. The SABA analogs selectively inhibited lipid biosynthesis in P. aeruginosa and exhibited minimal cytotoxicity to mammalian cells (50% cytotoxic concentration [CC50] ≥ 80 µM). The most potent SABA analogs had MICs of 0.5 to 7.0 µM (0.2 to 3.0 µg/ml) against an efflux-deficient Escherichia coli (ΔtolC) strain but had no detectable MIC against efflux-proficient E. coli or against P. aeruginosa (efflux deficient or proficient). Genetic, molecular genetic, and biochemical studies revealed that SABA analogs target the enzyme (AccC) catalyzing the biotin carboxylase half-reaction of the acetyl coenzyme A (acetyl-CoA) carboxylase step in the initiation phase of FASII in E. coli and P. aeruginosa. These results validate the capability and the sensitivity of this novel bioluminescent reporter screen to identify inhibitors of E. coli and P. aeruginosa FASII.

Synthesis and antifungal evaluation of head-to-head and head-to-tail bisamidine compounds.

Herein, we describe the antifungal evaluation of 43 bisamidine compounds, of which 26 are new, having the scaffold [Am]-[HetAr]-[linker]-[HetAr]-[Am], in which [Am] is a cyclic or acyclic amidine group, [linker] is a benzene, pyridine, pyrimidine, pyrazine ring, or an aliphatic chain of two to four carbon, and [HetAr] is a 5,6-bicyclic heterocycle such as indole, benzimidazole, imidazopyridine, benzofuran, or benzothiophene. In the head-to-head series the two [HetAr] units are oriented such that the 5-membered rings are connected through the linker, and in the head-to-tail series, one of the [HetAr] systems is connected through the 6-membered ring; additionally, in some of the head-to-tail compounds, the [linker] is omitted. Many of these compounds exhibited significant antifungal activity against Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, and Cryptococcus neoformans (MIC ⩽ 4 µg/ml). The most potent compounds, for example, P10, P19 and P34, are comparable in antifungal activities to amphotericin B (MIC 0.125 µg/ml). They exhibited rapid fungicidal activity (>3 log10 decrease in cfu/ml in 4h) at concentrations equivalent to 4× the MIC in time kill experiments. The bisamidines strongly inhibited DNA, RNA and cell wall biosynthesis in C. albicans in macromolecular synthesis assays. However, the half-maximal inhibitory concentration for DNA synthesis was approximately 30-fold lower than those for RNA and cell wall biosynthesis. Fluorescence microscopy of intact cells of C. albicans treated with a bisamidine exhibited enhanced fluorescence in the presence of DNA, demonstrating that the bisamidine was localized to the nucleus. The results of this study show that bisamidines are potent antifungal agents with rapid fungicidal activity, which is likely to be the result of their DNA-binding activity. Although it was difficult to obtain a broad-spectrum antifungal compound with low cytotoxicity, some of the compounds (e.g., P9, P14 and P43) exhibited favorable CC50 values against HeLa cells and maintained considerable antifungal activity.

Structure-activity relationships of a novel pyranopyridine series of Gram-negative bacterial efflux pump inhibitors.

Recently we described a novel pyranopyridine inhibitor (MBX2319) of RND-type efflux pumps of the Enterobacteriaceae. MBX2319 (3,3-dimethyl-5-cyano-8-morpholino-6-(phenethylthio)-3,4-dihydro-1H-pyrano[3,4-c]pyridine) is structurally distinct from other known Gram-negative efflux pump inhibitors (EPIs), such as 1-(1-naphthylmethyl)-piperazine (NMP), phenylalanylarginine-ß-naphthylamide (PAßN), D13-9001, and the pyridopyrimidine derivatives. Here, we report the synthesis and biological evaluation of 60 new analogs of MBX2319 that were designed to probe the structure activity relationships (SARs) of the pyranopyridine scaffold. The results of these studies produced a molecular activity map of the scaffold, which identifies regions that are critical to efflux inhibitory activities and those that can be modified to improve potency, metabolic stability and solubility. Several compounds, such as 22d-f, 22i and 22k, are significantly more effective than MBX2319 at potentiating the antibacterial activity of levofloxacin and piperacillin against Escherichia coli.

Characterization of a novel pyranopyridine inhibitor of the AcrAB efflux pump of Escherichia coli.

Members of the resistance-nodulation-division (RND) family of efflux pumps, such as AcrAB-TolC of Escherichia coli, play major roles in multidrug resistance (MDR) in Gram-negative bacteria. A strategy for combating MDR is to develop efflux pump inhibitors (EPIs) for use in combination with an antibacterial agent. Here, we describe MBX2319, a novel pyranopyridine EPI with potent activity against RND efflux pumps of the Enterobacteriaceae. MBX2319 decreased the MICs of ciprofloxacin (CIP), levofloxacin, and piperacillin versus E. coli AB1157 by 2-, 4-, and 8-fold, respectively, but did not exhibit antibacterial activity alone and was not active against AcrAB-TolC-deficient strains. MBX2319 (3.13 µM) in combination with 0.016 µg/ml CIP (minimally bactericidal) decreased the viability (CFU/ml) of E. coli AB1157 by 10,000-fold after 4 h of exposure, in comparison with 0.016 µg/ml CIP alone. In contrast, phenyl-arginine-ß-naphthylamide (PAßN), a known EPI, did not increase the bactericidal activity of 0.016 µg/ml CIP at concentrations as high as 100 µM. MBX2319 increased intracellular accumulation of the fluorescent dye Hoechst 33342 in wild-type but not AcrAB-TolC-deficient strains and did not perturb the transmembrane proton gradient. MBX2319 was broadly active against Enterobacteriaceae species and Pseudomonas aeruginosa. MBX2319 is a potent EPI with possible utility as an adjunctive therapeutic agent for the treatment of infections caused by Gram-negative pathogens.

Unsaturated fatty acid perturbation combats emerging triazole antifungal resistance in the human fungal pathogen Aspergillus fumigatus.

Contemporary antifungal therapies utilized to treat filamentous fungal infections are inhibited by intrinsic and emerging drug resistance. Consequently, there is an urgent need to develop novel antifungal compounds that are effective against drug-resistant filamentous fungi. Here, we utilized an Aspergillus fumigatus cell-based high-throughput screen to identify small molecules with antifungal activity that also potentiated triazole activity. The screen identified 16 hits with promising activity against A. fumigatus. A nonspirocyclic piperidine, herein named MBX-7591, exhibited synergy with triazole antifungal drugs and activity against pan-azole-resistant A. fumigatus isolates. MBX-7591 has additional potent activity against Rhizopus species and CO2-dependent activity against Cryptococcus neoformans. Chemical, genetic, and biochemical mode of action analyses revealed that MBX-7591 increases cell membrane saturation by decreasing oleic acid content. MBX-7591 has low toxicity in vivo and shows good efficacy in decreasing fungal burden in a murine model of invasive pulmonary aspergillosis. Taken together, our results suggest MBX-7591 is a promising hit with a novel mode of action for further antifungal drug development to combat the rising incidence of triazole-resistant filamentous fungal infections.IMPORTANCEThe incidence of infections caused by fungi continues to increase with advances in medical therapies. Unfortunately, antifungal drug development has not kept pace with the incidence and importance of fungal infections, with only three major classes of antifungal drugs currently available for use in the clinic. Filamentous fungi, also called molds, are particularly recalcitrant to contemporary antifungal therapies. Here, a recently developed Aspergillus fumigatus cell reporter strain was utilized to conduct a high-throughput screen to identify small molecules with antifungal activity. An emphasis was placed on small molecules that potentiated the activity of contemporary triazole antifungals and led to the discovery of MBX-7591. MBX-7591 potentiates triazole activity against drug-resistant molds such as A. fumigatus and has activity against Mucorales fungi. MBX-7591's mode of action involves inhibiting the conversion of saturated to unsaturated fatty acids, thereby impacting fungal membrane integrity. MBX-7591 is a novel small molecule with antifungal activity poised for lead development.

Detour of prenostodione synthesis towards pyrazolones for antibacterial activity.

Our attempts to prepare indolyl acid (3), enroute to prenostodione (2), from phenyl-hydrazine following a reported procedure of Fischer-Indole synthesis rather lead to ethyl 2-(5-oxo-1-phenyl-2,5-dihydro-1H-pyrazol-3-yl)acetate as a major product, which underwent facile condensation with aldehydes to provide the pyrazolones. Intrigued by the opportunity for the diversity oriented synthesis of substituted pyrazolones, we have developed a facile one pot approach for pyrazolones and screened for antibacterial activity and, herein, results are reported.

Luciferase-Based High-Throughput Screen with Aspergillus fumigatus to Identify Antifungal Small Molecules.

Only three classes of contemporary antifungal drugs are routinely utilized in the clinic against filamentous fungal pathogens such as Aspergillus fumigatus. High-throughput phenotypic screens to identify small molecules with activity against filamentous fungi remain challenging due to the hyphal, biofilm-like growth morphology of these important organisms. In this chapter, we describe a protocol for utilizing a bioluminescent A. fumigatus strain for identifying small molecules that potentiate the activity of the triazole antifungal drug fluconazole. The assay holds great promise for identifying small molecules with activity against filamentous fungal pathogens.

Investigating N-arylpyrimidinamine (NAPA) compounds as early-stage inhibitors against human cytomegalovirus.

Human cytomegalovirus (CMV) is a ubiquitous ß-herpesvirus that establishes latent asymptomatic infections in healthy individuals but can cause serious infections in immunocompromised people, resulting in increased risk of morbidity and mortality. The current FDA-approved CMV drugs target late stages of the CMV life-cycle. While these drugs are effective in most cases, they have serious drawbacks, including poor oral bioavailability, dose-limiting toxicity, and a low barrier to resistance. Given the clinical relevance of CMV-associated diseases, novel therapies are needed. Thus, a novel class of compounds that inhibits the early stages of the CMV life-cycle was identified and found to block infection of different strains in physiologically relevant cell types. This class of compounds, N-arylpyrimidinamine (NAPA), demonstrated potent anti-CMV activity against ganciclovir-sensitive and -resistant strains in in vitro replication assays, a selectivity index >30, and favorable in vitro ADME properties. Mechanism of action studies demonstrated that NAPA compounds inhibit an early step of virus infection. NAPA compounds are specific inhibitors of cytomegaloviruses and exhibited limited anti-viral activity against other herpesviruses. Collectively, we have identified a novel class of CMV inhibitor that effectively limits viral infection and proliferation.

trans-Translation inhibitors bind to a novel site on the ribosome and clear Neisseria gonorrhoeae in vivo.

Bacterial ribosome rescue pathways that remove ribosomes stalled on mRNAs during translation have been proposed as novel antibiotic targets because they are essential in bacteria and are not conserved in humans. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, the main ribosome rescue pathway in bacteria. Here, we report optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles, producing MBX-4132, which clears multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Single particle cryogenic-EM studies of non-stop ribosomes show that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein bL27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. These results show that trans-translation is a viable therapeutic target and reveal a new conformation within the bacterial ribosome that may be critical for ribosome rescue pathways.

Aryl rhodanines specifically inhibit staphylococcal and enterococcal biofilm formation.

Staphylococcus epidermidis and Staphylococcus aureus are the leading causative agents of indwelling medical device infections because of their ability to form biofilms on artificial surfaces. Here we describe the antibiofilm activity of a class of small molecules, the aryl rhodanines, which specifically inhibit biofilm formation of S. aureus, S. epidermidis, Enterococcus faecalis, E. faecium, and E. gallinarum but not the gram-negative species Pseudomonas aeruginosa or Escherichia coli. The aryl rhodanines do not exhibit antibacterial activity against any of the bacterial strains tested and are not cytotoxic against HeLa cells. Preliminary mechanism-of-action studies revealed that the aryl rhodanines specifically inhibit the early stages of biofilm development by preventing attachment of the bacteria to surfaces.

Pleiotropic Anti-Infective Effects of Defensin-Derived Antimicrobial Compounds.

We identified low-molecular weight compounds derived from the antimicrobial peptide human neutrophil peptide-1 that bind to Lipid II, an essential precursor of bacterial cell wall biosynthesis. These compounds act as antibacterials on multiple biosynthesis pathways with specificity against gram-positive organisms. Here, we have tested a small subset of our most promising leads against the bacterium Clostridium perfringens and sporozoites of Eimeria tenella, an intracellular protozoan parasite that causes intestinal disease in poultry. We found one compound, 1611-0203 (2-{2,3,5,6-tetrafluoro-4-[2,3,5,6-tetrafluoro-4-(2-hydroxyphenoxy)phenyl]phenoxy}phenol), specifically to inhibit growth of both agents out of all compounds tested. Additionally, compound 1611-0203 inhibits Staphylococcus aureus and Enterococcus spp. Mechanism-of-action studies further reveal that 1611-0203 affects cell wall biosynthesis and inhibits additional biosynthetic pathways. Combined, our results indicate that compounds such as 1611-0203 have therapeutic potential to act as anti-infectives against various organisms simultaneously.

Efectos anti infecciosos pleiotrópicos de los compuestos antimicrobianos derivados de la defensina. Se identificaron compuestos de bajo peso molecular derivados del péptido antimicrobiano, péptido neutrófilico humano 1 que se unen al lípido II, que es un precursor esencial de la biosíntesis de la pared celular bacteriana. Estos compuestos actúan como antibacterianos en múltiples vías de biosíntesis con especificidad contra organismos Gram positivos. En este estudio se probó un pequeño subconjunto de los proyectos más prometedores contra la bacteria Clostridium perfringens y los esporozoitos de Eimeria tenella, que es un parásito protozoario intracelular que causa enfermedad intestinal en aves comerciales. Se encontró un compuesto, 1611-0203 (2-{2,3,5,6-tetrafluoro-4-[2,3,5,6-tetrafluoro-4-(2-hidroxifenoxi)fenil]fenoxi}fenol), específicamente para inhibir el crecimiento de ambos agentes de todos los compuestos probados. Además, el compuesto 1611-0203 inhibe a Staphylococcus aureus y Enterococcus spp. Los estudios del mecanismo de acción revelan además que 1611-0203 afecta la biosíntesis de la pared celular e inhibe las vías biosintéticas adicionales. Estos resultados combinados indican que los compuestos como el 1611-0203 tienen potencial terapéutico para actuar como anti infecciosos contra varios organismos simultáneamente.

Towards Development of Small Molecule Lipid II Inhibitors as Novel Antibiotics.

Recently we described a novel di-benzene-pyrylium-indolene (BAS00127538) inhibitor of Lipid II. BAS00127538 (1-Methyl-2,4-diphenyl-6-((1E,3E)-3-(1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)pyryl-1-ium) tetrafluoroborate is the first small molecule Lipid II inhibitor and is structurally distinct from natural agents that bind Lipid II, such as vancomycin. Here, we describe the synthesis and biological evaluation of 50 new analogs of BAS00127538 designed to explore the structure-activity relationships of the scaffold. The results of this study indicate an activity map of the scaffold, identifying regions that are critical to cytotoxicity, Lipid II binding and range of anti-bacterial action. One compound, 6jc48-1, showed significantly enhanced drug-like properties compared to BAS00127538. 6jc48-1 has reduced cytotoxicity, while retaining specific Lipid II binding and activity against Enterococcus spp. in vitro and in vivo. Further, this compound showed a markedly improved pharmacokinetic profile with a half-life of over 13 hours upon intravenous and oral administration and was stable in plasma. These results suggest that scaffolds like that of 6jc48-1 can be developed into small molecule antibiotic drugs that target Lipid II.

Structure-activity exploration of a small-molecule Lipid II inhibitor.

We have recently identified low-molecular weight compounds that act as inhibitors of Lipid II, an essential precursor of bacterial cell wall biosynthesis. Lipid II comprises specialized lipid (bactoprenol) linked to a hydrophilic head group consisting of a peptidoglycan subunit (N-acetyl glucosamine [GlcNAc]-N-acetyl muramic acid [MurNAc] disaccharide coupled to a short pentapeptide moiety) via a pyrophosphate. One of our lead compounds, a diphenyl-trimethyl indolene pyrylium, termed BAS00127538, interacts with the MurNAc moiety and the isoprenyl tail of Lipid II. Here, we report on the structure-activity relationship of BAS00127538 derivatives obtained by in silico analyses and de novo chemical synthesis. Our results indicate that Lipid II binding and bacterial killing are related to three features: the diphenyl moiety, the indolene moiety, and the positive charge of the pyrylium. Replacement of the pyrylium moiety with an N-methyl pyridinium, which may have importance in stability of the molecule, did not alter Lipid II binding or antibacterial potency.

Static biofilm cultures of Gram-positive pathogens grown in a microtiter format used for anti-biofilm drug discovery.

An in vitro assay is presented for culturing staphylococcal biofilms and biofilms of nonmotile Gram-positive bacteria under static conditions in microtiter assay plates, and for the quantification of biofilm growth, using a simple staining procedure that measures amounts of bacterial cells and extracellular matrix. This basic assay can be adapted readily to study several aspects of biofilm formation, for high-throughput screening to identify small molecule inhibitors of biofilm formation or biofilm-defective mutants, and for quantifying the anti-biofilm activity of biofilm inhibitors.