John J. Allocco

Platensimycin is a selective FabF inhibitor with potent antibiotic properties

Bacterial infection remains a serious threat to human lives because of emerging resistance to existing antibiotics. Although the scientific community has avidly pursued the discovery of new antibiotics that interact with new targets, these efforts have met with limited success since the early 1960s. Here we report the discovery of platensimycin, a previously unknown class of antibiotics produced by Streptomyces platensis. Platensimycin demonstrates strong, broad-spectrum Gram-positive antibacterial activity by selectively inhibiting cellular lipid biosynthesis. We show that this anti-bacterial effect is exerted through the selective targeting of β-ketoacyl-(acyl-carrier-protein (ACP)) synthase I/II (FabF/B) in the synthetic pathway of fatty acids. Direct binding assays show that platensimycin interacts specifically with the acyl-enzyme intermediate of the target protein, and X-ray crystallographic studies reveal that a specific conformational change that occurs on acylation must take place before the inhibitor can bind. Treatment with platensimycin eradicates Staphylococcus aureus infection in mice. Because of its unique mode of action, platensimycin shows no cross-resistance to other key antibiotic-resistant strains tested, including methicillin-resistant S. aureus, vancomycin-intermediate S. aureus and vancomycin-resistant enterococci. Platensimycin is the most potent inhibitor reported for the FabF/B condensing enzymes, and is the only inhibitor of these targets that shows broad-spectrum activity, in vivo efficacy and no observed toxicity.

Toxoplasma gondii Cyclic GMP-Dependent Kinase: Chemotherapeutic Targeting of an Essential Parasite Protein Kinase

ABSTRACT The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (compound 1) has in vivo activity against the apicomplexan parasites Toxoplasma gondii and Eimeria tenella in animal models. The presumptive molecular target of this compound in E. tenella is cyclic GMP-dependent protein kinase (PKG). Native PKG purified from T. gondii has kinetic and pharmacologic properties similar to those of the E. tenella homologue, and both have been functionally expressed as recombinant proteins in T. gondii. Computer modeling of parasite PKG was used to predict catalytic site amino acid residues that interact with compound 1. The recombinant laboratory-generated mutants T. gondii PKG T761Q or T761M and the analogous E. tenella T770 alleles have reduced binding affinity for, and are not inhibited by, compound 1. By all other criteria, PKG with this class of catalytic site substitution is indistinguishable from wild-type enzyme. A genetic disruption of T. gondii PKG can only be achieved if a complementing copy of PKG is provided in trans, arguing that PKG is an essential protein. Strains of T. gondii, disrupted at the genomic PKG locus and dependent upon the T. gondii T761-substituted PKGs, are as virulent as wild type in mice. However, unlike mice infected with wild-type T. gondii that are cured by compound 1, mice infected with the laboratory-generated strains of T. gondii do not respond to treatment. We conclude that PKG represents the primary molecular target responsible for the antiparasitic efficacy of compound 1.

Broad spectrum antiprotozoal agents that inhibit histone deacetylase: structure-activity relationships of apicidin. Part 1.

Apicidin, a natural product recently isolated at Merck, inhibits both mammalian and protozoan histone deacetylases (HDACs). The conversion of apicidin, a nanomolar inhibitor of HDACs, into a series of side-chain analogues that display picomolar enzyme affinity is described within this structure-activity study.

PAP Inhibitor with In Vivo Efficacy Identified by Candida albicans Genetic Profiling of Natural Products

Natural products provide an unparalleled source of chemical scaffolds with diverse biological activities and have profoundly impacted antimicrobial drug discovery. To further explore the full potential of their chemical diversity, we survey natural products for antifungal, target-specific inhibitors by using a chemical-genetic approach adapted to the human fungal pathogen Candida albicans and demonstrate that natural-product fermentation extracts can be mechanistically annotated according to heterozygote strain responses. Applying this approach, we report the discovery and characterization of a natural product, parnafungin, which we demonstrate, by both biochemical and genetic means, to inhibit poly(A) polymerase. Parnafungin displays potent and broad spectrum activity against diverse, clinically relevant fungal pathogens and reduces fungal burden in a murine model of disseminated candidiasis. Thus, mechanism-of-action determination of crude fermentation extracts by chemical-genetic profiling brings a powerful strategy to natural-product-based drug discovery.

Evaluation of a Cyclic GMP-Dependent Protein Kinase Inhibitor in Treatment of Murine Toxoplasmosis: Gamma Interferon Is Required for Efficacy

ABSTRACT The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (compound 1) is a potent inhibitor of cyclic GMP-dependent protein kinases from Apicomplexan protozoa and displays cytostatic activity against Toxoplasma gondii in vitro. Compound 1 has now been evaluated against T. gondii infections in the mouse and appeared to protect the animals when given intraperitoneally at 50 mg/kg twice daily for 10 days. However, samples from brain, spleen, and lung taken from infected treated mice revealed the presence of parasites after cessation of administration of compound 1, indicating that a transient asymptomatic parasite recrudescence occurs in all survivors. The ability of mice to control Toxoplasma infection after compound 1 treatment has been terminated suggested that the mouse immune system plays a synergistic role with chemotherapy in controlling the infection. To explore this possibility, gamma interferon (IFN-γ)-knockout mice were infected with parasites and treated with compound 1, and survival was compared to that of normal mice. IFN-γ-knockout mice were protected against T. gondii throughout the treatment phase but died during the posttreatment phase in which peak recrudescence was observed in treated immunocompetent mice. These data suggest that an IFN-γ-dependent immune response was essential for controlling and resolving parasite recrudescence in mice treated with compound 1. In addition, when compound 1-cured immunocompetent mice were rechallenged with a lethal dose of T. gondii, all survived (n = 32). It appears that the cytostatic nature of compound 1 provides an “immunization” phase during chemotherapy which allows the mice to survive the recrudescence and any subsequent challenge with a lethal dose of T. gondii.

Kibdelomycin A, a congener of kibdelomycin, derivatives and their antibacterial activities.

Emergence of bacterial resistance has eroded the effectiveness of many life saving antibiotics leading to an urgent need for new chemical classes of antibacterial agents. We have applied a Staphylococcus aureus fitness test strategy to natural products screening to meet this challenge. In this paper we report the discovery of kibdelomycin A, a demethylated congener of kibdelomycin, the representative of a novel class of antibiotics produced by a new strain of Kibdelosporangium. Kibdelomycin A is a potent inhibitor of DNA gyrase and topoisomerase IV, inhibits DNA synthesis and shows whole cell antibiotic activity, albeit, less potently than kibdelomycin. Kibdelomycin C-33 acetate and tetrahydro-bisdechloro derivatives of kibdelomycin were prepared which helped define a basic SAR of the family.

MK-4815, a Potential New Oral Agent for Treatment of Malaria

ABSTRACT Malaria continues to have a significant impact on the health of the developing world. Efforts to combat this disease now focus on combination therapy in order to stem the emergence of resistant parasites. Continued efforts are needed to discover and develop new agents for use in combination antimalarial regimens. MK-4815 is a small molecule with antimalarial activity that was identified from a large pharmaceutical compound collection using a semiautomated version of a well-established in vitro assay for the erythrocytic stages of Plasmodium falciparum. In vitro studies indicate that the compound selectively accumulates in infected red blood cells and is most effective against the metabolically active late trophozoite/early schizont stages. A variety of drug-resistant field isolates of P. falciparum were found to be as sensitive to MK-4815 as the wild-type lines. MK-4815 is orally active in a P. berghei mouse model of acute malaria. In this model, where untreated animals succumb to infection 10 to 12 days postinfection, MK-4815 was completely curative when given as a single dose of 50 mg/kg, 2 doses of 25 mg/kg, or 4.5 doses of 12.5 mg/kg. In pharmacokinetic studies with mice and rhesus monkeys, MK-4815 demonstrated oral bioavailability and low clearance. In addition, MK-4815 is inexpensive to synthesize, an important characteristic for providing affordable antimalaria therapy to the developing world. The attractive biological and pharmaceutical profile of MK-4815 demonstrates its potential for use in combination with other agents in the fight against malaria.

Amylopectin Synthase of Eimeria tenella: Identification and Kinetic Characterization

ABSTRACT. A soluble enzyme amylopectin synthase (UDP‐glucose‐α 1,4‐glucan α‐4‐glucosyltransferase) which transfers glucose from uridine 5′‐diphosphate glucose (UDP‐glucose) to a primer to form α‐I,4‐glucosyl linkages has been identified in the extracts of unsporulated oocysts of Eimeria tenella. UDP‐glucose and not ADP‐glucose was the most active glucosyl donor. Corn amylopectin, rabbit liver glycogen, oyster glycogen and corn starch served as primers; the latter two were less efficient. The enzyme has an apparent pH optimum of 7.5 and exhibited typical Michaelis‐Menten kinetics with dependence on both the primer and substrate concentrations. The Michaelis constants (Km). with respect to UDP‐glucose, was 0.5 mM; and 0.25 mg/ml and 1.25 mg/ml with respect to amylopectin and rabbit liver glycogen. The product formed by the reaction was predominantly a glucan containing α‐1,4 linkages. The specificity of the enzyme suggests that this enzyme is similar to glycogen synthase in eukaryotes and has been designated as amylopectin synthase (UDP‐glucose‐α‐1,4‐glucosetransferase EC 2.4.1.11).

NITROPHENIDE (MEGASUL") BLOCKS EIMERIA TENELLA DEVELOPMENT BY INHIBITING THE MANNITOL CYCLE ENZYME MANNITOL-1-PHOSPHATE DEHYDROGENASE

Unsporulated oocysts of the protozoan parasite Eimeria tenella contain high levels of mannitol, which is thought to be the principal energy source for the process of sporulation. Biosynthesis and utilization of this sugar alcohol occurs via a metabolic pathway known as the mannitol cycle. Here, results are presented that suggest that 3-nitrophenyl disulfide (nitrophenide, Megasul™), an anticoccidial drug commercially used in the 1950s, inhibits mannitol-1-phosphate dehydrogenase (M1PDH), which catalyzes the committed enzymatic step in the mannitol cycle. Treatment of E. tenella-infected chickens with nitrophenide resulted in a 90% reduction in oocyst shedding. The remaining oocysts displayed significant morphological abnormalities and were largely incapable of further development. Nitrophenide treatment did not affect parasite asexual reproduction, suggesting specificity for the sexual stage of the life cycle. Isolated oocysts from chickens treated with nitrophenide exhibited a dose-dependent reduction in mannitol, suggesting in vivo inhibition of parasite mannitol biosynthesis. Nitrophenide-mediated inhibition of M1PDH was observed in vitro using purified native enzyme. Moreover, M1PDH activity immunoprecipitated from E. tenella-infected cecal tissues was significantly lower in nitrophenide-treated compared with untreated chickens. Western blot analysis and immunohistochemistry showed that parasites from nitrophenide-treated and untreated chickens contained similar enzyme levels. These data suggest that nitrophenide blocks parasite development at the sexual stages by targeting M1PDH. Thus, targeting of the mannitol cycle with drugs could provide an avenue for controlling the spread of E. tenella in commercial production facilities by preventing oocyst shedding.