Development of Novel Immunoprophylactic Agents against Multidrug-Resistant Gram-Negative Bacterial Infections.

The widespread emergence of antibiotic resistance, including multidrug resistance in Gram-negative (G-) bacterial pathogens, poses a critical challenge to the current antimicrobial armamentarium. Antibody-drug conjugates (ADCs), primarily used in anticancer therapy, offer a promising treatment alternative due to their ability to deliver a therapeutic molecule while simultaneously activating the host immune response. The Cloudbreak platform is being used to develop ADCs to treat infectious diseases, composed of a therapeutic targeting moiety (TM) attached via a noncleavable linker to an effector moiety (EM) to treat infectious diseases. In this proof-of-concept study, 21 novel dimeric peptidic molecules (TMs) were evaluated for activity against a screening panel of G- pathogens. The activities of the TMs were not impacted by existing drug resistance. Potent TMs were conjugated to the Fc fragment of human IgG1 (EM), resulting in 4 novel ADCs. These ADCs were evaluated for immunoprophylactic efficacy in a neutropenic mouse model of deep thigh infection. In colistin-sensitive infections, 3 of the 4 ADCs offered protection similar to that of therapeutically dosed colistin, while CTC-171 offered enhanced protection. The efficacy of these ADCs was unchanged in colistin-resistant infections. Together, these results indicate that the ADCs used here are capable of potent binding to G- pathogens regardless of lipopolysaccharide (LPS) modifications that otherwise lead to antibiotic resistance and support further exploration of ADCs in the treatment of infections caused by drug-resistant G- bacteria.

Ex vivo Rezafungin Adsorption and Clearance During Continuous Renal Replacement Therapy.

BACKGROUND/AIMS: To determine adsorption and transmembrane clearances (CLTM) of rezafungin, a novel long-acting echinocandin, in continuous venovenous hemofiltration (CVVH). METHODS: A validated ex vivo bovine blood CVVH model using polysulfone and AN69 hemodiafilters was used to evaluate urea and rezafungin CLTM at 3 different ultrafiltrate flow rates. Rezafungin adsorption to the CRRT apparatus was determined for each hemodiafilter. RESULTS: The sieving coefficient (SC) from CVVH with 3 different ultrafiltrate flow rates was 0 for both HF1400 and Multiflow-150 hemodiafilters, while urea SC was approximately 1 at all flow rates. Hemodiafilter type and ultrafiltrate flow rate did not influence CLTM. Rezafungin adsorption to the CVVH apparatus was not observed for either hemodiafilter. CONCLUSION: Rezafungin is not removed by CVVH by membrane adsorption or via CLTM. Ultrafiltrate flow rates and hemodiafilter types are unlikely to influence rezafungin CLTM. No dosage adjustment of rezafungin is likely required for critically ill patients receiving CVVH.

CD101: a novel long-acting echinocandin.

CD101 is a novel echinocandin drug being developed to treat severe fungal infections including invasive candidiasis. We have performed a series of studies to evaluate the antifungal properties of CD101 against both echinocandin-susceptible and -resistant Candida strains. Antifungal susceptibility testing performed on a collection of 95 Candida strains including 30 caspofungin-resistant isolates containing fks mutations demonstrated comparable antifungal potency of CD101 relative to micafungin (MCF) across different Candida species. Comparable kinetic inhibition of glucan synthase activity was also observed for CD101 and MCF on both wild-type (WT) and resistant fks mutant Candida strains. Similarly, both drugs yielded nearly identical values for a mutant prevention concentration. In a murine model of invasive candidiasis, CD101 displayed better or at least comparable efficacy relative to MCF in treating WT or fks mutant Candida albicans. An exceptional long-lived pharmacokinetic profile was observed in mice following a single dose of CD101. Collectively, CD101 has great potential not only in treating invasive Candida infections but also in preventing emergence of resistance to currently approved echinocandin drugs.

Preclinical Evaluation of the Stability, Safety, and Efficacy of CD101, a Novel Echinocandin.

Fungal infections pose a significant public health burden with high morbidity and mortality. CD101 is a novel echinocandin under development for the treatment and prevention of systemic Candida infections. Preclinical studies were conducted to evaluate the metabolic stability, plasma protein binding, pharmacokinetics, toxicity, and efficacy of CD101 at various dose levels. CD101 was stable to biotransformation in rat, monkey, and human liver microsomes and rat, monkey, dog, and human hepatocytes. In vitro studies suggest minimal interaction with recombinant cytochrome P450 enzymes (50% inhibitory concentrations [IC50s] of >10 µM). Similar to anidulafungin, CD101 bound avidly (>98%) to human, mouse, rat, and primate plasma proteins. In a 2-week repeat-dose comparison study, CD101 was well tolerated in rats (no effects on body weight, hematology, coagulation, or urinalysis). In contrast, administration of anidulafungin (at comparable exposure levels) resulted in reduced body weight, decreases in red blood cell, hemoglobin, hematocrit, mean cell volume, mean corpuscular hemoglobin, platelet, and reticulocyte counts, increases in neutrophil and eosinophil counts, polychromasia, and decreased activated partial thromboplastin time. Elevated plasma transaminases, total bilirubin, cholesterol, and globulin, dark and enlarged spleens, and single-cell hepatocyte necrosis were also observed for anidulafungin but not CD101. Hepatotoxicity may be due to the inherent chemical lability of anidulafungin generating potentially reactive intermediates. A glutathione trapping experiment confirmed the formation of a reactive species from anidulafungin, whereas CD101 did not exhibit instability or reactive intermediates. CD101 showed antifungal activity against Candida and Aspergillus infections in neutropenic mice. These preclinical studies demonstrated that CD101 is chemically and metabolically stable, well tolerated with no hepatotoxicity, and efficacious as an antifungal agent.

Isolation of apoptosis-inducing stilbenoids from four members of the Orchidaceae family.

High-throughput natural product research produced a suite of anticancer hits among several species of the Orchidaceae family (Oncidium microchilum, O. isthmi, and Myrmecophila humboldtii). A commercial Oncidium sp. was also examined as a convenient source of additional material. Isolation and structure elucidation led to the identification of fifteen stilbenoids including a new phenanthraquinone and two new dihydrostilbenes. NMR data for structure elucidation and dereplication were acquired utilizing a Bruker BioSpin TCI 1.7-mm MicroCryoProbe or a 5-µL CapNMR capillary microcoil. Several compounds inhibited proliferation of NCI-H460 and M14 cancer cell lines. All compounds were also examined for their ability to induce apoptosis. Apoptosis induction was determined by measuring caspase 3/7 activation and LDH release in a NCI-H460 cell line. Based on these results, a portion of the extract from a commercially available Oncidium sp. was chemically modified in an attempt to obtain additional phenanthraquinones.

Dammarane-type triterpene glycosides from Oncoba manii active against methicillin-resistant Staphylococcus aureus.

Drug-resistant bacteria are becoming more prevalent both in the community and in hospitals. In a search for new antibiotic leads, we used a high-throughput natural products chemistry approach to isolate one new (1) and two known (2, 3) dammarane-type triterpenes with mass-limited material from the African plant Oncoba manii. The new compound was determined by spectroscopic methods to be 1beta,2alpha,3beta,20(R)-tetrahydroxydammar-24-ene 3-O-alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranoside. Compounds 1 and 2 inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA).

Structure-based design of new dihydrofolate reductase antibacterial agents: 7-(benzimidazol-1-yl)-2,4-diaminoquinazolines.

A new series of dihydrofolate reductase (DHFR) inhibitors, the 7-(benzimidazol-1-yl)-2,4-diaminoquinazolines, were designed and optimized for antibacterial potency and enzyme selectivity. The most potent inhibitors in this series contained a five-membered heterocycle at the 2-position of the benzimidazole, leading to highly potent and selective compounds that exploit the differences in the size of a binding pocket adjacent to the NADPH cofactor between the bacterial and human DHFR enzymes. Typical of these compounds is 7-((2-thiazol-2-yl)benzimidazol-1-yl)-2,4 diaminoquinazoline, which is a potent inhibitor of S. aureus DHFR (Ki = 0.002 nM) with 46700-fold selectivity over human DHFR. This compound also has high antibacterial potency on Gram-positive bacteria with an MIC versus wild type S. aureus of 0.0125 µg/mL and a MIC versus trimethoprim-resistant S. aureus of 0.25 µg/mL. In vivo efficacy versus a S. aureus septicemia was demonstrated, highlighting the potential of this new series.

Identification of bacteria-selective threonyl-tRNA synthetase substrate inhibitors by structure-based design.

A series of potent and bacteria-selective threonyl-tRNA synthetase (ThrRS) inhibitors have been identified using structure-based drug design. These compounds occupied the substrate binding site of ThrRS and showed excellent binding affinities for all of the bacterial orthologues tested. Some of the compounds displayed greatly improved bacterial selectivity. Key residues responsible for potency and bacteria/human ThrRS selectivity have been identified. Antimicrobial activity has been achieved against wild-type Haemophilus influenzae and efflux-deficient mutants of Escherichia coli and Burkholderia thailandensis.

Tricyclic GyrB/ParE (TriBE) inhibitors: a new class of broad-spectrum dual-targeting antibacterial agents.

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.

Antibacterial clerodane diterpenes from Goldenrod (Solidago virgaurea).

Nine clerodane diterpenes, solidagoic acids C-I (1-7), cleroda-3,13(14)-dien-16,15:18,19-diolide (8) and cleroda-3,13(14)-dien-15,16:18,19-diolide (9) were isolated and characterised from the ethanol-ethyl acetate (1:1) extract of Solidago virgaurea. The structures were determined by NMR spectroscopic analysis. Several displayed moderate antibacterial activity against Staphylococcus aureus.

Stilbenes from the Orchid Phragmipedium sp.

Three species of Phragmipedium (Orchidaceae), P. calurum, P. longifolium, and P. hybrid (var. Sorcerer's Apprentice), were analyzed by high-throughput isolation. A total of 12 new (1-4, 6-10, 12, 14, 16) and five known compounds (5, 11, 13, 15, 17) were isolated from these orchids. Compounds 1-8 are stilbenes substituted with one or two 4-hydroxybenzyl moieties. This type of substitution on stilbenes is unusual and has been reported only twice. The structure elucidation was based on spectroscopic analysis.

Anti-HCV bioactivity of pseudoguaianolides from Parthenium hispitum.

Five new (1-5) and four known (6-9) C14-oxygenated 1alpha-hydroxy-11(13)-pseudoguaien-6beta,12-olides with potent inhibition of hepatitis C virus (HCV) replication were obtained from Parthenium hispitum via high-throughput natural product chemistry methods. A semipreparative HPLC system was used to purify these compounds. The miniaturization of the structure elucidation and dereplication for the mass-limited samples were performed primarily utilizing a capillary-scale NMR probe. Compounds 2-4 were found to possess in vitro anti-HCV activity in the subgenomic HCV replicon system containing luciferase reporter with significant inhibition above 90% at 2 microM concentration.

Antibacterial, partially acetylated oligorhamnosides from Cleistopholis patens.

Three new (1-3) and five known (4-8) partially acetylated oligorhamnoside derivatives were obtained from Cleistopholis patens via high-throughput natural products chemistry procedures. The rapid structure elucidation and dereplication were performed primarily utilizing a capillary-scale NMR probe and LR-/HRESIMS spectroscopic methods. Compounds 1, 2, and 6 were found to possess significant in vitro antibacterial activity against the Gram-positive bacteria methicillin-resistant Staphylococcus aureus ATCC 33591 and S. aureus 78-13607A with MICs of < or =16 microg/mL. Furthermore, 2 and 6 were found to show significant in vitro antibacterial activity against an expanded panel of Gram-positive pathogens including either ATCC strains or well-characterized clinical isolates from the global SENTRY Antimicrobial Surveillance Program.

Bacterial biofilm inhibitors from Diospyros dendo.

One new (1) and four known (2-5) ursene triterpenes with potent inhibition of the formation of the bacterial biofilm Pseudomonas aeruginosa PA01 were obtained from Diospyros dendo using a high-throughput natural products chemistry procedure. These compounds were isolated as mass-limited samples. The miniaturization of the structure elucidation and dereplication was performed primarily utilizing a capillary-scale NMR probe.