A Novel Oral GyrB/ParE Dual Binding Inhibitor Effective against Multidrug-Resistant Neisseria gonorrhoeae and Other High-Threat Pathogens.

Drug-resistant Neisseria gonorrhoeae is a serious global health concern. New drugs are needed that can overcome existing drug resistance and limit the development of new resistances. Here, we describe the small molecule tricyclic pyrimidoindole JSF-2414 [8-(6-fluoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-4-yl)-2-oxa-8-azaspiro[4.5]decan-3-yl)methanol], which was developed to target both ATP-binding regions of DNA gyrase (GyrB) and topoisomerase (ParE). JSF-2414 displays potent activity against N. gonorrhoeae, including drug-resistant strains. A phosphate pro-drug, JSF-2659, was developed to facilitate oral dosing. In two different animal models of Neisseria gonorrhoeae vaginal infection, JSF-2659 was highly efficacious in reducing microbial burdens to the limit of detection. The parent molecule also showed potent in vitro activity against high-threat Gram-positive organisms, and JSF-2659 was shown in a deep tissue model of vancomycin-resistant Staphylococcus aureus (VRSA) and a model of Clostridioides difficile-induced colitis to be highly efficacious and protective. JSF-2659 is a novel preclinical drug candidate against high-threat multidrug resistant organisms with low potential to develop new resistance.

Antitubercular Triazines: Optimization and Intrabacterial Metabolism.

The triazine antitubercular JSF-2019 was of interest due to its in vitro efficacy and the nitro group shared with the clinically relevant delamanid and pretomanid. JSF-2019 undergoes activation requiring F420H2 and one or more nitroreductases in addition to Ddn. An intrabacterial drug metabolism (IBDM) platform was leveraged to demonstrate the system kinetics, evidencing formation of NO⋅ and a des-nitro metabolite. Structure-activity relationship studies focused on improving the solubility and mouse pharmacokinetic profile of JSF-2019 and culminated in JSF-2513, relying on the key introduction of a morpholine. Mechanistic studies with JSF-2019, JSF-2513, and other triazines stressed the significance of achieving potent in vitro efficacy via release of intrabacterial NO⋅ along with inhibition of InhA and, more generally, the FAS-II pathway. This study highlights the importance of probing IBDM and its potential to clarify mechanism of action, which in this case is a combination of NO⋅ release and InhA inhibition.

Discovery of camphor-derived pyrazolones as 11ß-hydroxysteroid dehydrogenase type 1 inhibitors.

Starting from screening hit, (4S,7R)-1,7,8,8-tetramethyl-2-phenyl-1,2,4,5,6,7-hexahydro-4,7-methano-indazol-3-one (7), we optimized the potency and pharmacokinetic properties. This led to the identification of compounds with good in vivo activity in a mouse pharmacodynamic model of inhibition of 11ßHSD1.

Heterobiaryl purine derivatives as potent antiproliferative agents: inhibitors of cyclin dependent kinases. Part II.

C-6 Biarylmethylamino purine derivatives of roscovitine (1) inhibit cyclin dependent kinases and demonstrate potent antiproliferative activity. Replacement of the aryl rings of the C-6 biarylmethylamino group with heterobiaryl rings has provided compounds with significantly improved activity. In particular, derivatives 18 g and 9 c demonstrated 1000-fold and 1250-fold improvements, respectively, in the growth inhibition of HeLa cells compared to roscovitine (1).

Noncell-autonomous photoreceptor degeneration in a zebrafish model of choroideremia.

Choroideremia is an X-linked hereditary retinal degeneration resulting from mutations in the Rab escort protein-1 (REP1). The Rep1 protein facilitates posttranslational modification of Rab proteins, which regulate intracellular trafficking in the retinal pigment epithelium (RPE) and photoreceptors and are likely involved in the removal of outer segment disk membranes by the RPE. A critical question for potential treatment of choroideremia is whether photoreceptor degeneration results from autonomous defects in opsin transport within the photoreceptor or as a nonautonomous and secondary consequence of RPE degeneration. To address this question, we have characterized the retinal pathology in zebrafish rep1 mutants, which carry a recessive nonsense mutation in the REP1 gene. Zebrafish rep1 mutants exhibit degeneration of the RPE and photoreceptors and complete loss of visual function as measured by electroretinograms. In the mutant RPE, photoreceptor outer segment material was not effectively eliminated, and large vacuoles were observed. However, opsin trafficking in photoreceptors occurred normally. Mosaic analysis revealed that photoreceptor degeneration was nonautonomous and required contact with the mutant RPE as mutant photoreceptors were rescued in wild-type hosts and wild-type photoreceptors degenerated in mutant hosts. We conclude that mutations in REP1 disrupt cellular processes in the RPE, which causes photoreceptor death as a secondary consequence. These results suggest that therapies that correct the RPE may successfully rescue photoreceptor loss in choroideremia.

Cone survival despite rod degeneration in XOPS-mCFP transgenic zebrafish.

PURPOSE: In animal models of retinitis pigmentosa, rod photoreceptor degeneration eventually leads to loss of cone photoreceptors. The purpose of this study was to characterize a transgenic model of rod degeneration in zebrafish. METHODS: Zebrafish transgenic for XOPS-mCFP, a membrane-targeted form of cyan fluorescent protein driven by the Xenopus rhodopsin promoter, were generated by plasmid injection. Immunohistochemistry was used to detect cell type, proliferation, and TUNEL markers in larval and adult retinas. Rod- and cone-specific transcripts were detected by RT-PCR. Visual responses in transgenic adults were measured by electroretinogram. RESULTS: The XOPS promoter directed specific expression of mCFP in rods by 55 hours post fertilization (hpf). Rods in XOPS-mCFP heterozygotes began dying at 3.5 days post fertilization (dpf) and were almost completely absent by 5 dpf. A few rods were observed at the retinal margin, and numerous immature rods were observed in the outer nuclear layer (ONL) of transgenic adults. Apoptosis was increased in the ONL of larval and adult transgenic animals, and an elevation of rod precursor proliferation in adults was observed. ERG analysis confirmed that rod responses were absent in this line. Cone morphology and electrophysiology appeared normal in transgenic animals up to 7 months of age. CONCLUSIONS: The XOPS-mCFP transgene causes selective degeneration of rods without secondary loss of cones in animals up to 7 months of age. This raises important questions about the significance of rod-cone interactions in zebrafish and their potential as a model of human inherited retinal degenerations.

Ultraviolet- and short-wavelength cone contributions alter the early components of the ERG of young zebrafish.

The electroretinogram (ERG) is a commonly used measure to examine retinal processing in both basic and clinical research. The purpose of this study was to determine the retinal mechanisms responsible for the developmental differences found in the zebrafish ERG waveform. The ERG of young zebrafish possesses a voltage-negative response to ultraviolet- and short-wavelength stimuli, but not to middle- and long-wavelength stimuli; the ERG of adult zebrafish does not possess this response component. ERGs were obtained from young zebrafish before and after the introduction of either aspartate, or a combination of APB (DL-2-amino-4-phosphonobutyric acid) and PDA (cis-2,3-piperidinedicarboxylic acid) in order to suppress the responses of various types of retinal neurons. Log irradiance versus response amplitude functions of the ERG response to 200-ms stimuli of various wavelengths at various times following stimulus onset (70 and 120 ms) was derived as well as spectral sensitivity. Aspartate eliminated all voltage-positive responses regardless of stimulus wavelength; irradiance-response functions following aspartate were similar to the early responses of young control fish to ultraviolet- and short-wavelength stimuli. APB + PDA produced similar but not identical results as aspartate, suggesting that the combination of these agents does not completely eliminate all post-receptoral contributions to the ERG. Spectral sensitivity functions derived from aspartate-exposed subjects at various time measurements were dominated by contributions from ultraviolet- and short-wavelength-sensitive cone types. These wavelength-dependent ERG responses are similar to those found in humans with enhanced S-cone syndrome. Finally, ERG waveform differences across stimulus wavelength suggest that the circuitry of ultraviolet- and short-wavelength cone types is different to that of middle- and long-wavelength cone types in young zebrafish.

Assessing appetitive choice discrimination learning in zebrafish.

Within the last decade, the zebrafish (Danio rerio) has emerged as an important vertebrate model in developmental biology and medicine for problems typically associated with humans. However, where behavioral assays are needed, the utility of the zebrafish model has been limited by the narrow range of procedures so far developed to investigate zebrafish learning. The purpose of this study was to further develop and test procedures to study appetitive choice discrimination learning in zebrafish. Zebrafish were conditioned to swim into one of three chambers for food reinforcement. The correct (S+) chamber on a trial was signaled by the presence of a light stimulus in the chamber; the two negative (S-) chambers were dark. Each of the 15 fish tested learned the discrimination to a criterion of 80% correct in both of two consecutive sessions. Tests for stimulus control showed that discriminative behavior was indeed under the control of the S+ discriminandum. These results were discussed in relation to the recent report of zebrafish discrimination learning in a two-alternative task, and the importance of examining individual zebrafish learning curves.

Ethanol exposure alters zebrafish development: a novel model of fetal alcohol syndrome.

Prenatal exposure to alcohol has been shown to produce the overt physical and behavioral symptoms known as fetal alcohol syndrome (FAS) in humans. Also, it is believed that low concentrations and/or short durations of alcohol exposure can produce more subtle effects. The purpose of this study was to investigate the effects of embryonic ethanol exposure on the zebrafish (Danio rerio) in order to determine whether this species is a viable animal model for studying FAS. Fertilized embryos were reared in varying concentrations of ethanol (1.5% and 2.9%) and exposure times (e.g., 0-8, 6-24, 12-24, and 48-72 h postfertilization; hpf); anatomical measures including eye diameter and heart rate were compared across groups. Results found that at the highest concentration of ethanol (2.9%), there were more abnormal physical distortions and significantly higher mortality rates than any other group. Embryos exposed to ethanol for a shorter duration period (0-8 hpf) at a concentration of 1.5% exhibited more subtle effects such as significantly smaller eye diameter and lower heart rate than controls. These results indicate that embryonic alcohol exposure affects external and internal physical development and that the severity of these effects is a function of both the amount of ethanol and the timing of ethanol exposure. Thus, the zebrafish represents a useful model for examining basic questions about the effects of embryonic exposure to ethanol on development.

Visual processing of the zebrafish optic tectum before and after optic nerve damage.

Although the zebrafish has become an important model in visual neuroscience, little has been done to examine the processing of its higher visual centers. The purpose of this work was twofold. The first purpose was to examine the physiology of the zebrafish retinotectal system and its relationship to retinal physiology. Spectral sensitivity functions were derived from visually evoked tectal responses and these functions were compared to the functions of electroretinogram (ERG) responses obtained using the same stimulus conditions. The second purpose was to examine the recovery of visual functioning of the tectum following optic nerve damage. The optic nerves of adult zebrafish were damaged (crushed), and tectal visual processing was assessed following damage. The results showed that the spectral sensitivity functions based on the On-responses of the tectum and ERG were qualitatively similar. The functions based on each response type received similar cone contributions including both nonopponent and opponent contributions. However, the spectral sensitivity functions based on the Off-responses of the tectum and ERG differed. The results also showed that the zebrafish visual system is capable of neural regeneration. By 90 days following an optic nerve crush, the spectral sensitivity function based on the tectal On-response was similar to functions obtained from normal zebrafish. Although the tectal Off-response did recover, the spectral sensitivity based on the Off-response was not the same as the function of normal zebrafish. These results support the notion that different levels of the visual system process information differently and that the zebrafish visual system, like those of other lower vertebrates, is capable of functional regeneration.

Effects of restricted spectral rearing on the development of zebrafish retinal physiology.

Research has shown that rearing in abnormal lighting environments affects both visual behavior and retinal physiology in zebrafish larvae. These studies, however, used only constant dark and constant white light as the experimental rearing conditions. This study assessed the effects of rearing larvae in restricted spectral lighting environments on zebrafish retinal physiology. Larvae were reared in one of seven different lighting environments: cyclic white light (control group), constant blue light, constant green light, constant orange light, cyclic blue light, cyclic green light, and cyclic orange light. Assessment of retinal physiology was done using the electroretinogram (ERG). Results showed that rearing larvae in constant light conditions caused deficits in sensitivity to ultraviolet- and short-wavelength stimuli, but had little effect on sensitivity to middle- and long-wavelength stimuli. Rearing larvae in cyclic light did not cause differences in sensitivity to middle- and long-wavelength stimuli, but did cause extreme deficits in sensitivity to ultraviolet- and short-wavelength stimuli in the cyclic green and orange light-rearing conditions. Sensitivity of the cyclic blue light-rearing group was similar to the control group to stimuli of all wavelengths. The results support the notion that the light-rearing environment impacts the development of the ultraviolet- and short-wavelength cone mechanisms but has little impact on the development of the middle- and long-wavelength cone mechanisms; these effects coincide with the development of the various cone types. This study supports the notion that the zebrafish is a viable model for studying the effects of the lighting environment on visual development.

Effects of embryonic exposure to ethanol on zebrafish visual function.

Across a variety of species, including humans, it has been shown that embryos exposed to ethanol display eye abnormalities as well as deficiencies in visual physiology and behavior. The purpose of this study was to examine the effects of embryonic exposure to ethanol on visual function in zebrafish. Visual function was assessed physiologically, via electroretinogram (ERG) recordings, and behaviorally, by measuring visual acuity with the optomotor response. Zebrafish larvae were exposed to 1.5% ethanol at various times during development, including the period of maximal eye development. The results show that ethanol effects on visual function were most pronounced when exposure occurred during eye development. ERG recordings from ethanol-exposed larvae differed from normal subjects both in shape of the response waveform and in visual thresholds under both light and dark adaptation; the differences were more pronounced under lower levels of adaptation. Also, ethanol-exposed larvae displayed lower visual acuity as determined from the optomotor response. These results indicate embryonic ethanol exposure affects visual function particularly when exposure occurs during eye development. In addition, these findings illustrate the usefulness of the zebrafish as a viable animal model for studying Fetal Alcohol Syndrome (FAS).

APB differentially affects the cone contributions to the zebrafish ERG.

APB (DL-2-amino-4-phosphonobutyric acid) has been found to affect the retinal processing of many vertebrate species as evidenced by the suppression of the b-wave component of the electroretinogram (ERG). The present study examined the effects of APB on the cone contributions to the ERG response of adult zebrafish (Danio rerio). ERG responses were obtained from light-adapted adult zebrafish following intravitreal injection of either saline alone or saline with various concentrations of APB ranging from 10 microm to 500 microM. Visual stimuli were 200-ms flashes of various wavelengths and irradiances. Spectral sensitivity functions were calculated from the irradiance versus response amplitude functions of the a-, b-, and d-wave components of the ERG response. Saline had no effects on the ERG response. However, APB had differential effects on the sensitivity of the b- and d-wave components. The effects of APB on the b-wave component were most apparent in the ultraviolet and short-wavelength portions (320-440 nm) of the spectral sensitivity function, although the b-wave was not completely eliminated at these wavelengths. APB-treated subjects were found to possess the same cone mechanisms (L-M and M-S) in the middle- and long-wavelength areas of the spectrum as saline injected subjects, although absolute sensitivity was lower for the APB-injected subjects. Spectral sensitivity based on the d-wave response was affected by APB but only in the short-wavelength region. All results appear to be independent of the APB dose. These results support the notion that glutamate receptors play a specific role in zebrafish visual processing. In addition, the effects of APB support recent anatomical evidence that the zebrafish retina may possess different types of glutamate receptors.