Targeting a microbiota Wolbachian aminoacyl-tRNA synthetase to block its pathogenic host.

The interplay between humans and their microbiome is crucial for various physiological processes, including nutrient absorption, immune defense, and maintaining homeostasis. Microbiome alterations can directly contribute to diseases or heighten their likelihood. This relationship extends beyond humans; microbiota play vital roles in other organisms, including eukaryotic pathogens causing severe diseases. Notably, Wolbachia, a bacterial microbiota, is essential for parasitic worms responsible for lymphatic filariasis and onchocerciasis, devastating human illnesses. Given the lack of rapid cures for these infections and the limitations of current treatments, new drugs are imperative. Here, we disrupt Wolbachia's symbiosis with pathogens using boron-based compounds targeting an unprecedented Wolbachia enzyme, leucyl-tRNA synthetase (LeuRS), effectively inhibiting its growth. Through a compound demonstrating anti-Wolbachia efficacy in infected cells, we use biophysical experiments and x-ray crystallography to elucidate the mechanism behind Wolbachia LeuRS inhibition. We reveal that these compounds form adenosine-based adducts inhibiting protein synthesis. Overall, our study underscores the potential of disrupting key microbiota to control infections.

Wolbachia depletion blocks transmission of lymphatic filariasis by preventing chitinase-dependent parasite exsheathment.

Lymphatic filariasis is a vector-borne neglected tropical disease prioritized for global elimination. The filarial nematodes that cause the disease host a symbiotic bacterium, Wolbachia, which has been targeted using antibiotics, leading to cessation of parasite embryogenesis, waning of circulating larvae (microfilariae [mf]), and gradual cure of adult infection. One of the benefits of the anti-Wolbachia mode of action is that it avoids the rapid killing of mf, which can drive inflammatory adverse events. However, mf depleted of Wolbachia persist for several months in circulation, and thus patients treated with antibiotics are assumed to remain at risk for transmitting infections. Here, we show that Wolbachia-depleted mf rapidly lose the capacity to develop in the mosquito vector through a defect in exsheathment and inability to migrate through the gut wall. Transcriptomic and Western blotting analyses demonstrate that chitinase, an enzyme essential for mf exsheathment, is down-regulated in Wolbachia-depleted mf and correlates with their inability to exsheath and escape the mosquito midgut. Supplementation of in vitro cultures of Wolbachia-depleted mf with chitinase enzymes restores their ability to exsheath to a similar level to that observed in untreated mf. Our findings elucidate a mechanism of rapid transmission-blocking activity of filariasis after depletion of Wolbachia and adds to the broad range of biological processes of filarial nematodes that are dependent on Wolbachia symbiosis.

Development of Pyrazolopyrimidine Anti-Wolbachia Agents for the Treatment of Filariasis.

Anti-Wolbachia therapy has been identified as a viable treatment for combating filarial diseases. Phenotypic screening revealed a series of pyrazolopyrimidine hits with potent anti-Wolbachia activity. This paper focuses on the exploration of the SAR for this chemotype, with improvement of metabolic stability and solubility profiles using medicinal chemistry approaches. Organic synthesis has enabled functionalization of the pyrazolopyrimidine core at multiple positions, generating a library of compounds of which many analogues possess nanomolar activity against Wolbachia in vitro with improved DMPK parameters. A lead compound, 15f, was selected for in vivo pharmacokinetics (PK) profiling in mice. The combination of potent anti-Wolbachia activity in two in vitro assessments plus the exceptional oral PK profiles in mice puts this lead compound in a strong position for in vivo proof-of-concept pharmacodynamics studies and demonstrates the strong potential for further optimization and development of this series for treatment of filariasis in the future.

Anti-Wolbachia drugs for filariasis.

The mutualistic association between Wolbachia endosymbionts and their filarial nematode hosts has been exploited as a validated drug target delivering macrofilaricidal outcomes. Limitations of existing antibiotics to scale-up have driven the search for new drugs, which are effective in shorter regimens of 7 days or less. Here, we review the last 14 years of anti-Wolbachia drug discovery by the anti-Wolbachia (A·WOL) consortium, which has screened more than two million compounds, delivering thousands of hit compounds. Refined screening models integrated with robust pharmacokinetic/pharmacodynamic (PK/PD) driven optimisation and selection strategies have delivered the first two drug candidates specifically designed to target Wolbachia. AWZ1066S and ABBV-4083 are currently progressing through clinical trials with the aim of delivering safe and effective macrofilaricides to support the elimination of onchocerciasis and lymphatic filariasis.

Validation of the Three-Item Screener for Personality Disorders From the Inventory of Interpersonal Problems (IIP-3).

We previously developed a three-item screener for identifying respondents with any personality disorder (PD) using the Inventory of Interpersonal Problems (IIP). The current goal was to examine the convergent validity of the IIP-3 with other PD screeners and diagnostic tools and to investigate its relationship to measures of adult attachment and emotion regulation. The sample consisted of participants from five studies (total N = 852), with data from collateral informants available for a subsample (N = 353). Despite its brevity, the IIP-3 showed moderate to strong relationships with other longer PD screeners, with PD symptom scores from the Structured Interview for DSM-IV Personality (SIDP-IV), and with a global rating of PD severity. It was most sensitive to the stylistic aspects of PD typical of the traditional DSM cluster B (dramatic, expressive) PDs. These results emerged with data from both participants and informants, although correlations using informant data were generally smaller.

Using formative evaluation methods to improve clinical implementation efforts: Description and an example.

Formative evaluation, a rigorous assessment process to identify potential and actual influences on the implementation process, is a necessary first step prior to launching any implementation effort. Without formative evaluation, intervention studies may fail to translate into meaningful patient care or public health outcomes or across different contexts. Formative evaluation usually consists of qualitative methods, but may involve quantitative or mixed methods. A unique aspect of formative evaluation is that data are shared with the implementation team during the study in order to adapt and improve the process of implementation during the course of the study or improvement activity. In implementation science, and specifically within formative evaluation, it is imperative that a theory or conceptual model or framework guide the selection of the various individual, organizational or contextual factors to be assessed. Data from these theory-based constructs can translate into the development and specification of implementation strategies to support the uptake of the intervention. In this article, we describe different types of formative evaluations (developmental, implementation-focused, progress-focused, and interpretive), and then present a formative evaluation case study from a real-world implementation study within several academic pain clinics, guided by the Theory of Diffusion of Innovation.

Development of a High-Throughput Cytometric Screen to Identify Anti- Wolbachia Compounds: The Power of Public-Private Partnership.

The Anti- Wolbachia (A·WOL) consortium at the Liverpool School of Tropical Medicine (LSTM) has partnered with the Global High-Throughput Screening (HTS) Centre at AstraZeneca to create the first anthelmintic HTS for neglected tropical diseases (NTDs). The A·WOL consortium aims to identify novel macrofilaricidal drugs targeting the essential bacterial symbiont ( Wolbachia) of the filarial nematodes causing onchocerciasis and lymphatic filariasis. Working in collaboration, we have validated a robust high-throughput assay capable of identifying compounds that selectively kill Wolbachia over the host insect cell. We describe the development and validation process of this complex, phenotypic high-throughput assay and provide an overview of the primary outputs from screening the AstraZeneca library of 1.3 million compounds.

Preclinical development of an oral anti-Wolbachia macrolide drug for the treatment of lymphatic filariasis and onchocerciasis.

There is an urgent global need for a safe macrofilaricide drug to accelerate elimination of the neglected tropical diseases onchocerciasis and lymphatic filariasis. From an anti-infective compound library, the macrolide veterinary antibiotic, tylosin A, was identified as a hit against Wolbachia This bacterial endosymbiont is required for filarial worm viability and fertility and is a validated target for macrofilaricidal drugs. Medicinal chemistry was undertaken to develop tylosin A analogs with improved oral bioavailability. Two analogs, A-1535469 and A-1574083, were selected. Their efficacy was tested against the gold-standard second-generation tetracycline antibiotics, doxycycline and minocycline, in mouse and gerbil infection models of lymphatic filariasis (Brugia malayi and Litomosoides sigmodontis) and onchocerciasis (Onchocerca ochengi). A 1- or 2-week course of oral A-1535469 or A-1574083 provided >90% Wolbachia depletion from nematodes in infected animals, resulting in a block in embryogenesis and depletion of microfilarial worm loads. The two analogs delivered comparative or superior efficacy compared to a 3- to 4-week course of doxycycline or minocycline. A-1574083 (now called ABBV-4083) was selected for further preclinical testing. Cardiovascular studies in dogs and toxicology studies in rats and dogs revealed no adverse effects at doses (50 mg/kg) that achieved plasma concentrations >10-fold above the efficacious concentration. A-1574083 (ABBV-4083) shows potential as an anti-Wolbachia macrolide with an efficacy, pharmacology, and safety profile that is compatible with a short-term oral drug course for treating lymphatic filariasis and onchocerciasis.

Discovery of ABBV-4083, a novel analog of Tylosin A that has potent anti-Wolbachia and anti-filarial activity.

There is a significant need for improved treatments for onchocerciasis and lymphatic filariasis, diseases caused by filarial worm infection. In particular, an agent able to selectively kill adult worms (macrofilaricide) would be expected to substantially augment the benefits of mass drug administration (MDA) with current microfilaricides, and to provide a solution to treatment of onchocerciasis / loiasis co-infection, where MDA is restricted. We have identified a novel macrofilaricidal agent, Tylosin A (TylA), which acts by targeting the worm-symbiont Wolbachia bacterium. Chemical modification of TylA leads to improvements in anti-Wolbachia activity and oral pharmacokinetic properties; an optimized analog (ABBV-4083) has been selected for clinical evaluation.

Boron-Pleuromutilins as Anti- Wolbachia Agents with Potential for Treatment of Onchocerciasis and Lymphatic Filariasis.

A series of pleuromutilins modified by introduction of a boron-containing heterocycle on C(14) of the polycyclic core are described. These analogs were found to be potent anti- Wolbachia antibiotics and, as such, may be useful in the treatment of filarial infections caused by Onchocerca volvulus, resulting in Onchocerciasis or river blindness, or Wuchereria bancrofti and Brugia malayi and related parasitic nematodes resulting in lymphatic filariasis. These two important neglected tropical diseases disproportionately impact patients in the developing world. The lead preclinical candidate compound containing 7-fluoro-6-oxybenzoxaborole (15, AN11251) was shown to have good in vitro anti- Wolbachia activity and physicochemical and pharmacokinetic properties providing high exposure in plasma. The lead was effective in reducing the Wolbachia load in filarial worms following oral administration to mice.

Industrial scale high-throughput screening delivers multiple fast acting macrofilaricides.

Nematodes causing lymphatic filariasis and onchocerciasis rely on their bacterial endosymbiont, Wolbachia, for survival and fecundity, making Wolbachia a promising therapeutic target. Here we perform a high-throughput screen of AstraZeneca's 1.3 million in-house compound library and identify 5 novel chemotypes with faster in vitro kill rates (<2 days) than existing anti-Wolbachia drugs that cure onchocerciasis and lymphatic filariasis. This industrial scale anthelmintic neglected tropical disease (NTD) screening campaign is the result of a partnership between the Anti-Wolbachia consortium (A∙WOL) and AstraZeneca. The campaign was informed throughout by rational prioritisation and triage of compounds using cheminformatics to balance chemical diversity and drug like properties reducing the chance of attrition from the outset. Ongoing development of these multiple chemotypes, all with superior time-kill kinetics than registered antibiotics with anti-Wolbachia activity, has the potential to improve upon the current therapeutic options and deliver improved, safer and more selective macrofilaricidal drugs.

Screening for Personality Disorders: A Three-Item Screener From the Inventory of Interpersonal Problems (IIP-3).

To encourage screening for personality disorders (PDs), we developed (in previous work) self-report scales for PDs using the Inventory of Interpersonal Problems (IIP). The combined score from three of the scales-inter-personal sensitivity, interpersonal ambivalence, and aggression-requiring 15 items (IIP-15) did the best job of distinguishing between respondents with any versus no PD. The goals for the present work were (a) to cross-validate the IIP-15 by examining its performance using receiver operating characteristics (ROC) analyses in a new sample (N = 410), and (b) to investigate the utility of a brief three-item variant (IIP-3). The present results again documented the good operating characteristics of the IIP-15. Sensitivity, specificity, and positive and negative predictive values were all above. 70. The operating characteristics of the IIP-3 were nearly as good despite its brevity and support its use as an initial screen for PDs.

AWZ1066S, a highly specific anti-Wolbachia drug candidate for a short-course treatment of filariasis.

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect ∼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis.

Intermittent v. continuous energy restriction: differential effects on postprandial glucose and lipid metabolism following matched weight loss in overweight/obese participants.

The intermittent energy restriction (IER) approach to weight loss involves short periods of substantial (>70 %) energy restriction (ER) interspersed with normal eating. Studies to date comparing IER to continuous energy restriction (CER) have predominantly measured fasting indices of cardiometabolic risk. This study aimed to compare the effects of IER and CER on postprandial glucose and lipid metabolism following matched weight loss. In all, twenty-seven (thirteen male) overweight/obese participants (46 (sem 3) years, 30·1 (sem 1·0) kg/m2) who were randomised to either an IER intervention (2638 kJ for 2 d/week with an overall ER of 22 (sem 0·3) %, n 15) or a CER intervention (2510 kJ below requirements with overall ER of 23 (sem 0·8) %) completed the study. Postprandial responses to a test meal (over 360 min) and changes in anthropometry (fat mass, fat-free mass, circumferences) were assessed at baseline and upon attainment of 5 % weight loss, following a 7-d period of weight stabilisation. The study found no statistically significant difference in the time to attain a 5 % weight loss between groups (median 59 d (interquartile range (IQR) 41-80) and 73 d (IQR 48-128), respectively, P=0·246), or in body composition (P≥0·437). For postprandial measures, neither diet significantly altered glycaemia (P=0·266), whereas insulinaemia was reduced comparatively (P=0·903). The reduction in C-peptide tended (P=0·057) to be greater following IER (309 128 (sem23 268) to 247781 (sem20 709) pmol×360 min/l) v. CER (297 204 (sem25 112) to 301 655 (sem32 714) pmol×360 min/l). The relative reduction in TAG responses was greater (P=0·045) following IER (106 (sem30) to 68 (sem 15) mmol×360 min/l) compared with CER (117 (sem 43) to 130 (sem 31) mmol×360 min/l). In conclusion, these preliminary findings highlight underlying differences between IER and CER, including a superiority of IER in reducing postprandial lipaemia, which now warrant targeted mechanistic evaluation within larger study cohorts.

Corrigendum: Minocycline as a re-purposed anti-Wolbachia macrofilaricide: superiority compared with doxycycline regimens in a murine infection model of human lymphatic filariasis.

This corrects the article DOI: 10.1038/srep23458.

Author Correction: Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Albendazole and antibiotics synergize to deliver short-course anti-Wolbachia curative treatments in preclinical models of filariasis.

Elimination of filariasis requires a macrofilaricide treatment that can be delivered within a 7-day period. Here we have identified a synergy between the anthelmintic albendazole (ABZ) and drugs depleting the filarial endosymbiont Wolbachia, a proven macrofilaricide target, which reduces treatment from several weeks to 7 days in preclinical models. ABZ had negligible effects on Wolbachia but synergized with minocycline or rifampicin (RIF) to deplete symbionts, block embryogenesis, and stop microfilariae production. Greater than 99% Wolbachia depletion following 7-day combination of RIF+ABZ also led to accelerated macrofilaricidal activity. Thus, we provide preclinical proof-of-concept of treatment shortening using antibiotic+ABZ combinations to deliver anti-Wolbachia sterilizing and macrofilaricidal effects. Our data are of immediate public health importance as RIF+ABZ are registered drugs and thus immediately implementable to deliver a 1-wk macrofilaricide. They also suggest that novel, more potent anti-Wolbachia drugs under development may be capable of delivering further treatment shortening, to days rather than weeks, if combined with benzimidazoles.

Identification and prioritization of novel anti-Wolbachia chemotypes from screening a 10,000-compound diversity library.

Lymphatic filariasis and onchocerciasis are two important neglected tropical diseases (NTDs) that cause severe disability. Control efforts are hindered by the lack of a safe macrofilaricidal drug. Targeting the Wolbachia bacterial endosymbionts in these parasites with doxycycline leads to a macrofilaricidal outcome, but protracted treatment regimens and contraindications restrict its widespread implementation. The Anti-Wolbachia consortium aims to develop improved anti-Wolbachia drugs to overcome these barriers. We describe the first screening of a large, diverse compound library against Wolbachia. This whole-organism screen, streamlined to reduce bottlenecks, produced a hit rate of 0.5%. Chemoinformatic analysis of the top 50 hits led to the identification of six structurally diverse chemotypes, the disclosure of which could offer interesting avenues of investigation to other researchers active in this field. An example of hit-to-lead optimization is described to further demonstrate the potential of developing these high-quality hit series as safe, efficacious, and selective anti-Wolbachia macrofilaricides.

An Item Bank for Abuse of Prescription Pain Medication from the Patient-Reported Outcomes Measurement Information System (PROMIS®).

OBJECTIVE: There is a need to monitor patients receiving prescription opioids to detect possible signs of abuse. To address this need, we developed and calibrated an item bank for severity of abuse of prescription pain medication as part of the Patient-Reported Outcomes Measurement Information System (PROMIS ® ). METHODS: Comprehensive literature searches yielded an initial bank of 5,310 items relevant to substance use and abuse, including abuse of prescription pain medication, from over 80 unique instruments. After qualitative item analysis (i.e., focus groups, cognitive interviewing, expert review, and item revision), 25 items for abuse of prescribed pain medication were included in field testing. Items were written in a first-person, past-tense format, with a three-month time frame and five response options reflecting frequency or severity. The calibration sample included 448 respondents, 367 from the general population (ascertained through an internet panel) and 81 from community treatment programs participating in the National Drug Abuse Treatment Clinical Trials Network. RESULTS: A final bank of 22 items was calibrated using the two-parameter graded response model from item response theory. A seven-item static short form was also developed. The test information curve showed that the PROMIS ® item bank for abuse of prescription pain medication provided substantial information in a broad range of severity. CONCLUSION: The initial psychometric characteristics of the item bank support its use as a computerized adaptive test or short form, with either version providing a brief, precise, and efficient measure relevant to both clinical and community samples.

Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis.

Lymphatic filariasis (LF) and onchocerciasis are priority neglected tropical diseases targeted for elimination. The only safe drug treatment with substantial curative activity against the filarial nematodes responsible for LF (Brugia malayi, Wuchereria bancrofti) or onchocerciasis (Onchocerca volvulus) is doxycycline. The target of doxycycline is the essential endosymbiont, Wolbachia. Four to six weeks doxycycline therapy achieves >90% depletion of Wolbachia in worm tissues leading to blockade of embryogenesis, adult sterility and premature death 18-24 months post-treatment. Long treatment length and contraindications in children and pregnancy are obstacles to implementing doxycycline as a public health strategy. Here we determine, via preclinical infection models of Brugia malayi or Onchocerca ochengi that elevated exposures of orally-administered rifampicin can lead to Wolbachia depletions from filariae more rapidly than those achieved by doxycycline. Dose escalation of rifampicin achieves >90% Wolbachia depletion in time periods of 7 days in B. malayi and 14 days in O. ochengi. Using pharmacokinetic-pharmacodynamic modelling and mouse-human bridging analysis, we conclude that clinically relevant dose elevations of rifampicin, which have recently been determined as safe in humans, could be administered as short courses to filariasis target populations with potential to reduce anti-Wolbachia curative therapy times to between one and two weeks.