Biology subjects

Lee, R. E.

Publications and source records attributed to Lee, R. E..

11 recordsLinked to original sources

bioRxiv2025

Inhibitors of trehalose-6-phosphate synthase activity in fungal pathogens compromise thermal tolerance pathways

Infections caused by fungal pathogens such as Candida and Cryptococcus are associated with high mortality rates, partly due to limitations in the current antifungal arsenal. This highlights the need for antifungal drug targets with novel mechanisms of action. The trehalose biosynthesis pathway is a promising antifungal drug target because trehalose biosynthesis is essential for virulence in Cryptococcus neoformans and Candida albicans and is also a mediator of fungal stress responses, such as thermotolerance. To exploit its untapped antifungal potentials, we screened the St. Jude 3-point pharmacophore library to identify small molecule inhibitors of the first enzyme in the trehalose biosynthesis pathway, trehalose-6-phosphate synthase (Tps1). Structure-guided optimization of a potent hit, SJ6675, yielded a water-soluble inhibitor named 4456dh. Employing biochemical, structural and cell-based assays, we demonstrate that 4456dh inhibits Tps1 enzymatic activity, suppresses trehalose synthesis and exerts a fungicidal effect. Notably, the structure of Tps1 in complex with 4456 reveals that 4456 occupies the substrate binding pocket. Importantly, 4456dh renders normally thermotolerant fungal pathogens unable to survive at elevated temperatures, which is critical as we investigate the emergence of fungi from the environment due to a warming climate. Overall, this work develops the water-soluble 4456dh as an early-stage antifungal drug that has a distinct mechanism of action compared to existing clinical antifungals. ImportanceThe rise of fungal infections in recent years is alarming due to an increase in the vulnerable immunocompromised population, global temperature increase and limited antifungal treatment options. One of the major hurdles in developing new drugs is the identification of fungal-specific antifungal drug targets due to highly conserved cellular machinery between fungi and humans. Here, we describe a small molecule inhibitor, 4456dh, of the trehalose biosynthesis pathway. This pathway is present in fungi but not humans. Trehalose plays a critical role in stress responses such as thermotolerance in fungal pathogens and is essential for their virulence. We show that treatment with 4456dh blocks production of trehalose and renders fungal cells inviable. Thus far, 4456dh is active against two fungal pathogens of critical importance suggesting a broad-spectrum activity.

microbiology↗

bioRxiv2024

Spectinamide MBX-4888A exhibits favorable lesion and tissue distribution and promotes treatment shortening in advanced murine models of tuberculosis

The spectinamides are novel, narrow-spectrum semisynthetic analogs of spectinomycin, modified to avoid intrinsic efflux by Mycobacterium tuberculosis. Spectinamides, including lead MBX-4888A (Lee-1810), exhibit promising therapeutic profiles in mice, as single drugs and as partner agents with other anti-tuberculosis antibiotics including rifampin and/or pyrazinamide. To demonstrate that this translates to more effective cure, we first confirmed the role of rifampin, with or without pyrazinamide, as essential to achieve effective bactericidal responses and sterilizing cure in the current standard of care regimen in chronically infected C3HeB/FeJ mice compared to BALB/c mice. Thus, demonstrating added value in testing clinically relevant regimens in murine models of increasing pathologic complexity. Next we show that MBX-4888A, given by injection with the front-line standard of care regimen, is treatment shortening in multiple murine tuberculosis infection models. The positive treatment responses to MBX-4888A combination therapy in multiple mouse models including mice exhibiting advanced pulmonary disease can be attributed to favorable distribution in tissues and lesions, retention in caseum, along with favorable effects with rifampin and pyrazinamide under conditions achieved in necrotic lesions. This study also provides an additional data point regarding the safety and tolerability of spectinamide MBX-4888A in long-term murine efficacy studies.

microbiology↗

bioRxiv2023

Reduced male fertility of an Antarctic mite following extreme heat stress could prompt localized population declines

Climate change is leading to substantial global thermal changes, which are particularly pronounced in polar regions. Few studies have examined the impact of heat stress on reproduction in Antarctic terrestrial arthropods, specifically how brief, extreme events may alter survival. We observed that sublethal heat stress reduces male fecundity in an Antarctic mite, yielding females that produced fewer viable eggs. Females and males collected from microhabitats with high temperatures showed a similar reduction in fertility. This impact is temporary, as indicated by recovery of male fecundity following return to cooler, stable conditions. The diminished fecundity is likely due to a drastic reduction in the expression of male-associated factors that occur in tandem with a substantial increase in the expression of heat shock proteins. Cross mating between mites from different sites confirmed that heat-exposed populations have impaired male fertility. However, the impact on fertility declines with time when the mites are allowed to recover under less stressful conditions, suggesting that the negative effects are transient. Modeling indicated that heat stress is likely to reduce population growth and that short bouts of non-lethal heat stress could have substantial effects on local populations of Antarctic arthropods.

physiology↗

bioRxiv2022

Viral Airway Injury Promotes Cell Engraftment in an In Vitro Model of Cystic Fibrosis Cell Therapy

Cell therapy is a potential treatment for cystic fibrosis (CF). However, cell engraftment into the airway epithelium is challenging. Here, we model cell engraftment in vitro using the air-liquid interface (ALI) culture system by injuring well-differentiated CF ALI cultures and delivering non-CF cells at the time of peak injury. Engraftment efficiency was quantified by measuring chimerism by droplet digital PCR and functional ion transport in Ussing chambers. Using this model, we found that human bronchial epithelial cells (HBECs) engraft more efficiently when they are cultured by conditionally reprogrammed cell (CRC) culture methods. Cell engraftment into the airway epithelium requires airway injury, but the extent of injury needed is unknown. We compared three injury models and determined that severe injury with partial epithelial denudation facilitates long-term cell engraftment and functional CFTR recovery up to 20% of wildtype function. The airway epithelium promptly regenerates in response to injury, creating competition for space and posing a barrier to effective engraftment. We examined competition dynamics by time-lapse confocal imaging and found that delivered cells accelerate airway regeneration by incorporating into the epithelium. Irradiating the repairing epithelium granted engrafting cells a competitive advantage by diminishing resident stem cell proliferation. Intentionally causing severe injury to the lungs of people with CF would be dangerous. However, naturally occurring events like viral infection can induce similar epithelial damage with patches of denuded epithelium. We found that viral preconditioning promoted effective engraftment of cells primed for viral resistance.

cell biology↗

bioRxiv2022

K978C CFTR restores essential epithelial function with greater efficiency than wildtype CFTR when expressed in CF airway cells.

Class Ia/b CFTR variants cause severe cystic fibrosis (CF) lung disease in ~10% of CF patients and are untreatable with small molecule pharmaceuticals. Genetic replacement strategies offer a potential cure for all patients but so far, have displayed limited efficiency in vivo. We hypothesised that increasing protein abundance and/or activity of introduced CFTRs would more effectively restore function to CF bronchial epithelial cells (CFBE) in the presence of CF sputum (CFS) than wildtype (WT)-CFTR. We investigated codon optimised CFTR (hCAI), increased open probability CFTR (K978C) and codon optimised plus K978C (h^K978C) as candidates for gene therapy. Transfection of HEK293T with hCAI and h^K978C produced ~10-fold more CFTR protein than WT or K978C CFTRs. hCAI and h^K978C also displayed ~4-fold greater anion transport than WT in a halide-sensitive YFP quenching assay. However, functionality of modified CFTR cDNAs expressed in CFBE were profoundly different. 10% transduction of CFBE with K978C, compared to 22% transduction with WT, restored Cl- transport to similar levels as that recorded from non-CF cells. K978C increased ASL height and pH more effectively than WT-CFTR, while hCAI and h^K978C had limited impact. Further investigation indicated that codon optimised CFTRs mis-localised in CFBE and compromised vectoral Cl- transport. These data provide further evidence that codon optimised CFTR cDNAs may be unsuitable for gene therapy practices that employ high activity promoters. However, increased activity CFTR cDNAs such as K978C, that potentially mimic the effect of potentiators, may provide more potent recovery of function than WT-CFTR cDNA in CF airways. Significance StatementCystic fibrosis (CF) disease is associated with genetic malfunction of the Cl- channel CFTR, leading to dehydration and decreased pH in the fluid lining the airways. Replacement of CFTR by gene therapy/gene editing offers potential therapeutic benefit but efficiency is poor. We show that gain of activity K978C CFTR under the control of a high activity promoter fully restored Cl- transport, hydration and pH to CF bronchial epithelial cells (CFBE) in the presence of CF sputum and more efficiently than wild type CFTR. Codon optimised forms of CFTR were much less effective and proteins were mis-localised/mis-processed in CFBE. Thus, K978C could offer improved therapeutic potential.

molecular biology↗

bioRxiv2022

A genome-wide atlas of antibiotic susceptibility targets and pathways to tolerance.

Detailed knowledge on how bacteria evade antibiotics and eventually develop resistance could open avenues for novel therapeutics and diagnostics. It is thereby key to develop a comprehensive genome-wide understanding of how bacteria process antibiotic stress, and how modulation of the involved processes affects their ability to overcome said stress. Here we undertake a comprehensive genetic analysis of how the major human pathogen Streptococcus pneumoniae responds to 20 antibiotics. We built a genome-wide atlas of drug susceptibility determinants and generate a genetic interaction network that connects cellular processes and genes of unknown function, which we show can be used as therapeutic targets. Pathway analysis reveals a genome-wide "tolerome", defined by cellular processes that can make a bacterium less susceptible, and often tolerant, in an antibiotic specific manner. Importantly, modulation of these processes confers fitness benefits during active infections under antibiotic selection. Moreover, screening of sequenced clinical isolates demonstrates that mutations in tolerome genes readily evolve and are frequently associated with resistant strains, indicating such mutations may be an important harbinger for the emergence of antibiotic resistance.

microbiology↗

bioRxiv2021

Robust W1282X-CFTR rescue by a small molecule GSPT1 degrader

With the approval of elexacaftor/tezacaftor/ivacaftor (trade name Trikafta), the vast majority of people with cystic fibrosis (CF) are eligible for CF transmembrane conductance regulator (CFTR) modulator therapy. Remaining individuals have premature termination codons or rare CFTR variants with limited treatment options. Although clinical modulator response can be reliably predicted using primary airway epithelial cells, primary cells carrying rare CFTR variants are scarce. To overcome this obstacle, these cells can be expanded by overexpression of mouse Bmi-1 and human TERT (hTERT). We therefore used this approach to develop two non-CF and three CF (F508del/F508del, F508del/S492F, W1282X/W1282X) nasal cell lines and two W1282X/W1282X bronchial cell lines. Bmi-1/hTERT cell lines recapitulated primary cell morphology and ion transport function. The F508del/F508del and F508del/S492F cell lines robustly responded to Trikafta, which was mirrored in the parent primary cells and the cell donors clinical response. CC-90009, a novel cereblon E3 ligase modulator targeting the GSPT1 protein, rescued ~20% of wildtype CFTR function in our panel of W1282X/W1282X cell lines and primary cells. Intriguingly, CC-90009 also diminished epithelial sodium channel function. These studies demonstrate that Bmi-1/hTERT cell lines faithfully mirror primary cell responses to CFTR modulators and illustrate novel therapeutic approaches for the W1282X CFTR variant.

pathology↗

bioRxiv2021

Rapid stress hardening in the Antarctic midge improves male fertility by increasing courtship success and preventing the decline of accessory gland proteins following cold exposure

Rapid hardening is a process that quickly improves an animals performance following exposure to a potentially damaging stress. Features of reproduction can be improved by rapid hardening, but little is known about how rapid hardening may contribute to physiological responses in the cold environment of Antarctica. In this study of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae), we examine how rapid hardening in response to dehydration (RDH) or cold (RCH) improves male pre- and post-copulatory function related to fertility when the insects are subsequently subjected to a damaging cold exposure. Neither RDH nor RCH improved survival in response to lethal cold stress, but male activity following sublethal cold exposure was enhanced. Both RCH and RDH improved mating success of males compared to those subjected directly to a sublethal bout of cold. Egg viability decreased following direct exposure to sublethal cold, but improved following RCH and RDH. Sublethal cold exposure reduced expression of four accessory gland proteins, while expression remained high in males exposed to RCH. Though rapid hardening may be cryptic in males, this study shows that it can be revealed by pre- and post-copulatory interactions with females.

physiology↗

bioRxiv2020

SARS-CoV-2 D614G Variant Exhibits Enhanced Replication ex vivo and Earlier Transmission in vivo

The D614G substitution in the S protein is most prevalent SARS-CoV-2 strain circulating globally, but its effects in viral pathogenesis and transmission remain unclear. We engineered SARS-CoV-2 variants harboring the D614G substitution with or without nanoluciferase. The D614G variant replicates more efficiency in primary human proximal airway epithelial cells and is more fit than wildtype (WT) virus in competition studies. With similar morphology to the WT virion, the D614G virus is also more sensitive to SARS-CoV-2 neutralizing antibodies. Infection of human ACE2 transgenic mice and Syrian hamsters with the WT or D614G viruses produced similar titers in respiratory tissue and pulmonary disease. However, the D614G variant exhibited significantly faster droplet transmission between hamsters than the WT virus, early after infection. Our study demonstrated the SARS-CoV2 D614G substitution enhances infectivity, replication fitness, and early transmission.

microbiology↗

bioRxiv2020

Brief exposure to a diverse range of environmental stress enhances stress tolerance in the polyextremophilic Antarctic midge, Belgica antarctica

Insects use rapid acclimation to enhance their tolerance of abiotic stresses within minutes to hours. These responses are critical adaptations for insects and other small ectotherms to tolerate drastic changes in temperature, hydration, or other factors that can fluctuate precipitously with ambient conditions or as a result of behavior. Rapid cold-hardening, where insects use brief exposure to modest chilling as a cue to enhance their cold tolerance, is the most thoroughly-studied of these responses and relatively little is known about rapid acclimation that is either triggered by or enhances tolerance of other abiotic stresses. Here, we used larvae of the Antarctic midge, Belgica antarctica, a polar extremophile that routinely experiences numerous stresses in nature, to investigate how 2 h exposure to modest environmental stresses affect stress tolerance in insects. Brief pretreatment by various stresses, including hyperosmotic challenge, hypoosmotic challenge, acidity, basicity, and UV irradiation enhanced stress tolerance in B. antarctica larvae relative to untreated controls. These results indicate that numerous environmental cues can trigger rapid acclimation in insects and that these responses can enhance tolerance of multiple stresses.

physiology↗

bioRxiv2019

Multi-level analysis of reproduction in an Antarctic midge identifies female and male accessory gland products that are altered by larval stress and impact progeny viability

The Antarctic midge, Belgica antarctica, is a wingless, non-biting midge endemic to Antarctica. Larval development requires at least two years, but adult life lasts only two weeks. The nonfeeding adults mate in swarms and females die shortly after oviposition. Eggs are suspended in a gel of unknown composition that is expressed from the female accessory gland. This project characterizes molecular mechanisms underlying reproduction in this midge by examining differential gene expression in whole males, females, and larvae, as well as in male and female accessory glands. Functional studies were used to assess the role of the gel encasing the eggs, as well as the impact of stress on reproductive biology. RNA-seq analyses revealed sex- and development-specific gene sets along with those associated with the accessory glands. Proteomic analyses were used to define the composition of the egg-containing gel, which is generated during multiple developmental stages and derived from both the accessory gland and other female organs. Functional studies indicate the gel provides a larval food source and thermal and dehydration buffer, all of which are critical for viability. Larval dehydration stress directly reduces production of storage proteins and key accessory gland components, a feature that impacts adult reproductive success. Modeling reveals that bouts of dehydration may significantly impact population growth. This work lays a foundation for further examination of reproduction in midges and provides new information related to general reproduction in dipterans. A key aspect is that reproduction and stress dynamics, currently understudied in polar organisms, are likely to prove critical for determining how climate change will alter survivability.

genomics↗