Molecular determinants of phospholipid treatment to reduce intracellular cholesterol accumulation in NPC1 deficiency.

Niemann-Pick type C (NPC) disease, caused by mutations in the NPC1 or NPC2 genes, leads to abnormal intracellular cholesterol accumulation in late endosomes/lysosomes (LE/LY). Exogenous enrichment with lysobisphosphatidic acid (LBPA), also known as bis-monoacylglycerol phosphate or BMP, either directly or via the LBPA precursor phosphatidylglycerol (PG), has been investigated as a therapeutic intervention to reduce cholesterol accumulation in NPC disease. Here we report the effects of stereoisomer configuration and acyl chain composition of LBPA on cholesterol clearance in NPC1-deficient cells. We find that S,R, S,S, and S,R LBPA stereoisomers behaved similarly, with all 3 compounds leading to comparable reductions in filipin staining in two NPC1-deficient human fibroblast cell lines. Examination of several LBPA molecular species containing one or two mono- or polyunsaturated acyl chains showed that all LBPA species containing one 18:1 chain significantly reduced cholesterol accumulation, whereas the shorter chain species di-14:0 LBPA had little effect on cholesterol clearance in NPC1 deficient cells. Since cholesterol accumulation in NPC1 deficient cells can also be cleared by PG incubation, we used non-hydrolyzable PG analogues to determine whether conversion to LBPA is required for sterol clearance, or whether PG itself is effective. The results showed that non-hydrolyzable PG species were not appreciably converted to LBPA and showed virtually no cholesterol clearance efficacy in NPC1 deficient cells, supporting the notion that LBPA is the active agent promoting LE/LY cholesterol clearance. Overall these studies are helping to define the molecular requirements for potential therapeutic use of LBPA as an option for addressing NPC disease.

The "Doorstop Pocket" In Thioredoxin Reductases─An Unexpected Druggable Regulator of the Catalytic Machinery.

Pyridine nucleotide-disulfide oxidoreductases are underexplored as drug targets, and thioredoxin reductases (TrxRs) stand out as compelling pharmacological targets. Selective TrxR inhibition is challenging primarily due to the reliance on covalent inhibition strategies. Recent studies identified a regulatory and druggable pocket in Schistosoma mansoni thioredoxin glutathione reductase (TGR), a TrxR-like enzyme, and an established drug target for schistosomiasis. This site is termed the "doorstop pocket" because compounds that bind there impede the movement of an aromatic side-chain necessary for the entry and exit of NADPH and NADP+ during enzymatic turnover. This discovery spearheaded the development of new TGR inhibitors with efficacies surpassing those of current schistosomiasis treatment. Targeting the "doorstop pocket" is a promising strategy, as the pocket is present in all members of the pyridine nucleotide-disulfide oxidoreductase family, opening new avenues for exploring therapeutic approaches in diseases where the importance of these enzymes is established, including cancer and inflammatory and infectious diseases.

Non-covalent inhibitors of thioredoxin glutathione reductase with schistosomicidal activity in vivo.

Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target. TGR inhibitors identified to date are irreversible and/or covalent inhibitors with unacceptable off-target effects. In this work, we identify noncovalent TGR inhibitors with efficacy against schistosome infections in mice, meeting the criteria for lead progression indicated by WHO. Comparisons with previous in vivo studies with praziquantel suggests that these inhibitors outperform the drug of choice for schistosomiasis against juvenile worms.

Neuronal complexity is attenuated in preclinical models of migraine and restored by HDAC6 inhibition.

Migraine is the sixth most prevalent disease worldwide but the mechanisms that underlie migraine chronicity are poorly understood. Cytoskeletal flexibility is fundamental to neuronal-plasticity and is dependent on dynamic microtubules. Histone-deacetylase-6 (HDAC6) decreases microtubule dynamics by deacetylating its primary substrate, α-tubulin. We use validated mouse models of migraine to show that HDAC6-inhibition is a promising migraine treatment and reveal an undiscovered cytoarchitectural basis for migraine chronicity. The human migraine trigger, nitroglycerin, produced chronic migraine-associated pain and decreased neurite growth in headache-processing regions, which were reversed by HDAC6 inhibition. Cortical spreading depression (CSD), a physiological correlate of migraine aura, also decreased cortical neurite growth, while HDAC6-inhibitor restored neuronal complexity and decreased CSD. Importantly, a calcitonin gene-related peptide receptor antagonist also restored blunted neuronal complexity induced by nitroglycerin. Our results demonstrate that disruptions in neuronal cytoarchitecture are a feature of chronic migraine, and effective migraine therapies might include agents that restore microtubule/neuronal plasticity.

Migraines are a common brain disorder that affects 14% of the world's population. For many people the main symptom of a migraine is a painful headache, often on one side of the head. Other symptoms include increased sensitivity to light or sound, disturbed vision, and feeling sick. These sensory disturbances are called aura and they often occur before the headache begins. One particularly debilitating subset of migraines are chronic migraines, in which patients experience more than 15 headache days per month. Migraine therapies are often only partially effective or poorly tolerated, making it important to develop new drugs for this condition, but unfortunately, little is known about the molecular causes of migraines. To bridge this gap, Bertels et al. used two different approaches to cause migraine-like symptoms in mice. One approach consisted on giving mice nitroglycerin, which dilates blood vessels, produces hypersensitivity to touch, and causes photophobia in both humans and mice. In the second approach, mice underwent surgery and potassium chloride was applied onto the dura, a thick membrane that surrounds the brain. This produces cortical spreading depression, an event that is linked to migraine auras and involves a wave of electric changes in brain cells that slowly propagates across the brain, silencing brain electrical activity for several minutes. Using these approaches, Bertels et al. studied whether causing chronic migraine-like symptoms in mice is associated with changes in the structures of neurons, focusing on the effects of migraines on microtubules. Microtubules are cylindrical protein structures formed by the assembly of smaller protein units. In most cells, microtubules assemble and disassemble depending on what the cell needs. Neurons need stable microtubules to establish connections with other neurons. The experiments showed that provoking chronic migraines in mice led to a reduction in the numbers of connections between different neurons. Additionally, Bertels et al. found that inhibiting HDAC6 (a protein that destabilizes microtubules) reverses the structural changes in neurons caused by migraines and decreases migraine symptoms. The same effects are seen when a known migraine treatment strategy, known as CGRP receptor blockade, is applied. These results suggest that chronic migraines may involve decreased neural complexity, and that the restoration of this complexity by HDAC6 inhibitors could be a potential therapeutic strategy for migraine.

Detection of Ovarian Cancer Using Samples Sourced from the Vaginal Microenvironment.

Mass spectrometry (MS) offers high levels of specificity and sensitivity in clinical applications, and we have previously been able to demonstrate that matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS is capable of distinguishing two-component cell mixtures at low limits of detection. Ovarian cancer is notoriously difficult to detect due to the lack of diagnostic techniques available to the medical community. By sampling a local microenvironment, such as the vaginal canal and cervix, a MS based method is presented for monitoring disease progression from proximal samples to the diseased tissue. A murine xenograft model of high grade serous ovarian carcinoma (HGSOC) was used for this study, and vaginal lavages were obtained from mice on a weekly basis throughout disease progression and subjected to our MALDI-TOF MS workflow followed by statistical analyses. Proteins in the 4-20 kDa region of the mass spectrum yielded a fingerprint that we could consistently measure over time that correlated with disease progression. These fingerprints were found to be largely stable across all mice, with the protein fingerprint converging toward the end point of the study. MALDI-TOF MS serves as a unique analytical technique for measuring a sampled vaginal microenvironment in a specific and sensitive manner for the detection of HGSOC in a murine model.

Ectopic suicide inhibition of thioredoxin glutathione reductase.

Selective suicide inhibitors represent a seductively attractive approach for inactivation of therapeutically relevant enzymes since they are generally devoid of off-target toxicity in vivo. While most suicide inhibitors are converted to reactive species at enzyme active sites, theoretically bioactivation can also occur in ectopic (secondary) sites that have no known function. Here, we report an example of such an "ectopic suicide inhibition", an unprecedented bioactivation mechanism of a suicide inhibitor carried out by a non-catalytic site of thioredoxin glutathione reductase (TGR). TGR is a promising drug target to treat schistosomiasis, a devastating human parasitic disease. Utilizing hits selected from a high throughput screening campaign, time-resolved X-ray crystallography, molecular dynamics, mass spectrometry, molecular modeling, protein mutagenesis and functional studies, we find that 2-naphtholmethylamino derivatives bound to this novel ectopic site of Schistosoma mansoni (Sm)TGR are transformed to covalent modifiers and react with its mobile selenocysteine-containing C-terminal arm. In particular, one 2-naphtholmethylamino compound is able to specifically induce the pro-oxidant activity in the inhibited enzyme. Since some 2-naphtholmethylamino analogues show worm killing activity and the ectopic site is not conserved in human orthologues, a general approach to development of novel and selective anti-parasitic therapeutics against schistosoma is proposed.

Whole Cell MALDI Fingerprinting Is a Robust Tool for Differential Profiling of Two-Component Mammalian Cell Mixtures.

MALDI fingerprinting was first described two decades ago as a technique to identify microbial cell lines. Microbial fingerprinting has since evolved into an automated platform for microorganism identification and classification, which is now routinely used in clinical and environmental sectors. The extension of fingerprinting to mammalian cells has yet to progress partly due to compartmentalization of eukaryotic cells and overall higher cellular complexity. A number of publications on mammalian whole cell fingerprinting suggest that the method could be useful for classification of different cell types, cell states, and monitoring cell differentiation. We report the optimization of MALDI fingerprinting workflow parameters for mammalian cells and its application for differential profiling of mammalian cell lines and two-component cell line mixtures. Murine fallopian tube cells and high-grade ovarian carcinoma cell lines and their mixtures are used as model mammalian cell lines. Two-component cell mixtures serve to determine the method's feasibility for complex biological samples as the ability to detect cancer cells in a mixed cell population. The level of detection of cancer cells in the two-component mixture by principle component analysis (PCA) starts to deteriorate at 5% but with application of a different statistical approach, Wilcoxon rank sum test, the level of detection was determined to be 1%. The ability to differentiate heterogeneous cell mixtures will help further extend whole cell MALDI fingerprinting to complex biological systems. Graphical Abstract.

A novel indigoid anti-tuberculosis agent.

The structure of a novel indigoid component was characterized by X-ray crystallography. This compound exhibited excellent anti-tuberculosis activity against Mycobacterium tuberculosis H37Rv in whole cell culture showing a submicromolar minimum inhibitory concentration (MIC). A synthesis of this molecule was designed and carried out to produce sufficient material for further testing. The in vitro profile, structure, and first synthesis of this indigoid component is reported.

Structure-activity relationships of macrolides against Mycobacterium tuberculosis.

Existing 14, 15 and 16-membered macrolide antibiotics, while effective for other bacterial infections, including some mycobacteria, have not demonstrated significant efficacy in tuberculosis. Therefore an attempt was made to optimize this class for activity against Mycobacterium tuberculosis through semisyntheses and bioassay. Approximately 300 macrolides were synthesized and screened for anti-TB activity. Structural modifications on erythromycin were carried out at positions 3, 6, 9, 11, and 12 of the 14-membered lactone ring; as well as at position 4'' of cladinose and position 2' of desosamine. In general, the synthesized macrolides belong to four subclasses: 9-oxime, 11,12-carbamate, 11,12-carbazate, and 6-O-substituted derivatives. Selected compounds were assessed for mammalian cell toxicity and in some cases were further assessed for CYP3A4 inhibition, microsome stability, in vivo tolerance and efficacy. The activity of 11,12-carbamates and carbazates as well as 9-oximes is highly influenced by the nature of the substitution at these positions. For hydrophilic macrolides, lipophilic substitution may result in enhanced potency, presumably by enhanced passive permeation through the cell envelope. This strategy, however, has limitations. Removal of the C-3 cladinose generally reduces the activity. Acetylation at C-2' or 4'' maintains potency of C-9 oximes but dramatically decreases that of 11,12-substituted compounds. Further significant increases in the potency of macrolides for M. tuberculosis may require a strategy for the concurrent reduction of ribosome methylation.