Benchmarking a highly selective USP30 inhibitor for enhancement of mitophagy and pexophagy

The deubiquitylase USP30 is an actionable target considered for treatment of conditions associated with defects in the PINK1-PRKN pathway leading to mitophagy. We provide a detailed cell biological characterization of a benzosulphonamide molecule, compound 39, that has previously been reported to inhibit USP30 in an in vitro enzymatic assay. The current compound offers increased selectivity over previously described inhibitors. It enhances mitophagy and generates a signature response for USP30 inhibition after mitochondrial depolarization. This includes enhancement of TOMM20 and SYNJ2BP ubiquitylation and phosphoubiquitin accumulation, alongside increased mitophagy. In dopaminergic neurons, generated from Parkinson disease patients carrying loss of function PRKN mutations, compound 39 could significantly restore mitophagy to a level approaching control values. USP30 is located on both mitochondria and peroxisomes and has also been linked to the PINK1-independent pexophagy pathway. Using a fluorescence reporter of pexophagy expressed in U2OS cells, we observe increased pexophagy upon application of compound 39 that recapitulates the previously described effect for USP30 depletion. This provides the first pharmacological intervention with a synthetic molecule to enhance peroxisome turnover.

Profiling of D-alanine production by the microbial isolates of rat gut microbiota

D-alanine (D-Ala) and several other D-amino acids (D-AAs), unusual amino acids present in mammals, act as hormones and neuromodulators in nervous and endocrine systems. Unlike the endogenously synthesized D-serine in animals, D-Ala may be from exogenous sources, e.g., diet and intestinal microorganisms. However, it is unclear if the capability to produce D-Ala and other D-AAs varies among different microbial strains in the gut. We isolated individual microorganisms of rat gut microbiota and profiled their D-AA secretion in vitro, focusing on D-Ala. Serial dilutions of intestinal content from adult male rats were plated on agar to obtain clonal cultures. Using MALDI-TOF MS for rapid strain typing, we identified 38 unique isolates, grouped into 11 species based on 16S rRNA gene sequences. We then used two-tier screening to profile bacterial D-AA secretion, combining a D-amino acid oxidase-based enzymatic assay for rapid assessment of overall D-AA amount, followed by chiral LC-MS/MS to quantify individual D-AAs, revealing 19 out of the 38 isolated strains as D-AA producers. LC-MS/MS analysis of the eight top D-AA producers showed high levels of D-Ala in all strains tested, with substantial inter- and intra-species variations. Though results from enzymatic assay and LC-MS/MS analysis aligned well, LC-MS/MS further revealed the existence of D-glutamate and D-aspartate, which are poor substrates for enzymatic assay. We observed large inter- and intra-species variation of D-AA secretion profiles from rat gut microbiome species, demonstrating the importance of chemical profiling of gut microbiota in addition to sequencing, furthering the idea that microbial metabolites modulate host physiology.

A Novel LC–MS/MS-Based Method for the Diagnosis of ADA2 Deficiency from Dried Plasma Spot

Adenosine Deaminase 2 Deficiency (DADA2) (OMIM: 607575) is a monogenic, autoinflammatory disease caused by the loss of functional homozygous or heterozygous mutations in the ADA 2 gene (previously CECR1, Cat Eye Syndrome Chromosome Region 1). A timely diagnosis is crucial to start Anti-TNF therapies that are efficacious in controlling the disease. The confirmation of DADA2 is based on DNA sequencing and enzymatic assay. It is, thus, very important to have robust and reliable assays that can be rapidly utilized in specialized laboratories that can centralize samples from other centers. In this paper, we show a novel enzymatic assay based on liquid chromatography-tandem mass spectrometry that allows the accurate determination of the ADA2 enzyme activity starting from very small amounts of plasma spotted on filter paper (dried plasma spot). The method allows significantly distinguishing healthy controls from affected patients and carriers and could be of help in implementing the diagnostic workflow of DADA2.

Enzymatic determination of D-alanine with L-alanine dehydrogenase and alanine racemase

An enzymatic assay system of D-Ala, which is reported to affect the taste, was constructed using alanine racemase and L-alanine dehydrogenase. D-Ala is converted to L-Ala by alanine racemase and then deaminated by L-alanine dehydrogenase with the reduction of NAD+ to NADH, which is determined with water-soluble tetrazolium. Using the assay system, the D-Ala contents of seven crustaceans were determined.

A design and optimization of a high throughput valve based microfluidic device for single cell compartmentalization and analysis

The need for high throughput single cell screening platforms has been increasing with advancements in genomics and proteomics to identify heterogeneity, unique cell subsets or super mutants from thousands of cells within a population. For real-time monitoring of enzyme kinetics and protein expression profiling, valve-based microfluidics or pneumatic valving that can compartmentalize single cells is advantageous by providing on-demand fluid exchange capability for several steps in assay protocol and on-chip culturing. However, this technique is throughput limited by the number of compartments in the array. Thus, one big challenge lies in increasing the number of microvalves to several thousand that can be actuated in the microfluidic device to confine enzymes and substrates in picoliter volumes. This work explores the design and optimizations done on a microfluidic platform to achieve high-throughput single cell compartmentalization as applied to single-cell enzymatic assay for protein expression quantification. Design modeling through COMSOL Multiphysics was utilized to determine the circular microvalve’s optimized parameters, which can close thousands of microchambers in an array at lower sealing pressure. Multiphysical modeling results demonstrated the relationships of geometry, valve dimensions, and sealing pressure, which were applied in the fabrication of a microfluidic device comprising of up to 5000 hydrodynamic traps and corresponding microvalves. Comparing the effects of geometry, actuation media and fabrication technique, a sealing pressure as low as 0.04 MPa was achieved. Applying to single cell enzymatic assay, variations in granzyme B activity in Jurkat and human PBMC cells were observed. Improvement in the microfluidic chip’s throughput is significant in single cell analysis applications, especially in drug discovery and treatment personalization.

Mangifera indica Extracts as Novel PKM2 Inhibitors for Treatment of Triple Negative Breast Cancer

Pyruvate kinase (PK), a key enzyme that determines glycolytic activity, has been known to support the metabolic phenotype of tumor cells, and specific pyruvate kinase isoform M2 (PKM2) has been reported to fulfill divergent biosynthetic and energetic requirements of cancerous cells. PKM2 is overexpressed in several cancer types and is an emerging drug target for cancer during recent years. Therefore, this study was carried out to identify PKM2 inhibitors from natural products for cancer treatment. Based on the objectives of this study, firstly, plant extract library was established. In order to purify protein for the establishment of enzymatic assay system, pET-28a-HmPKM2 plasmid was transformed to E. coli BL21 (DE3) cells for protein expression and purification. After the validation of enzymatic assay system, plant extract library was screened for the identification of inhibitors of PKM2 protein. Out of 51 plant extracts screened, four extracts Mangifera indica (leaf, seed, and bark) and Bombex ceiba bark extracts were found to be inhibitors of PKM2. In the current study, M. indica (leaf, seed, and bark) extracts were further evaluated dose dependently against PKM2. These extracts showed different degrees of concentration-dependent inhibition against PKM2 at 90-360 μg/ml concentrations. We have also investigated the anticancer potential of these extracts against MDA-MB231 cells and generated dose-response curves for the evaluation of IC50 values. M. indica (bark and seed) extracts significantly halted the growth of MDA-MB231 cells with IC50 values of 108 μg/ml and 33 μg/ml, respectively. Literature-based phytochemical analysis of M. indica was carried out, and M. indica-derived 94 compounds were docked against three binding sites of PKM2 for the identification of PKM2 inhibitors. The results of in silico based screening have unveiled various PKM2 modulators; however, further studies are recommended to validate their PKM2 inhibitory potential via in vitro biochemical assay. The results of this study provide novel findings for possible mechanism of action of M. indica (bark and seed) extracts against TNBC via PKM2 inhibition suggesting that M. indica might be of therapeutic interest for the treatment of TNBC.

Uji Aktivitas Inhibitor Tirosinase Ekstrak Kulit Buah Cokelat (Theobroma cacao L.) dan Formulasinya dalam Bentuk Sediaan Nanoemulsi

Limbah kulit buah cokelat diketahui mengandung berbagai senyawa aktif seperti polifenol dan flavonoid sehingga berpotensi memiliki aktivitas inhibitor tirosinase. Untuk meningkatkan kemampuan penetrasi ekstrak pada penghantaran topikal akan dikembangkan menjadi sediaan nanoemulsi. Tujuan dari penelitian ini untuk menguji aktivitas inhibitor tirosinase ekstrak kulit buah cokelat dan memformulasikannya menjadi sediaan nanoemulsi yang memiliki sifat fisik yang baik. Simplisia diekstraksi dengan metode maserasi menggunakan etanol 70% dan selanjutnya diuji aktivitas inhibitor tirosinasenya dengan metode dopakrom berbasis colorimetric enzymatic assay. Sediaan nanoemulsi ekstrak kulit buah cokelat dibuat dengan menggunakan minyak biji anggur, tween 80 sebagai surfaktan, dan gliserin sebagai kosurfaktan untuk selanjutnya dikarakterisasi secara fisik. Hasil uji menunjukkan ekstrak kulit buah cokelat memiliki aktivitas inhibitor tirosinase dengan nilai IC50 199,98 ppm. Sediaan nanoemulsi mengandung ekstrak kulit buah cokelat penampilan fisik yang jernih dan homogen, pH 6,21±0,02, viskositas 1070 ± 24,5 cps, sifat alir Newtonian, dengan ukuran globul 108 ±15 nm. Sediaan nanoemulsi memiliki stabilitas fisik yang baik berdasarkan uji sentrifugasi, heating cooling, dan freeze thaw. Disimpulkan bahwa ekstrak kulit buah cokelat terbukti memiliki aktivits inhibitor tyrosinase dan telah berhasil diformulasikan menjadi sediaan nanoemulsi dengan sifat fisik dan stabilitas yang baik.