The use of beta-D-glucanase as a substitute for Novozyme 234 in immunofluorescence and protoplasting

Novozym 234 has been used for many years to prepare protoplasts of Aspergillus nidulans and other fungi for transformation. It has also been very useful in immunofluorescence studies for partially digesting walls of fixed hyphae or germlings to allow antibodies to penetrate into the cytoplasm. In recent years, the availability of Novozym 234 has become problematic, and we have searched for combinations of available enzymes that are suitable for protoplasting and immunofluorescence studies in A. nidulans

MCAK associates with the tips of polymerizing microtubules.

International audienceMCAK is a member of the kinesin-13 family of microtubule (MT)-depolymerizing kinesins. We show that the potent MT depolymerizer MCAK tracks (treadmills) with the tips of polymerizing MTs in living cells. Tip tracking of MCAK is inhibited by phosphorylation and is dependent on the extreme COOH-terminal tail of MCAK. Tip tracking is not essential for MCAK's MT-depolymerizing activity. We propose that tip tracking is a mechanism by which MCAK is preferentially localized to regions of the cell that modulate the plus ends of MTs

A highly potent anti-VISTA antibody KVA12123 - a new immune checkpoint inhibitor and a promising therapy against poorly immunogenic tumors

BackgroundImmune checkpoint therapies have led to significant breakthroughs in cancer patient treatment in recent years. However, their efficiency is variable, and resistance to immunotherapies is common. VISTA is an immune-suppressive checkpoint inhibitor of T cell response belonging to the B7 family and a promising novel therapeutic target. VISTA is expressed in the immuno-suppressive tumor microenvironment, primarily by myeloid lineage cells, and its genetic knockout or antibody blockade restores an efficient antitumor immune response.MethodsFully human monoclonal antibodies directed against VISTA were produced after immunizing humanized Trianni mice and sorting and sequencing natively-linked B cell scFv repertoires. Anti-VISTA antibodies were evaluated for specificity, cross-reactivity, monocyte and T cell activation, Fc-effector functions, and antitumor efficacy using in vitro and in vivo models to select the KVA12123 antibody lead candidate. The pharmacokinetics and safety profiles of KVA12123 were evaluated in cynomolgus monkeys.ResultsHere, we report the development of a clinical candidate anti-VISTA monoclonal antibody, KVA12123. KVA12123 showed high affinity binding to VISTA through a unique epitope distinct from other clinical-stage anti-VISTA monoclonal antibodies. This clinical candidate demonstrated high specificity against VISTA with no cross-reactivity detected against other members of the B7 family. KVA12123 blocked VISTA binding to its binding partners. KVA12123 induced T cell activation and demonstrated NK-mediated monocyte activation. KVA12123 treatment mediated strong single-agent antitumor activity in several syngeneic tumor models and showed enhanced efficacy in combination with anti-PD-1 treatment. This clinical candidate was engineered to improve its pharmacokinetic characteristics and reduce Fc-effector functions. It was well-tolerated in preclinical toxicology studies in cynomolgus monkeys, where hematology, clinical chemistry evaluations, and clinical observations revealed no indicators of toxicity. No cytokines associated with cytokine release syndrome were elevated.ConclusionThese results establish that KVA12123 is a promising drug candidate with a distinct but complementary mechanism of action of the first generation of immune checkpoint inhibitors. This antibody is currently evaluated alone and in combination with pembrolizumab in a Phase 1/2 open-label clinical trial in patients with advanced solid tumors

Dual-targeting GroEL/ES chaperonin and protein tyrosine phosphatase B (PtpB) inhibitors: A polypharmacology strategy for treating Mycobacterium tuberculosis infections

Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis – actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease – which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria

Novel Screen to Assess Bactericidal Activity of Compounds Against Non-replicating Mycobacterium abscessus

Mycobacterium abscessus infections are increasing worldwide. Current drug regimens are largely ineffective, yet the current development pipeline for M. abscessus is alarmingly sparse. Traditional discovery efforts for M. abscessus assess the capability of a new drug to inhibit bacterial growth under nutrient-rich growth conditions, but this does not predict the impact when used in the clinic. The disconnect between in vitro and in vivo activity is likely due to the genetic and physiological adaptation of the bacteria to the environmental conditions encountered during infection; these include low oxygen tension and nutrient starvation. We sought to fill a gap in the drug discovery pipeline by establishing an assay to identify novel compounds with bactericidal activity against M. abscessus under non-replicating conditions. We developed and validated a novel screen using nutrient starvation to generate a non-replicating state. We used alamarBlue® to measure metabolic activity and demonstrated this correlates with bacterial viability under these conditions. We optimized key parameters and demonstrated reproducibility. Using this assay, we determined that niclosamide was bactericidal against non-replicating bacilli, highlighting its potential to be included in M. abscessus regimens. In contrast, most other drugs currently used in the clinic for M. abscessus infections, were completely inactive, potentially explaining their poor efficacy. Thus, our assay allows for rapid identification of bactericidal compounds in a model using conditions that are more relevant in vivo. This screen can be used in a high-throughput way to identify novel agents with properties that promise an increase in efficacy, while also shortening treatment times

High content, high-throughput screening for small molecule inducers of NF-κB translocation.

NF-κB is an important mediator of immune activity and its activation is essential in mounting immune response to pathogens. Here, we describe the optimization and implementation of a high-throughput screening platform that utilizes high content imaging and analysis to monitor NF-κB nuclear translocation. We screened 38,991 compounds from three different small molecule libraries and identified 103 compound as hits; 31% of these were active in a dose response assay. Several of the molecules lacked cytotoxicity or had a selectivity index of more than 2-fold. Our image-based approach provides an important first step towards identifying small molecules with immunomodulatory activity

Image analysis pipeline.

<p>(A) Low and high pixel intensity threshold setting in DAPI channel to identify cells. Uneven shapes and features created with high threshold setting. Uniform features created with low threshold intensity. (B) Stepwise pipeline used to create nuclei, whole cell and cytoplasm masks. Fill holes operation done from threshold settings to create even features. Nuclei mask created from fill holes operation by shrinking features by a number of pixel intensities and size. Extended nuclei mask created from nuclei mask by increasing features by a few pixel intensities. Whole cell mask created from nuclei mask by increasing features be a number of pixel intensities. Cytoplasm mask created from both extended nuclei mask and whole cell mask (C) Different cell compartments identified using the pipeline. Yellow, blue and light green representing nuclei, cytoplasm and extended nuclei respectively.</p