Engineering a tunable micropattern-array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ.

The molecular heterogeneity of extracellular vesicles (EVs) and the co-isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single-EV and particle (siEVP) protein and RNA assay ( PRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The PRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ.

Pharmaceutical modulation of the proteolytic profile of Transforming Growth Factor Beta induced protein (TGFBIp) offers a new avenue for treatment of -corneal dystrophy.

Corneal dystrophies are a group of genetically inherited disorders with mutations in the gene affecting the Bowman's membrane and the corneal stroma. The mutant TGFBIp is highly aggregation-prone and is deposited in the cornea. Depending on the type of mutation the protein deposits may vary (amyloid, amorphous powdery aggregate or a mixed form of both), making the cornea opaque and thereby decreases visual acuity.

Fragment screening for drug leads by weak affinity chromatography (WAC-MS).

Fragment-based drug discovery is an important tool for design of small molecule hit-to-lead compounds against various biological targets. Several approved drugs have been derived from an initial fragment screen and many such candidates are in various stages of clinical trials. Finding fragment hits, that are suitable for optimisation by medicinal chemists, is still a challenge as the binding between the small fragment and its target is weak in the range of mM to µM of K and irrelevant non-specific interactions are abundant in this area of transient interactions.

Design and Synthesis of Ligand Efficient Dual Inhibitors of Janus Kinase (JAK) and Histone Deacetylase (HDAC) Based on Ruxolitinib and Vorinostat.

Concomitant inhibition of multiple oncogenic pathways is a desirable goal in cancer therapy. To achieve such an outcome with a single molecule would simplify treatment regimes. Herein the core features of ruxolitinib (1), a marketed JAK1/2 inhibitor, have been merged with the HDAC inhibitor vorinostat (2), leading to new molecules that are bispecific targeted JAK/HDAC inhibitors.

Analysis of Protein Target Interactions of Synthetic Mixtures by Affinity-LC/MS.

Analysis of interactions between molecules is of fundamental importance in life science research. In this study, we applied weak affinity chromatography, based on high-performance liquid chromatography and mass spectrometry, as a powerful tool for direct analysis of the components of a chemical reaction mixture for their binding to a target protein. As a demonstration of the potential of this method, we analyzed the binding of the compounds of the reaction mixture to the chaperone heat shock protein 90 (Hsp90).

Lipodisks integrated with weak affinity chromatography enable fragment screening of integral membrane proteins.

Membrane proteins constitute the largest class of drug targets but they present many challenges in drug discovery. Importantly, the discovery of potential drug candidates is hampered by the limited availability of efficient methods for screening drug-protein interactions. In this work we present a novel strategy for rapid identification of molecules capable of binding to a selected membrane protein.

Comparison of weak affinity chromatography and surface plasmon resonance in determining affinity of small molecules.

In this study, we compared affinity data from surface plasmon resonance (SPR) and weak affinity chromatography (WAC), two established techniques for determination of weak affinity (mM-μM) small molecule-protein interactions. In the current comparison, thrombin was used as target protein. In WAC the affinity constant (KD) was determined from retention times, and in SPR it was determined by Langmuir isotherm fitting of steady-state responses.

Weak affinity chromatography for evaluation of stereoisomers in early drug discovery.

In early drug discovery (e.g., in fragment screening), recognition of stereoisomeric structures is valuable and guides medicinal chemists to focus only on useful configurations.

High-throughput fragment screening by affinity LC-MS.

Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed.

Weak affinity chromatography as a new approach for fragment screening in drug discovery.

Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM-μM in dissociation constant (K(D))) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments.

Toward high-throughput drug screening on a chip-based parallel affinity separation platform.

High-throughput screening of compound libraries, including the study of fragments, has become one of the cornerstones in modern drug discovery research. During this process hits are defined that may be developed into valuable leads and eventually into possible drug candidates. In this paper, we have demonstrated that parallel zonal weak affinity chromatography in microcolumns on a chip offers a possible screening format for weakly binding ligands toward a protein target.