A lymphatic-absorbed multi-targeted kinase inhibitor for myelofibrosis therapy.

Activation of compensatory signaling nodes in cancer often requires combination therapies that are frequently plagued by dose-limiting toxicities. Intestinal lymphatic drug absorption is seldom explored, although reduced toxicity and sustained drug levels would be anticipated to improve systemic bioavailability. A potent orally bioavailable multi-functional kinase inhibitor (LP-182) is described with intrinsic lymphatic partitioning for the combined targeting of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways without observable toxicity. We demonstrate selectivity and therapeutic efficacy through reduction of downstream kinase activation, amelioration of disease phenotypes, and improved survival in animal models of myelofibrosis. Our further characterization of synthetic and physiochemical properties for small molecule lymphatic uptake will support continued advancements in lymphatropic therapy for altering disease trajectories of a myriad of human disease indications.

Rapid LC-MS Based High-Throughput Screening Method, Affording No False Positives or False Negatives, Identifies a New Inhibitor for Carbonic Anhydrase.

Developing effective high-throughput screening (HTS) methods is of paramount importance in the early stage of drug discovery. While rugged and robust assays may be easily developed for certain enzymes, HTS assays designed to identify ligands that block protein binding are much more challenging to develop; attenuating the number of false positives and false negatives under high-throughput screening conditions is particularly difficult. We describe an MS-based HTS workflow that addresses these challenges. The assay mitigates false positives by selectively identifying positive hits exclusively when a ligand at the binding site of interest is displaced; it mitigates false negatives by detecting a reporter compound that ionizes well, not by detecting the ligand binder, which may not ionize. The method was validated by detecting known binders of three proteins, pepsin, maltose binding protein (MBP), and carbonic anhydrase (CA) in the presence of hundreds of non-binders. We also identified a novel CA binder, pifithrin-µ, which could not have been identified by any other MS-based assay because of its poor ionization efficiency. This new method addresses many of the challenges that are currently encountered during high-throughput screening.

HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications.

A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have Ki values of 0.2 µM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods. Graphical Abstract ᅟ.