Monopolar spindle kinase 1 (MPS1/TTK) is a key element of the mitotic checkpoint and clinically evaluated as a target in the treatment of aggressive tumors such as triple-negative breast cancer. While long drug−target residence times have been suggested to be beneficial in the context of therapeutic MPS1 inhibition, no irreversible inhibitors have been reported. Here we present the design and characterization of the first irreversible covalent MPS1 inhibitor, RMS-07, targeting a poorly conserved cysteine in the kinase's hinge region. RMS-07 shows potent MPS1 inhibitory activity and selectivity against all protein kinases with an equivalent cysteine but also in a broader kinase panel. We demonstrate potent cellular target engagement and pronounced activity against various cancer cell lines. The covalent binding mode was validated by mass spectrometry and an X-ray crystal structure. This proof of MPS1 covalent ligandability may open new avenues for the design of MPS1-specific chemical probes or drugs.

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The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Monopolar spindle kinase 1 (MPS1/TTK) is a key element of the mitotic checkpoint securing proper chromosome segregation. It is being evaluated as a target in the treatment of aggressive tumors such as triple-negative breast cancer with several reversible inhibitors currently undergoing clinical trials. While long drug–target residence times have been suggested to be beneficial in the context of therapeutic MPS1 inhibition, no irreversible inhibitors are known. Here we present the design and characterization of the first irreversible covalent MPS1 inhibitor <b>RMS-07</b> targeting a cysteine (Cys604) in the kinase's hinge region present only in few other protein kinases. The compound showed excellent MPS1 inhibitory potency and high selectivity against all protein kinases harboring an equivalent cysteine as well as in a larger differential scanning fluorimetry-based screening panel. Covalent binding was confirmed by mass spectrometry and X-ray crystal structure. We expect this tool compound to open new avenues for the design of MPS1-specific covalent chemical probes or drugs.

word count: 198. Abstract 27 28 The principle and techniques applied in DNA extraction play a pivotal role in the obtention 29 of a considerable and purified yield of genetic material. In this work, we evaluate the 30 efficiency of seven protocols chosen from literature for the DNA extraction of Hypostomus 31 commersoni (Valenciennes, 1836), an important component of South American freshwater 32 ichthyofauna. We also proposed and evaluate an improved protocol, based on the 33 combination of variables from the selected methodologies that demonstrated higher 34 potential for extracting H. commersoni DNA. The quality of samples was assessed through 35 spectrophotometry, gel electrophoresis and PCR-RAPD amplification. The efficiency of 36 DNA extraction was influenced both by the method applied and target-tissue of choice. 37 Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive 38 spectrum of incubation in lysis buffer for up to 36 hours after sample collection, without 39 maceration, using fresh tissues and in the presence of high concentration of Proteinase K. 40 In these conditions, samples were successfully amplified using a set of primers from 41 Operon Technologies. To date, there is no record of description for the parameters 42 analyzed in this work, neither the description of RAPD markers for H. commersoni, 43 evidencing the importance of our findings. 44 45 Key-words: DNA extraction; PCR-RAPD; eye tissue; fresh fish tissue; proteinase K.46 47 48 52 A crucial step for high quality genetic analysis is considered to be a satisfactory DNA 53 extraction. Nevertheless, this purpose is not always easy to achieve, since different 54 biological compounds, such as lipids and proteins, can act as contaminants and interfere in 55 the final quality of the product [1]. The principles and techniques applied in DNA 56 extraction play a pivotal role in obtaining a considerable and purified yield of this molecule 57 [2]. 58A variety of methods has been established to isolate DNA from biological materials 59 [3,6]. Regardless the method of choice, the procedure usually consists of three steps: lysis, 60 purification and DNA recovery, being the first the most critical stage, since is at this point 61 that the chances of causing DNA damages are higher [7]. 62The quality and amount of available DNA strand template have a major influence 63 on genetic analyzes based on PCR (Polymerase Chain Reaction), for example [8,9]. 64 Therefore, an ideal extraction technique should optimize DNA yield, minimize DNA 65 degradation, and be efficient in terms of cost, time, labor, and supplies [10]. 66With the availability of a plethora of molecular analysis machinery, there is an 67 exponential demand for high quality samples, capable to generate great results with 68 minimum of errors. To meet these requirements, commercial kits were developed, aiming 69 for DNA extractions of excellence [11][12][13]. 70