Centrosomal kinase Nek2 is overexpressed in different cancers, yet how it contributes toward tumorigenesis remains poorly understood. dNek2 overexpression in a Drosophila melanogaster model led to upregulation of Drosophila Wnt ortholog wingless (Wg), and alteration of cell migration markers—Rho1, Rac1 and E-cadherin (Ecad)—resulting in changes in cell shape and tissue morphogenesis. dNek2 overexpression cooperated with receptor tyrosine kinase and mitogen-activated protein kinase signaling to upregulate activated Akt, Diap1, Mmp1 and Wg protein to promote local invasion, distant seeding and metastasis. In tumor cell injection assays, dNek2 cooperated with Ras and Src signaling to promote aggressive colonization of tumors into different adult fly tissues. Inhibition of the PI3K pathway suppressed the cooperation of dNek2 with other growth pathways. Consistent with our fly studies, overexpression of human Nek2 in A549 lung adenocarcinoma and HEK293T cells led to activation of the Akt pathway and increase in β-catenin protein levels. Our computational approach identified a class of Nek2-inhibitory compounds and a novel drug-like pharmacophore that reversed the Nek2 overexpression phenotypes in flies and human cells. Our finding posits a novel role for Nek2 in promoting metastasis in addition to its currently defined role in promoting chromosomal instability. It provides a rationale for the selective advantage of centrosome amplification in cancer.

Human cathepsin L belongs to the cathepsin family of proteolytic enzymes with primarily an endopeptidase activity. Although its primary functions were originally thought to be only of a housekeeping enzyme that degraded intracellular and endocytosed proteins in lysosome, numerous recent studies suggest that it plays many critical and specific roles in diverse cellular settings. Not surprisingly, the dysregulated function of cathepsin L has manifested itself in several human diseases, making it an attractive target for drug development. Unfortunately, several redundant and isoform-specific functions have recently emerged, adding complexities to the drug discovery process. To address this, a series of chemical biology tools have been developed that helped define cathepsin L biology with exquisite precision in specific cellular contexts. This review elaborates on the recently developed small molecule inhibitors and probes of human cathepsin L, outlining their mechanisms of action, and describing their potential utilities in dissecting unknown function.Molecules 2020, 25, 698 2 of 41 also now well established and has been a subject of elegant reviews elsewhere [25][26][27]. Unfortunately, several overlapping and redundant functions of cathepsin L have also emerged [5,[28][29][30]. It is therefore critically important that its functional biology in both normal and disease-specific cell types be clearly annotated before significant resources are directed in drug discovery endeavors. In this review, we will briefly outline the biogenesis of cathepsin L, describe its post-translational processing and trafficking, and highlight key structural features required for formation of an active and mature cathepsin L. A brief summary of cathepsin L biology and its role in human diseases is provided next. Finally, a detailed and up-to-date report on existing cathepsin L-targeting small molecule inhibitors and functional probes with their mechanistic details is described.Human cathepsin L gene, CTHL (Uniprot primary accession number: P07711), located at the 9q21-q22 position of chromosome 9, encodes for a total of 333 amino acid peptide sequence (M.W. = 37,564 kDa) [31]. The coding space includes the regions of a N-terminal signal peptide, two pro-peptides, and two mature peptides comprising of a heavy (H) chain and a light (L) chain ( Figure 1). After transcription, the signal peptide is co-translationally removed in polysome and the resulting 41 kDa pro-cathepsin L is translocated to endoplasmic reticulum where it undergoes N-linked glycosylation with mannose rich sugars. The mannose sugars on the pro-cathepsin L are then phosphorylated in cis Golgi by UDP-N-acetylglucosamine:N-acetylglucosaminephosphotransferase enzyme [32]. The mannose-6-phosphate (M6P) receptors located on the surface of the trans Golgi network recognize the M6P-pro-cathepsin peptide and deliver the pro-cathepsin L peptide to the lysosome via the endolysosomal pathway. The weakly acidic environment of endosome/lysosome releases M6P receptors and the phosphate ...