A Kunitz-type inhibitor from tick salivary glands: A promising novel antitumor drug candidate

Abstract

Salivary glands are vital structures responsible for successful tick feeding. The saliva of ticks contains numerous active molecules that participate in several physiological processes. A Kunitz-type factor Xa (FXa) inhibitor, similar to the tissue factor pathway inhibitor (TFPI) precursor, was identified in the salivary gland transcriptome of Amblyomma sculptum ticks. The recombinant mature form of this Kunitz-type inhibitor, named Amblyomin-X, displayed anticoagulant, antiangiogenic, and antitumor properties. Amblyomin-X is a protein that inhibits FXa in the blood coagulation cascade and acts via non-hemostatic mechanisms, such as proteasome inhibition. Amblyomin-X selectively induces apoptosis in cancer cells and promotes tumor regression through these mechanisms. Notably, the cytotoxicity of Amblyomin-X seems to be restricted to tumor cells and does not affect non-tumorigenic cells, tissues, and organs, making this recombinant protein an attractive molecule for anticancer therapy. The cytotoxic activity of Amblyomin-X on tumor cells has led to vast exploration into this protein. Here, we summarize the function, action mechanisms, structural features, pharmacokinetics, and biodistribution of this tick Kunitz-type inhibitor recombinant protein as a promising novel antitumor drug candidate.

Keywords: TFPI-like; amblyomin-X; antitumor / cytotoxic activity; bioactive molecule; proteasome inhibitor.

FIGURE 1

The salivary gland transcriptome of the Amblyomma sculptum tick was characterized and analyzed by expressed sequence tags (EST). The study revealed the presence of protein-related transcripts involved in the hemostatic process, especially proteases and inhibitors. A Kunitz-type inhibitor similar to the tissue factor pathway inhibitor (TFPI) precursor was identified. This inhibitor, Amblyomin-X, was obtained as a recombinant protein and presented anticoagulant, antiangiogenic, and antitumor properties. Furthermore, Amblyomin-X demonstrated selectivity for tumor cells. Figure created in BioRender.com.

FIGURE 2

Structural features of Amblyomin-X’s Kunitz domain. (A) Rainbow colored Amblyomin-X structure model (blue to red). (B) Structure superimposition of the Kunitz domains of Amblyomin-X (rainbow), TFPI-1 KD2 (white), and TFPI-2 KD1 (gray). Structures are shown in two orientations: the same as A (left) and rotated (right) as displayed in (C) (C) Electrostatic potential of the molecular surfaces of the indicated Kunitz domains. Some important residues for TFPI-1 KD2 interaction with FXa (Mesquita Pasqualoto et al., 2014) are indicated. Surfaces negatively charged are shown in red, non-charged in white, and positively charged in blue. For Amblyomin-X homology modeling, boophilin (PDB ID: 2ODY) was used as a template. For TFPI-1 KD2 and TFPI-2 KD1, PDB IDs are 1TFX and 1ZR0, respectively.

FIGURE 3

Comparison of Amblyomin-X’s KD with TAP and Ixolaris. Amblyomin-X’s KD homology model (colored gray in A and E, in the same orientation as Figure 2A) was aligned with NMR structures of TAP (PDB: 1TCP) (A), Ixolaris (PDB: 6NAN) (D), and Ixolaris K1 (F), and K2 (H), colored in rainbow (blue to red). Electrostatic potential of the molecular surfaces of (B) TAP, (C) KD of Amblyomin-X, (E) Ixolaris, (G) Ixolaris K1, and (I) Ixolaris K2 are shown. Structure visualizations were created in PyMOL v.2.5.1.

FIGURE 4

Mechanism of action of Amblyomin-X on tumor cells. The recombinant protein is internalized by endocytosis and transported by a dynein-assisted mechanism. Amblyomin-X inhibits proteasome activity, which results in aggresome formation via the non-exclusive ubiquitin pathway, ER stress, and mitochondrial dysfunction. Unlike other proteasome inhibitors, Amblyomin-X promotes dynein-assisted autophagy inhibition and mTOR localization. Therefore, the aggresomes are not cleared. These factors are crucial for apoptosis machinery to trigger a cell response. Figure created in BioRender.com.