Biological Activities of Secretory RNases: Focus on Their Oligomerization to Design Antitumor Drugs

Abstract

Ribonucleases (RNases) are a large number of enzymes gathered into different bacterial or eukaryotic superfamilies. Bovine pancreatic RNase A, bovine seminal BS-RNase, human pancreatic RNase 1, angiogenin (RNase 5), and amphibian onconase belong to the pancreatic type superfamily, while binase and barnase are in the bacterial RNase N1/T1 family. In physiological conditions, most RNases secreted in the extracellular space counteract the undesired effects of extracellular RNAs and become protective against infections. Instead, if they enter the cell, RNases can digest intracellular RNAs, becoming cytotoxic and having advantageous effects against malignant cells. Their biological activities have been investigated either in vitro, toward a number of different cancer cell lines, or in some cases in vivo to test their potential therapeutic use. However, immunogenicity or other undesired effects have sometimes been associated with their action. Nevertheless, the use of RNases in therapy remains an appealing strategy against some still incurable tumors, such as mesothelioma, melanoma, or pancreatic cancer. The RNase inhibitor (RI) present inside almost all cells is the most efficacious sentry to counteract the ribonucleolytic action against intracellular RNAs because it forms a tight, irreversible and enzymatically inactive complex with many monomeric RNases. Therefore, dimerization or multimerization could represent a useful strategy for RNases to exert a remarkable cytotoxic activity by evading the interaction with RI by steric hindrance. Indeed, the majority of the mentioned RNases can hetero-dimerize with antibody derivatives, or even homo-dimerize or multimerize, spontaneously or artificially. This can occur through weak interactions or upon introducing covalent bonds. Immuno-RNases, in particular, are fusion proteins representing promising drugs by combining high target specificity with easy delivery in tumors. The results concerning the biological features of many RNases reported in the literature are described and discussed in this review. Furthermore, the activities displayed by some RNases forming oligomeric complexes, the mechanisms driving toward these supramolecular structures, and the biological rebounds connected are analyzed. These aspects are offered with the perspective to suggest possible efficacious therapeutic applications for RNases oligomeric derivatives that could contemporarily lack, or strongly reduce, immunogenicity and other undesired side-effects.

Keywords: RNase oligomers; antitumor activity; cytotoxicity; domain swapping; ribonucleases.

Figure 1

Chemical cross-linkers mostly used with RNases and main immuno-RNases oligomeric derivatives. (A) Diimidoesters (171); (B) Mechanism of reaction of RNases with diimidoesters; (C) glutaraldehyde; (D) trifunctional maleimide (172); (E) divinylsulfone (DVS) (173); (F) difluorodinitrobenzene (DFDNB) (174); (G) Immuno-HP-RNase-heterodimer: the HP moiety (black) and the Erb2 one (gray) (175); (H) HP-RNase diabody (176); (I) Immuno-ONC-heterodimer (177); (J) ONC (rap)-diabody (177).

Figure 2

Scheme for the 3D-DS protein association mechanism. The closed interface present in the native monomer and reconstituted in the domain-swapped dimer, and the open interface(s) forming only in the dimer are indicated (241), as well as the composite functional unit (FU) (235) of the dimer inside the dashed line.

Figure 3

Structures of RNase A, of its tandem dimer, and of its domain-swapped oligomers. (A) RNase A; (B) covalent tandem dimer (232); (C) crystal structure of the N-swapped dimer, N_D (pdb 1A2W) (233); (D) crystal structure of the C-swapped dimer, C_D (pdb 1F0V) (234); (E) N + C-swapped trimer model, NC_T (197, 235, 236); (F) crystal structure of the totally C-swapped cyclic trimer, C_T (pdb 1JS0) (236); (G) N + C + N-tetramer linear model (197, 235); (H) C + N + C-tetramer linear model (197, 235), (I) N + C + N-tetramer bent model (237); (J) N + C + C + C-tetramer model (235).

Figure 4

Structures of BS-RNase and of its tetrameric derivatives. (A) BS-RNase unswapped native dimer isoform, M=M, about 30% of the total (278, 279); (B) N-swapped native dimer isoform, M×M, about 70% of the total (19, 278); (C) totally N-swapped cyclic tetramer model plus schematic model (280); (D) N + C + N-swapped tetramer (199); (E) PALQ-BS RNase mutant non-covalent dimer and (F) its comparison with the N-swapped BS-RNase wild type (281).

Figure 5

Structures of the human pancreatic RNase 1 and of the dimers of two of its mutants. (A) HP-RNase 1; (B) crystal structure of the N-swapped dimer of PM8 (PM5 + P101Q) mutant (pdb 1H8X) (303); (C) des-N-swapped dimer (304).

Figure 6

Structures and models of non-mammalian RNases and of their oligomers. (A) Amphibian onconase (ONC); (B) N-swapped ONC dimer model (200); (C) crystal structure of the N-swapped cyclic trimer of bacterial barnase (pdb 1YVS) (315); (D,E) two alternative models for the bacterial natively dimeric unswapped binase, stabilized by electrostatic interactions at the subunits' interface (90).