OnconaseTM , a homolog of ribonuclease A (RNase A) with low ribonucleolytic activity, is cytotoxic and has efficacy as a cancer chemotherapeutic. Here variants of RNase A were used to probe the interplay between ribonucleolytic activity and evasion of the cytosolic ribonuclease inhibitor protein (RI) in the cytotoxicity of ribonucleases. K41R/G88R RNase A is a less active catalyst than G88R RNase A but, surprisingly, is more cytotoxic. Like Onconase TM , the K41R/G88R variant has a low affinity for RI, which apparently compensates for its low ribonucleolytic activity. In contrast, K41A/G88R RNase A, which has the same affinity for RI as does the K41R/ G88R variant, is not cytotoxic. The nontoxic K41A/G88R variant is a much less active catalyst than is the toxic K41R/G88R variant. These data indicate that maintaining sufficient ribonucleolytic activity in the presence of RI is a requirement for a homolog or variant of RNase A to be cytotoxic. This principle can guide the design of new chemotherapeutics based on homologs and variants of RNase A.Ribonuclease A (RNase A; EC 3.1.27.5 (1)) was perhaps the most studied enzyme of the twentieth century. Now homologs of RNase A are becoming important new chemotherapeutics (2-4). For example, low levels of bovine seminal ribonuclease (BS-RNase) 1 are cytotoxic (5, 6). More significantly, Onconase TM (ONC), which is isolated from the frog Rana pipiens (7), is currently in Phase III human clinical trials for the treatment of malignant mesothelioma. In addition, ONC inhibits HIV-1 replication in chronically infected human cells (8). Understanding the mechanism of ribonuclease cytotoxicity is vital for the further development of ribonucleases as chemotherapeutics.Ribonuclease-mediated cytotoxicity is known to depend on several factors. Ribonucleases must enter the cell and reach the cytosol, where RNA degradation leads ultimately to cell death (9, 10). Indeed, injecting ribonucleases directly into Xenopus oocytes increases their cytotoxicity (11,12). In the cytosol, ribonucleases encounter the ribonuclease inhibitor protein (RI). RI constitutes Ն0.01% of protein in the cytosol (13,14) and inactivates ribonucleases by forming a tight complex that prevents RNA substrates from entering the active site (Fig. 1A) (15).Ribonucleolytic activity is requisite for the cytotoxicity of BS-RNase and ONC (16,17). Yet despite its relatively high ribonucleolytic activity, RNase A is not cytotoxic (9, 18). The cytotoxicity of BS-RNase and ONC has been attributed to the ability of BS-RNase A and ONC to evade RI (19,20). RI is a potent inhibitor of RNase A with K i near 10 Ϫ14 M (21, 22). In contrast, ONC (estimated K i Ն 10 Ϫ6 M (23)) and BS-RNase (24) escape inhibition by RI.BS-RNase and ONC use different strategies to evade RI. BS-RNase forms a homodimer, which is stabilized by two intersubunit disulfide bridges. This dimeric form has a much lower affinity for RI than does the free monomer (24). ONC evades RI as a monomer. Only 3 of the 24 RNase A residues that contact RI are conserved in ONC...

Onconase® (ONC) is an amphibian ribonuclease that is in clinical trials as a cancer chemotherapeutic agent. ONC is a homolog of ribonuclease A (RNase A). RNase A can be made toxic to cancer cells by replacing Gly 88 with an arginine residue, thereby enabling the enzyme to evade the endogenous cytosolic ribonuclease inhibitor protein (RI). Unlike ONC, RNase A contains a KFERQ sequence (residues 7-11), which signals for lysosomal degradation. Here, substitution of Arg 10 of the KFERQ sequence has no effect on either the cytotoxicity of G88R RNase A or its affinity for RI. In contrast, K7A/G88R RNase A is nearly 10-fold more cytotoxic than G88R RNase A and has more than 10-fold less affinity for RI. Up-regulation of the KFERQ-mediated lysosomal degradation pathway has no effect on the cytotoxicity of these ribonucleases. Thus, KFERQ-mediated degradation does not limit the cytotoxicity of RNase A variants. Moreover, only two amino acid substitutions (K7A and G88R) are shown to endow RNase A with cytotoxic activity that is nearly equal to that of ONC.

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To better understand electronic effects on the diastereoselectivity of nucleophilic additions to the carbonyl group, a series of 2-X-4-tert-butylcyclohexanones (X = H, CH(3), OCH(3), F, Cl, Br) were reacted with LiAlH(4). Reduction of ketones with equatorial substituents yields increasing amounts of axial alcohol in the series for X [H < CH(3) < Br < Cl < F << OCH(3)]. These data cannot be explained by steric or chelation effects or by the theories of Felkin-Anh or Cieplak. Instead, an electrostatic argument is introduced: due to repulsion between the nucleophile and the X group, axial approach becomes energetically less favorable with an increase in the component of the dipole moment anti to the hydride approach trajectory. The ab initio calculated diastereoselectivities were close to the experimental values but did not reproduce the relative selectivity ordering among substituents. For reduction of ketones with axial substituents, increasing amounts of axial alcohol are seen in the series for X [Cl < Br < CH(3) < OCH(3) < H < F]. After some minor adjustments are made, this ordering is consistent with both the electrostatic model and Felkin-Anh theory. Cieplak theory cannot account for these data regardless of adjustments. Ab initio calculated diastereoselectivities were reasonably accurate for the nonpolar substituents but were poor for the polar substituents.