Turning Navitoclax into a PROTAC Senolytic with a Better Safety Profile

It is interesting to compare today's open access paper on converting the senolytic drug navitoclax into a PROTAC with recent efforts to improve navitoclax by conjugation with galactose. In both cases the objective is to reduce side-effects, but the strategies are quite different. Navitoclax is arguably the worst of the viable first generation senolytic drugs capable of selectively destroying senescent cells in old tissues. Senescent cells accumulate with age and cause great harm via their inflammatory signaling. Removing them has been shown to extend healthy life, reverse aspects of aging, and turn back a wide range of age-related diseases in animal models. Nonetheless, navitoclax is a toxic and unpleasant chemotherapeutic, and in addition to being poorly selective in comparison to more recent senolytic compounds, it also kills platelets to produce thrombocytopenia.

Galactose conjugation is pretty straightforward: link navitoclax with galactose to produce a molecule that is innocuous until the β-galactosidase found in large amounts in senescent cells cleaves away the galactose to reveal navitoclax. Thus the activity of navitoclax is largely restricted to senescent cells, reducing its off-target effects. Making a PROTAC, a proteolysis targeting chimera, is somewhat more complex and needs less of the structure of navitoclax - it is more of an evolution of the compound to produce a new small molecule. In this case, the part of navitoclax capable of binding to Bcl-xl is connected to sequences that cause a cell to degrade the Bcl-xl protein, an activity that will force a senescent cell into apoptosis. This only takes place if the cell has the right molecular machinery to kick off that degradation process, however. Platelets do not have that machinery, and so the PROTAC derived from navitoclax will not activate in platelets. The platelets survive, and thrombocytopenia is evaded.

Using proteolysis-targeting chimera technology to reduce navitoclax platelet toxicity and improve its senolytic activity

ABT263 (also known as navitoclax), a Bcl-2 and Bcl-xl dual inhibitor, is one of the most potent and broad-spectrum senolytic agents identified to date. Bcl-xl inhibition with ABT263 and other small molecular inhibitors induces platelet apoptosis and results in severe thrombocytopenia, which prevents the use of ABT263 and other Bcl-xl specific inhibitors in the clinic - even for cancer patients - because platelets solely depend on Bcl-xl for survival. By contrast, Bcl-2 is dispensable for thrombopoiesis and platelet survival in mice and humans and inhibition of Bcl-2 with ABT199 (also known as venetoclax) does not induce thrombocytopenia. We hypothesize that we can reduce ABT263 on-target toxicity and generate a safer senolytic agent by converting ABT263 into a platelet-sparing Bcl-xl proteolysis-targeting chimera (PROTAC).

PROTACs are bivalent small molecules containing a ligand that recognizes a target protein linked to another ligand that recruits a specific E3 ubiquitin ligase. PROTAC binding induces proximity-induced ubiquitination of the target protein and its subsequent degradation by proteasomes. Importantly, because PROTACs rely on E3 ligases to induce protein degradation, it is possible to achieve cell/tissue selectivity, even when the target proteins are ubiquitously expressed as long as they target the proteins to an E3 ligase that is cell- or tissue-specific.

Here, we report the use of PROTAC technology to reduce ABT263 on-target toxicity by converting ABT263 into PZ15227 (PZ), a Bcl-xl specific PROTAC (Bcl-xl-P), which targets Bcl-xl to the E3 ligase cereblon (CRBN) that is poorly expressed in platelets. We find that PZ is less toxic to platelets but equally or slightly more potent against senescent cells compared with ABT263. These findings provide an approach to reduce on-target toxicity of toxic senolytic agents. With further improvement, Bcl-xl-Ps have the potential to be developed into safer and more effective senolytics than ABT263.


Elimination of senescent cells by β-galactosidase-targeted prodrug attenuates inflammation and restores physical function in aged mice
Herein, we developed a prodrug strategy to design a new compound based on the increased activity of lysosomal β-galactosidase (β-gal), a primary characteristic of senescent cells.Our prodrug SSK1 is specifically activated by β-gal ...
We further compared the specificity of SSK1 with other three reported senolytic drugs (ABT263, dasatinib plus quercetin, and fisetin) on HEFs, human preadipocytes and HUVECs, which were previously used to study senolytics. In our study, SSK1 selectively killed senescent HEFs induced by different stimuli . ABT263 could also specifically kill senescent HEFs induced by different stresses but in a narrow concentration range .
On the other hand, the combination of dasatinib and quercetin (D + Q) showed limited selectivity as it killed both senescent and non-senescent HEFs , and fisetin only killed certain senescent HEFs at high concentration. SSK1 also specifically killed senescent human preadipocytes , while ABT263 and fisetin had limited elimination effect on this cell type . D + Q could kill senescent human preadipocytes at high concentration with an affection of non-senescent cells . Additionally, the experiments on HUVECs showed that SSK1 selectively eliminated senescent cells with a broader concentration spectrum compared with ABT263 , whereas D + Q showed no selectivity to senescent and non-senescent cells .

These results demonstrated that SSK1 has higher specificity and potency to eliminate senescent cells.

Posted by: checkthisout at May 2nd, 2020 2:35 AM

Now the question is what will be the clinical significance of such conjugation. Another question is why still there not dozens of good senolytic candidates

Posted by: cuberat at May 4th, 2020 12:08 PM

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