Rationalizing PROTAC-Mediated Ternary Complex Formation Using Rosetta

The collection of refined models does not include highly-strained PROTAC conformations.

“Upon evaluating the internal energy of these conformers, we found that their distribution strongly overlaps with the distribution of energies from the conformers built into the ternary models (Figure S3): thus, the collection of refined models does not include highly-strained PROTAC conformations.”

The number of ternary models normalized to the number of linker conformers has readily converged when 1000 linker conformers are included.

“Our analysis therefore normalizes the number of ternary models to the number of linker conformers: we find that this value has readily converged when 1000 linker conformers are included (Figure S2).”

RMSD values less than 0.4 Å yielded binding modes suitable for further refinement.

“Through preliminary exploration we found that RMSD values less than 0.4 Å yielded binding modes suitable for further refinement, and thus we adopted this as a cutoff to determine which models to pass along for further refinement.”

A representative model for the ZXH-3-26 ternary complex shows adventitious protein-protein interactions.

“A representative model for one such ternary complex model (using the PROTAC ZXH-3-26) shows an example of the adventitious protein-protein interactions observed in these models (Figure 2c).”

The latter study's series of very potent degraders yield high FFC values when explored through the computational approach.

“That said, the latter study reports a series of very potent degraders 62, and indeed these yield high FFC values when explored through our computational approach (Table S1).”

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• AssumptionRests on an assumption: The number of models that emerge from filtering (FFC) is a measure of how efficiently the PROTAC induces ternary complex formation.

The collection of models contributing to FFC includes the crystal structure binding mode for the dBET70 ternary complex.

“The crystal structure for the dBET70 ternary complex has been solved 54, and gratifyingly our collection of models contributing to FFC does include this particular binding mode (Figure 2d).”

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• AssumptionRests on an assumption: The number of models that emerge from filtering (FFC) is a measure of how efficiently the PROTAC induces ternary complex formation.

Linkers with higher FFC values correspond exactly to those with enhanced cellular degradation of Brd4BD1.

“We observe that linkers with higher FFC values (i.e., the linker is compatible with more of the conformational ensemble from docking) correspond exactly to those with enhanced cellular degradation of Brd4BD1, presumably because these PROTACs more effectively induce ternary complex formation in cells.”

• AssumptionRests on an assumption: The ensemble of 5000 docked models captures the diversity of potential ways in which Brd4 and CRBN might form adventitious interactions.
• AssumptionRests on an assumption: The number of models that emerge from filtering (FFC) is a measure of how efficiently the PROTAC induces ternary complex formation.

The linker itself can contribute favorably to ternary complex formation in some cases by making additional interactions with one or both proteins.

“The linker itself can also contribute favorably to ternary complex formation in some cases, by making additional interactions with one or both proteins.”

For the five PROTACs comprising the 'ZXH' series, the numbers of poses arising from filtering (FFC) match very closely the trend in experimentally-observed cellular degradation of the target protein.

“Remarkably, for the five PROTACs comprising the “ZXH” series, the numbers of poses arising from filtering (which we report as FFC, the Fraction of Fully-compatible Complexes) match very closely the trend in experimentally-observed cellular degradation of the target protein (Table 1).”

• AssumptionRests on an assumption: The ensemble of 5000 docked models captures the diversity of potential ways in which Brd4 and CRBN might form adventitious interactions.
• AssumptionRests on an assumption: The number of models that emerge from filtering (FFC) is a measure of how efficiently the PROTAC induces ternary complex formation.

Even amongst the three kinases considered here, there are no clusters of conserved surface residues outside the ATP-binding site.

“Even amongst the three kinases considered here, there are no clusters of conserved surface residues outside the ATP-binding site (Figure 3f).”

• AssumptionRests on an assumption: The ATP-binding sites of canonical protein kinases share sequence and structural conservation, while the surface outside this site varies dramatically over the kinase family.

By analysis of the resulting collections of models, it is possible to retrospectively explain relationships between linker length/composition and cellular activity.

“By analysis of the resulting collections of models, we demonstrate that it is possible to retrospectively explain relationships between linker length/composition and cellular activity.”

The paper developed a new computational protocol for structure-based modeling of PROTAC-mediated ternary complexes.

“We report here the development of a new computational protocol for structure-based modeling of PROTAC-mediated ternary complexes.”

The protein-protein interactions in the ternary complex arise from the less-conserved regions on the kinase surface, which differ even among evolutionarily-related kinases.

“Importantly, the protein-protein interactions in the ternary complex arise from the less-conserved regions on the kinase surface, which differ even among evolutionarily-related kinases.”

• AssumptionRests on an assumption: The ATP-binding sites of canonical protein kinases share sequence and structural conservation, while the surface outside this site varies dramatically over the kinase family.

By exploring ternary complexes of PROTACs built upon the same kinase-binding warhead, surface differences on the conserved kinase fold can change the preferred binding modes for a given PROTAC.

“By exploring ternary complexes of PROTACs built upon the same kinase-binding warhead, already we find that surface differences on the conserved kinase fold can change the preferred binding modes for a given PROTAC.”

Strong degradation preferences arise despite the shared global fold of all three kinase domains.

“As might be expected for such a molecular recognition event, details matter: this is underscored by the fact that strong degradation preferences arise despite the shared global fold of all three kinase domains.”

The results from the computational protocol agreed well with cellular degradation data.

“Because cellular degradation is more commonly probed, however, it was more straightforward to draw these data from the literature; we were therefore delighted and surprised to discover how well the results from our computational protocol agreed with this ultimately more-relevant endpoint.”

For CRBN-recruiting PROTAC2, FFC values are high for c-Met and EphA2, consistent with the cellular effects of treatment with this PROTAC.

“By contrast, CRBN-recruiting PROTAC2 yields high FFC for c-Met and EphA2: this too is consistent with the cellular effects of treatment with this PROTAC (Figure 3c).”

• AssumptionRests on an assumption: The number of models that emerge from filtering (FFC) is a measure of how efficiently the PROTAC induces ternary complex formation.

Members of a structurally-conserved protein family can be recruited by the same PROTAC through vastly different binding modes.

“and that members of a structurally-conserved protein family can be recruited by the same PROTAC through vastly different binding modes.”

Although the warhead engages the structurally-conserved ATP-binding site in the same manner and the kinase fold is conserved, models of the ternary complex can be remarkably diverse.

“In summary, then, we find that although the warhead engages the structurally-conserved ATP-binding site in the same manner, and the kinase fold is conserved, our models of the ternary complex can be remarkably diverse.”

Recognition by VHL involves non-conserved regions of the c-Met surface.

“Importantly, recognition by VHL involves non-conserved regions of the c-Met surface (Figure 3e), consistent with the suggestion that protein-protein interactions involving these regions may explain the nascent selectivity of PROTACs built from promiscuous warheads.”

• AssumptionRests on an assumption: The ATP-binding sites of canonical protein kinases share sequence and structural conservation, while the surface outside this site varies dramatically over the kinase family.

For PROTAC1, multiple binding modes were observed for c-Met.

“For PROTAC1, which gave high FFC values for c-Met, multiple binding modes were observed for this kinase (Figure 3d); this was analogous to the observations from the Brd4BD1 case study presented earlier.”

In the kinase example presented, regions outside the ATP-binding site proved critical for driving PROTAC selectivity.

“Given the importance in the ternary complex of protein-protein interactions between the target protein and the E3 ligase, it will be essential that both proteins are accurately modeled beyond just their PROTAC binding sites; in the kinase example presented here, for example, regions outside the ATP-binding site proved critical for driving PROTAC selectivity.”

PROTACs induce target protein degradation by drawing the target protein into a ternary complex with an E3 ligase.

“If these two groups are connected via a carefully-chosen chemical linker, the resulting compound can induce formation of a ternary complex that includes the target protein, the PROTAC, and the E3 ligase. The resulting proximity between target protein and E3 ligase can then lead to ubiquitination of the target protein, and ultimately its degradation 1.”

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Foretinib itself inhibits at least a quarter of the human kinome.

“Using a competitive binding assay, foretinib itself was first shown to inhibit at least a quarter of the human kinome 20.”

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There are not yet enough published crystal structures of ternary complexes to allow for a thorough and rigorous evaluation of the method.

“While there have been several published crystal structures of ternary complexes 35, 52, 54, there are not yet enough to allow for a thorough and rigorous evaluation of our method.”

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The observation of diverse conformational arrangements is consistent with the 'plasticity' originally ascribed to the complexes in this series.

“This observation is consistent with the surprising “plasticity” originally ascribed to the complexes in this series 54.”

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The correspondence between higher FFC values and enhanced cellular degradation is presumably because these PROTACs more effectively induce ternary complex formation in cells.

“We observe that linkers with higher FFC values (i.e., the linker is compatible with more of the conformational ensemble from docking) correspond exactly to those with enhanced cellular degradation of Brd4BD1, presumably because these PROTACs more effectively induce ternary complex formation in cells.”

• AssumptionRests on an assumption: The number of models that emerge from filtering (FFC) is a measure of how efficiently the PROTAC induces ternary complex formation.

Crystal structures of foretinib in complex with c-Met, EphA2, and STK10 have been solved.

“Crystal structures of foretinib in complex with three different kinases have been solved to date (c-Met, EphA2, and STK10) 64, 65”

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Multiple binding modes are likely to contribute to the ternary complexes formed by these PROTACs.

“This viewpoint is wholly consistent with our observation that multiple binding modes are likely to contribute to the ternary complexes formed by these PROTACs.”

The extent to which different kinases were degraded was not simply a reflection of how tightly the PROTAC bound the kinase, since inhibition constants were not correlated with cellular degradation.

“Moreover, the extent to which different kinases were degraded was not simply a reflection related to how tightly the PROTAC bound the kinase, since inhibition constants were not correlated with cellular degradation 20.”

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Many single-point mutations to either Brd4 or CRBN outside their ligand-binding sites lead to modest changes that enhance or reduce ternary complex formation, without finding mutations that abrogate the interaction completely.

“It is further consistent with the fact that many single-point mutations to either Brd4 or to CRBN (outside of their respective ligand-binding sites) lead to modest changes that enhance or reduce ternary complex formation, without finding mutations that abrogate the interaction completely 54.”

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Randomly splitting the original collection of docked models into five subsets confirms the statistical significance of the differences between calculated FFC values for the 'ZXH' series.

“By randomly splitting the original collection of docked models into five subsets, we next evaluated the variance arising from sampling in each of the predicted FFC values for the “ZXH” series of PROTACs: this analysis confirms the statistical significance of the differences between calculated FFC values (see Methods) (Figure 2a).”

Sparse information from mutagenesis experiments is typically insufficient to unambiguously point to a specific binding mode in the ternary complex.

“Beyond the fact that we observe ensembles of models that are fully consistent with the geometric and energetic requirements of ternary complex formation (rather than a single static model for each), this observation also underscores the fact that sparse information from mutagenesis experiments are typically insufficient to unambiguously point to a specific binding mode in the ternary complex.”

The computational strategy implicitly neglects binding modes in which the protein-protein interaction does not contribute favorably to the ternary complex.

“By starting with an ensemble of docked models for the protein components, our computational strategy implicitly neglects binding modes in which the protein-protein interaction does not contribute favorably to the ternary complex.”

This leads to PROTAC selectivity that can differ from that of the warhead in surprising and unexpected ways.

“This in turn leads to PROTAC selectivity that can differ from that of the warhead in surprising and unexpected ways.”

Interpreting PROTAC activity is best approached using an ensemble of structures of the ternary complex rather than a single static conformation.

“We further find that interpreting PROTAC activity is best approached using an ensemble of structures of the ternary complex rather than a single static conformation”

For this series at least, cell permeability may not be the primary limitation of PROTAC activity.

“This suggests that, for this series at least, cell permeability may not be the primary limitation of PROTAC activity.”

In an anti-cooperative scenario, addition of the PROTAC still recruits the E3 ligase to the target protein.

“In an anti-cooperative scenario, addition of the PROTAC will still recruit the E3 ligase to the target protein: anti-cooperativity simply means that a higher concentration of PROTAC will be needed, relative to a non-cooperative or cooperative PROTAC with the same warhead and the E3-recruiting moiety.”

The computational results do not directly incorporate any knowledge of a PROTAC's cell penetrance, efflux, kinetics of ternary complex formation, ubiquitination efficiency, or rate of proteasomal degradation.

“Importantly, our computational results do not directly incorporate any knowledge of a PROTAC’s cell penetrance, efflux, kinetics of ternary complex formation, ubiquitination efficiency once the ternary complex is formed, or rate of proteasomal degradation of the ubiquitinated target protein.”

The computational method may underestimate cellular activity of anti-cooperative PROTACs.

“Thus, while our computational method may underestimate cellular activity of anti-cooperative PROTACs, we anticipate that even in these cases this approach can be used to facilitate optimization of activity through design of linkers that promote cooperativity.”

The computational approach focuses on binding modes in which the two proteins already have some intrinsic affinity for one another and are therefore likely to exhibit positive cooperativity.

“Instead, our approach focuses on binding modes in which the two proteins already have some intrinsic affinity for one another, and are therefore likely to exhibit (positive) cooperativity.”

This emergent selectivity will depend on the E3 ligase that is recruited, as different E3 ligases will engage these non-biological binding partners in different ways.

“This emergent selectivity will naturally depend as well on the E3 ligase that is to be recruited, as different E3 ligases will inevitably engage these non-biological binding partners in different ways.”

The results do not suggest that a particular FFC value can be used as a cutoff to determine whether or not a PROTAC will have activity.

“The results we present here do not suggest that a particular FFC value can be used as a cutoff to determine whether or not a PROTAC will have activity; rather, comparisons between the FFC values of related systems are more valuable.”

Even for anti-cooperative PROTACs, the approach can be used to facilitate optimization of activity through design of linkers that promote cooperativity.

“Thus, while our computational method may underestimate cellular activity of anti-cooperative PROTACs, we anticipate that even in these cases this approach can be used to facilitate optimization of activity through design of linkers that promote cooperativity.”

The dual selectivity of palbociclib is unsurprising, given complete sequence conservation between CDK6 and CDK4 at all amino acid positions that contact this ATP-competitive inhibitor.

“Support for this concept draws from recently-reported PROTACs built on the dual CDK6/CDK4 inhibitor palbociclib 69. The dual selectivity of palbociclib is unsurprising, given that there is complete sequence conservation between these two kinases at all amino acid positions that contact this ATP-competitive inhibitor.”

• cites [69] Fry D. W., 2004, Mol. Cancer Ther. sourceNot checked yet

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Binding modes in which the arrangement of the protein components is unfavorable, leading to anti-cooperativity, may not be present in the docked ensemble and could be missed in subsequent analysis.

“Conversely, binding modes in which the arrangement of the protein components are unfavorable – leading to anti-cooperativity – may not be present in our docked ensemble, and thus could be missed in our subsequent analysis.”

At least in the cases explored here, a PROTAC's activity is determined primarily by its ability to induce ternary complex formation and not these other factors.

“This implies that – at least in the cases we explore here – a PROTAC’s activity is determined primarily by its ability to induce ternary complex formation and not these other factors.”

Conclusions drawn from a single static model (such as a crystal structure) may be misleading.

“Accordingly, conclusions drawn from a single static model (such a crystal structure) may be misleading; we therefore eagerly await insights from cryo-electron microscopy, which is exceptionally well-positioned to yield definitive answers regarding the range of motion in PROTAC ternary complexes.”

The models can rationalize the surprising observation that the target-selectivity of a given PROTAC is not simply transferred from the target-binding warhead.

“Further, these models can also rationalize the surprising observation that the target-selectivity of a given PROTAC is not simply transferred from the target-binding warhead: rather, interactions with the E3 ligase in the ternary complex can dramatically shift the selectivity of the PROTAC.”

The underlying differences in kinase/E3 binding preferences are presumably captured in the initial docked ensembles, even before the PROTAC linker has been specified.

“While these differences are observed in the context of two PROTACs’ surprising degradation selectivity, already the underlying differences in the kinase/E3 binding preferences are presumably captured in the initial docked ensembles, even before the PROTAC linker has been specified.”

Interactions with the E3 ligase in the ternary complex can dramatically shift the selectivity of the PROTAC.

“Further, these models can also rationalize the surprising observation that the target-selectivity of a given PROTAC is not simply transferred from the target-binding warhead: rather, interactions with the E3 ligase in the ternary complex can dramatically shift the selectivity of the PROTAC.”

Recent versions of Rosetta include a dramatically-improved treatment of small molecules in the energy function that is necessary for refining linker conformations in the ternary complexes.

“In addition to other essential functionalities (the “jump_all” flag), recent versions of Rosetta also include a dramatically-improved treatment of small molecules in the energy function that is necessary for refining linker conformations in the ternary complexes 78.”

• cites [78] Park H., 2020, bioRxivNot checked yet

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