Room-temperature crystallography reveals altered binding of small-molecule fragments to PTP1B
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Based on two room-temperature X-ray crystallographic screens of PTP1B phosphatase against two sets of chemical fragments, and by comparing the results from a previous cryo screen, the authors report the important observation that, in addition to overlapping but non-identical sets of hits compared to the cryo screen, the room-temperature screens lead to significant differences in terms of binding sites and poses for some of the hits. The study provides compelling support for the use of room-temperature X-ray crystallography in early-stage drug discovery and highlights that temperature should be used as a parameter in efforts to extract additional insight from such analyses.
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Abstract
Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously, we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy et al., 2018). Here, we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of a diverse library of ligands to date, and enabling a direct interrogation of the effect of data collection temperature on protein-ligand interactions. We show that at RT, fewer ligands bind, and often more weakly – but with a variety of temperature-dependent differences, including unique binding poses, changes in solvation, new binding sites, and distinct protein allosteric conformational responses. Overall, this work suggests that the vast body of existing cryo-temperature protein-ligand structures may provide an incomplete picture, and highlights the potential of RT crystallography to help complete this picture by revealing distinct conformational modes of protein-ligand systems. Our results may inspire future use of RT crystallography to interrogate the roles of protein-ligand conformational ensembles in biological function.
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Author Response
Reviewer #1 (Public Review):
This paper presents the results of two fragment screens of PTP1B using room-temperature (RT) crystallography, and compares these results with a previously published fragment screen of PTP1b using cryo-temperature crystallography. The RT screen identified fewer fragment hits and lower occupancy compared to the cryo screen, consistent with prior publications on other proteins. The authors attempted to identify additional hits by applying two additional layers of data processing, which resulted in a doubling in the number of possible hits in one of the screens. Because I am not an expert in panDDA modeling, however, I am unable to evaluate the reproducibility and potential potency of these fragment hits as protein binders or their potential use as starting points for follow-up chemistry.
The…
Author Response
Reviewer #1 (Public Review):
This paper presents the results of two fragment screens of PTP1B using room-temperature (RT) crystallography, and compares these results with a previously published fragment screen of PTP1b using cryo-temperature crystallography. The RT screen identified fewer fragment hits and lower occupancy compared to the cryo screen, consistent with prior publications on other proteins. The authors attempted to identify additional hits by applying two additional layers of data processing, which resulted in a doubling in the number of possible hits in one of the screens. Because I am not an expert in panDDA modeling, however, I am unable to evaluate the reproducibility and potential potency of these fragment hits as protein binders or their potential use as starting points for follow-up chemistry.
The fragment library used in this study was larger than those used in previously published RT crystallography experiments. Among the cryo hits that bound in RT, most fragments bound in the same manner as they did in cryo, while some bound in altered orientations or conformations, and two bound at different locations in RT compared to cryo. This level of variability is not surprising. However, one fragment was observed to bind covalently to lysines in RT, even though it showed no density in the cryo crystallization attempt. It is unclear from the provided information whether this fragment decayed during storage or if the higher temperatures accelerated the covalent chemistry. The authors also observed temperature-dependent changes in the solvation shell, and modifications to the protein structure upon fragment binding, including a distal modification.
We thank the reviewer for the thorough summary of our manuscript.
Regarding reproducibility of fragment hits, cryo structures are more variable than RT structures for proteins themselves (Keedy et al., Structure, 2014). Thus the variability of repeated cryo-temperature crystallography experiments is a relevant consideration when comparing cryo to RT structures for protein-ligand interactions. However, to our knowledge, no published papers have explored this issue. Our previous cryo fragment screen (Keedy, Hill, et al., eLife, 2018), as with many others, was focused on breadth (many fragments), not depth (replicates). Unpublished work by some of the authors of the present study suggests that fragment poses are robust in replicate cryo experiments; however, future studies focused on fragment reproducibility in terms of binding occupancy, pose, and site at cryo temperature would be useful contributions to the field.
Regarding follow-up chemistry, there is growing evidence from multiple successful fragment-based inhibitor design studies (COVID Moonshot Consortium et al., bioRxiv, 2022; Gahbauer, Correy, et al., PNAS, 2023; etc.) that, although fragments usually bind too weakly to impact function on their own, they offer rich information to seed the design of high-affinity, potent functional modulators of proteins. As our study is the first to report many structures of fragments bound to proteins at RT, we cannot yet comment as to whether they offer unique advantages over cryo fragments in downstream fragment-based drug design efforts, but this is an open area for future study.
Regarding the covalent lysine binder, we agree with the reviewer on this point; our manuscript includes a note to this effect. Unfortunately we were unable to obtain the original fragment sample for mass spectrometry analysis. Returning to the point above about follow-up chemistry, the path forward for this fragment hit is promising and clear, and includes confirming chemistry using the original nominal compound vs. what is observed in the electron density, fragment linking and/or expansion, functional assays, and structural biology, all hopefully leading to a potent covalent inhibitor of wildtype PTP1B.
The current version of the paper is somewhat repetitive in its presentation of the results and could be clearer in its presentation of the variations and comparisons of the two different protocols. It would be helpful to have a more concise summary of the differences between the two protocols in the current paper, as well as a discussion of how they compare to the protocol used in the previously published cryo-temperature fragment screen.
We agree that it would be helpful to cut down on any redundant text and more straightforwardly compare/contrast the different room-temperature screen methods vs. the previous cryo-temperature screen method. To address this suggestion, we deleted the Discussion paragraph about the strengths and weaknesses of the two methods relative to serial approaches, deleted the text in the Introduction that introduces the two screens, and placed new text at the start of the Results section in the subsection titled “Two crystallographic fragment screens at room temperature” to provide a concise summary in one location of the manuscript.
While I appreciate the speculative nature of the discussion at the end of the paper, the evidence presented by the authors does not instil confidence that these results will correspond to meaningful binders that could be used to train future machine learning models. However, depending on the intended use, it may be acceptable to train ML models to predict expected densities under typical experimental conditions.
Indeed, this part of the Discussion is speculative, and seeks to place our results into a possible broader context. The definition of “meaningful binders” in the context of fragment screening is a difficult one. As noted above in response to the comment about follow-up chemistry, one important measure of meaningfulfulness is the ability to successfully seed structure-based design of analogs that have potent functional effects, and many fragments do meet this definition. Regarding potential applications to machine learning, we agree it is not self-evident that structural data for small-molecule fragments will be readily translatable to AI/ML methods aimed at larger compounds. The reviewer’s point about predicting densities is an intriguing one, and is in line with the fragment screening ethos, including existing experimental as well as computational (e.g. Greisman, Willmore, Yeh*, et al., bioRxiv, 2022) approaches to mapping ligandable surface sites and regions. The number of RT structures we report here is high relative to most crystallography studies, but still is likely insufficient to explore questions about AI/ML training, and at any rate would be beyond the scope of the current report. However, it seems equally true that AI/ML methods trained on structures based on data from nonphysiological cryogenic conditions, with associated structural artifacts, may have some (previously unrecognized) limitations, and thus RT crystal structures can play a useful role in AI/ML training sets in the future. We have added new text to the Discussion paragraph in question to convey these points.
Reviewer #2 (Public Review):
The authors set out to understand how a room-temperature X-Ray crystallography-based chemical-fragment screen against a drug target may differ from a cryo screen. They carried out two room-temperature screens and compared the results with that of a cryo screen they previously performed. With a substantial set of crystallographic evidence they showed that the modes of protein-fragment binding are affected by temperature. The conclusion of the work is compelling. It suggests that temperature provides another dimension in X-ray crystallography-based fragment screening. In a practical sense, it suggests that room-temperature fragment screen is a promising new avenue for hit identification in drug discovery and for obtaining insights into the fragment binding. Room-temperature screening carries unique advantage over cryo screening. This work is confirmative to the notion, which seems not yet universally considered, that very weak protein-small molecule binding may be inherently fluid structurally, and that crystal structures of such weak binding, especially cryo structures, cannot be taken for granted without cross validation.
We thank the reviewer for their clear summary and positive comments about our manuscript.
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eLife assessment
Based on two room-temperature X-ray crystallographic screens of PTP1B phosphatase against two sets of chemical fragments, and by comparing the results from a previous cryo screen, the authors report the important observation that, in addition to overlapping but non-identical sets of hits compared to the cryo screen, the room-temperature screens lead to significant differences in terms of binding sites and poses for some of the hits. The study provides compelling support for the use of room-temperature X-ray crystallography in early-stage drug discovery and highlights that temperature should be used as a parameter in efforts to extract additional insight from such analyses.
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Reviewer #1 (Public Review):
This paper presents the results of two fragment screens of PTP1B using room-temperature (RT) crystallography, and compares these results with a previously published fragment screen of PTP1b using cryo-temperature crystallography. The RT screen identified fewer fragment hits and lower occupancy compared to the cryo screen, consistent with prior publications on other proteins. The authors attempted to identify additional hits by applying two additional layers of data processing, which resulted in a doubling in the number of possible hits in one of the screens. Because I am not an expert in panDDA modeling, however, I am unable to evaluate the reproducibility and potential potency of these fragment hits as protein binders or their potential use as starting points for follow-up chemistry.
The fragment library …
Reviewer #1 (Public Review):
This paper presents the results of two fragment screens of PTP1B using room-temperature (RT) crystallography, and compares these results with a previously published fragment screen of PTP1b using cryo-temperature crystallography. The RT screen identified fewer fragment hits and lower occupancy compared to the cryo screen, consistent with prior publications on other proteins. The authors attempted to identify additional hits by applying two additional layers of data processing, which resulted in a doubling in the number of possible hits in one of the screens. Because I am not an expert in panDDA modeling, however, I am unable to evaluate the reproducibility and potential potency of these fragment hits as protein binders or their potential use as starting points for follow-up chemistry.
The fragment library used in this study was larger than those used in previously published RT crystallography experiments. Among the cryo hits that bound in RT, most fragments bound in the same manner as they did in cryo, while some bound in altered orientations or conformations, and two bound at different locations in RT compared to cryo. This level of variability is not surprising. However, one fragment was observed to bind covalently to lysines in RT, even though it showed no density in the cryo crystallization attempt. It is unclear from the provided information whether this fragment decayed during storage or if the higher temperatures accelerated the covalent chemistry. The authors also observed temperature-dependent changes in the solvation shell, and modifications to the protein structure upon fragment binding, including a distal modification.
The current version of the paper is somewhat repetitive in its presentation of the results and could be clearer in its presentation of the variations and comparisons of the two different protocols. It would be helpful to have a more concise summary of the differences between the two protocols in the current paper, as well as a discussion of how they compare to the protocol used in the previously published cryo-temperature fragment screen.
While I appreciate the speculative nature of the discussion at the end of the paper, the evidence presented by the authors does not instil confidence that these results will correspond to meaningful binders that could be used to train future machine learning models. However, depending on the intended use, it may be acceptable to train ML models to predict expected densities under typical experimental conditions.
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Reviewer #2 (Public Review):
The authors set out to understand how a room-temperature X-Ray crystallography-based chemical-fragment screen against a drug target may differ from a cryo screen. They carried out two room-temperature screens and compared the results with that of a cryo screen they previously performed. With a substantial set of crystallographic evidence they showed that the modes of protein-fragment binding are affected by temperature. The conclusion of the work is compelling. It suggests that temperature provides another dimension in X-ray crystallography-based fragment screening. In a practical sense, it suggests that room-temperature fragment screen is a promising new avenue for hit identification in drug discovery and for obtaining insights into the fragment binding. Room-temperature screening carries unique advantage …
Reviewer #2 (Public Review):
The authors set out to understand how a room-temperature X-Ray crystallography-based chemical-fragment screen against a drug target may differ from a cryo screen. They carried out two room-temperature screens and compared the results with that of a cryo screen they previously performed. With a substantial set of crystallographic evidence they showed that the modes of protein-fragment binding are affected by temperature. The conclusion of the work is compelling. It suggests that temperature provides another dimension in X-ray crystallography-based fragment screening. In a practical sense, it suggests that room-temperature fragment screen is a promising new avenue for hit identification in drug discovery and for obtaining insights into the fragment binding. Room-temperature screening carries unique advantage over cryo screening. This work is confirmative to the notion, which seems not yet universally considered, that very weak protein-small molecule binding may be inherently fluid structurally, and that crystal structures of such weak binding, especially cryo structures, cannot be taken for granted without cross validation.
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