Bruton’s tyrosine kinase (BTK) is targeted in the treatment of B-cell disorders including leukemias and lymphomas. Currently approved BTK inhibitors, including Ibrutinib, a first-in-class covalent inhibitor of BTK, bind directly to the kinase active site. While effective at blocking the catalytic activity of BTK, consequences of drug binding on the global conformation of full-length BTK are unknown. Here, we uncover a range of conformational effects in full-length BTK induced by a panel of active site inhibitors, including large-scale shifts in the conformational equilibria of the regulatory domains. Additionally, we find that a remote Ibrutinib resistance mutation, T316A in the BTK SH2 domain, drives spurious BTK activity by destabilizing the compact autoinhibitory conformation of full-length BTK, shifting the conformational ensemble away from the autoinhibited form. Future development of BTK inhibitors will need to consider long-range allosteric consequences of inhibitor binding, including the emerging application of these BTK inhibitors in treating COVID-19. Treatments for blood cancers, such as leukemia and lymphoma, rely heavily on chemotherapy, using drugs that target a vulnerable aspect of the cancer cells. B-cells, a type of white blood cell that produces antibodies, require a protein called Bruton’s tyrosine kinase, or BTK for short, to survive. The drug ibrutinib (Imbruvica) is used to treat B-cell cancers by blocking BTK. The BTK protein consists of several regions. One of them, known as the kinase domain, is responsible for its activity as an enzyme . The other regions of BTK, known as regulatory modules, control this activity. In BTK’s inactive form, the regulatory modules attach to the kinase domain, blocking the regulatory modules from interacting with other proteins. When BTK is activated, it changes its conformation so the regulatory regions detach and become available for interactions with other proteins, at the same time exposing the active kinase domain.Ibrutinib and other BTK drugs in development bind to the kinase domain to block its activity. However, it is not known how this binding affects the regulatory modules. Previous efforts to study how drugs bind to BTK have used a version of the protein that only had the kinase domain, instead of the full-length protein.Now, Joseph et al. have studied full-length BTK and how it binds to five different drugs. The results reveal that ibrutinib and another drug called dasatinib both indirectly disrupt the normal position of the regulatory domains pushing BTK toward a conformation that resembles the activated state. By contrast, the three other compounds studied do not affect the inactive structure. Joseph et al. also examined a mutation in BTK that confers resistance against ibrutinib. This mutation increases the activity of BTK by disrupting the inactive structure, leading to B cells surviving better.Understanding how drug resistance mechanisms can work will lead to better drug treatment strategies for cancer. BTK is also a target in other diseases such as allergies or asthma and even COVID-19. If interactions between partner proteins and the regulatory domain are important in these diseases, then they may be better treated with drugs that maintain the regulatory modules in their inactive state. This research will help to design drugs that are better able to control BTK activity. Bruton’s tyrosine kinase (BTK) is a non-receptor tyrosine kinase belonging to the TEC family . BTK is BTK has become an important pharmacological target  due to iDespite the success of Ibrutinib in the clinic, drug resistance is a major ongoing challenge. Based on previous kinase-targeted therapies, it has become clear that acquisition of drug resistance occurs via a variety of mechanisms including loss of drug binding, activation of the kinase, or the activation of downstream substrates . Of the Although an abundance of data including pharmacology, associated toxicities, metabolic processing, clearance, potency, and efficacy are available for the full-length BTK protein , the majTo expand our mechanistic understanding of drug resistance and the effect of different inhibitors on the full-length BTK protein, we developed solution-based Nuclear Magnetic Resonance (NMR) and Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) tools to probe local and long-range conformational consequences of resistance mutation and drug occupancy in the BTK active site. A panel of five BTK inhibitors, Ibrutinib, GDC-0853 (Fenebrutinib), CGI1746, CC-292 , and Dasatinib, was assembled based on availability of crystallographic data and commercial availability of each drug. Additionally, drug choices were prioritized to represent a range of kinase structures  and different inhibitor binding modes . All inhibitors in this study, except Dasatinib, have been developed as BTK-specific inhibitors.Our findings provide the first molecular insights into the conformational response of full-length BTK toward a spectrum of small-molecule active-site inhibitors. The inhibitors fall into two categories, differentiated by the presence or absence of an inhibitor-induced effect on the regulatory regions distant from the active site. We find that Ibrutinib and Dasatinib have profound effects on the entire BTK protein that extend beyond the active site. In contrast, GDC-0853, CGI1746, and CC-292 do not alter the conformational state of the regulatory domains. Additionally, we explore the mechanism of action of the T316A resistance mutation. This single remote amino acid mutation destabilizes the autoinhibited conformation of full-length BTK. We propose that an increase in the population of active enzyme contributes to the decreased Ibrutinib sensitivity observed in vivo. Taken together, these data provide a framework for developing new strategies to target BTK in the face of acquired drug resistance and reveal significant differences in the overall conformational preferences of full-length BTK bound to distinct small molecule inhibitors. We anticipate that further development of BTK inhibitors will rely on such information to fully understand, and ultimately control, the consequences of drug interactions within the full-length BTK protein.A conserved structural switch in the αC-helix of protein kinases plays a crucial role in controlling catalytic activity . The con1H-15N TROSY HSQC spectra . As previously reported (iv). The greater W395 upfield shift induced by Ibrutinib compared to apo full-length BTK (red versus gray dashed line) may be due to local chemical shift changes in the presence of Ibrutinib and/or further stabilization of the αC-out conformer.Next, we acquired brutinib , iv. As reported , full-lereported . Ibrutinreported , iv. Theiv, peak denoted with asterisk). This resonance is broadened beyond detection in the apo full-length BTK spectrum due to intermediate exchange and so its appearance upon addition of Ibrutinib suggests that dynamics in the SH3 domain region have been altered. We have previously found that the W251 indole resonance is also visible in spectra of BTK Y223A (iv) suggests that drug binding to the kinase active site shifts the conformational preference of the regulatory SH3 domain away from its autoinhibitory contacts on the distal side of the kinase domain. This observation prompted further exploration of the consequences of other BTK active site inhibitors on full-length BTK to determine whether this is a shared feature of active site inhibition of BTK.Ibrutinib binding to full-length BTK also results in appearance of a new peak corresponding to the indole NH resonance of a different tryptophan, W251 in the SH3 domain  and the activation loop is not visible in the electron density . Superpo1H-15N TROSY HSQC spectra of apo BTK LKD to BTK LKD bound to the five different active site inhibitors  mutant confirms the W395 assignments  conformation is stabilized in the kinase domain, while the regulatory domains are at least partially released from the distal surface of the kinase domain to populate an open ‘active’ conformation.Inhibitor binding to full-length BTK causes clear effects on conformation and dynamics as measured by HDX-MS, and there are stark differences between the different inhibitors  and 6. Cnterface  and 6. Snterface . TogetheThe allosteric effects of Ibrutinib binding to the BTK active site are quite distinct from the effects induced by GDC-0853 and CGI1746  and 6. DThe kinase domain conformation of the BTK:CC-292 complex shares structural similarity with the Ibrutinib, GDC-0853, and CGI1746 BTK complex crystal structures but the solution behavior of the CC-292 complex points to differences. While CC-292 is covalently attached to BTK C481 and contacts the hinge region of the kinase, it does not extend further into the kinase active site setting it apart from the other BTK inhibitors . ConsistHDX-MS of the BTK:Dasatinib complex shows that this complex exhibits the largest differences compared to apo full-length BTK  and 6. IRefocusing on the clinically important BTK inhibitor, Ibrutinib, we turn next to evaluating the cause of Ibrutinib resistance upon mutation of BTK T316 to alanine. Unlike the majority of Ibrutinib resistance mutations, including the most common C481S mutation in the kinase domain active site , BTK T31To more fully understand the consequences of the T316A mutation on BTK and to gain insight into how a mutation that is remote from the active site confers resistance to Ibrutinib, we applied NMR and HDX-MS approaches to the full-length BTK T316A mutant. Comparing the extent of deuterium uptake for wild-type BTK versus the T316A mutant shows small increases in exchange in the non-catalytic regulatory SH3-SH2 region upon mutation of T316 to alanine . Mapping1H-15N TROSY HSQC spectrum of the full-length BTK T316A to wild-type BTK shows a change in the conformational equilibrium upon mutation  population. Comparing the mutation . Peak inmutation . Consistmutation , the parmutation . To diremutation . BTK C48mutation . Thus, t50 values previously reported for Ibrutinib  in deuterium uptake are observed between wild-type BTK and the BTK T316A mutant over a range of Ibrutinib concentrations . Thus, oThe essential role that kinases play in cell signaling make them attractive pharmacological targets . The vasThe BTK T316A resistance mutant retains the ability to be covalently modified by Ibrutinib. Thus, when the BTK T316A mutant encounters Ibrutinib it is likely ‘trapped’ by the covalent drug in a manner similar to wild-type BTK. However, the BTK T316A mutation is itself activating; loss of the T316 sidechain destabilizes a key down-regulatory interaction within BTK and shifts the conformational equilibrium away from the autoinhibited state  and 8a. Our findings now show that mutation is not the only mechanism by which the BTK conformational ensemble shifts away from the ‘closed’ autoinhibited state. Binding of either Ibrutinib or Dasatinib triggers the same shift in the conformational equilibrium of the regulatory domains away from the autoinhibited conformation creating the opportunity for BTK regulatory domain interactions with exogenous ligands despite the inhibited kinase domain. This conformational shift could promote kinase-independent signaling events  and/or cOf the five active site inhibitors studied here only CGI1746, CC-292, and GDC-0853 inhibit kinase activity without disrupting the autoinhibitory conformation of the full-length kinase. Ibrutinib and Dasatinib alter the conformational equilibrium of the multidomain kinase decreasing the population of the autoinhibited kinase even though the drugs block and inactivate the kinase domain. The effect of an active site inhibitor on the overall conformational preference of a multidomain kinase must originate in the kinase domain. Multiple mobile elements including the αC-helix, DFG motif, and the activation loop switch the kinase domain between its inactive and active conformations . A numbeFor the SRC family, the αC-in configuration has been correlated with release of the regulatory domains from the autoinhibited state . Other sTo understand why Ibrutinib and Dasatinib disrupt the autoinhibited conformation of full-length BTK while GDC-0853, CGI1746, and CC-292 do not, we turn back to the crystal structures of BTK LKD bound to each inhibitor . ProbingIn accessing the back pocket of the active site, Ibrutinib and Dasatinib alter the back pocket around the regulatory (R-) spine  residue Structural studies that combine data from X-ray crystallography, solution NMR and HDX-MS provide a detailed and more complete picture to identify key regulatory interactions at work in protein kinases. In addition, use of the full-length kinase reveals unexpected differences between BTK inhibitors that are missed in crystallographic analyses of fragments. For BTK, this work has demonstrated that the clinically important inhibitor Ibrutinib imposes conformational adjustments on the distant non-catalytic domains of BTK, which may be functionally relevant in vivo. BTK inhibitors that do not induce a conformational shift toward the ‘open’ form of BTK may improve the efficacy of disease treatment where the kinase-independent functions of BTK play an important role. Additionally, targeting downstream BTK substrates such as PLCγ2 or combination therapies of Ibrutinib together with reversible active site/allosteric inhibitors that have a prolonged serum half-life may reduce the type of resistance embodied by the T316A mutation. The sustained presence of reversible inhibitors in the serum may block the activity of newly synthesized BTK T316A and prevent escape from Ibrutinib. Recognizing the conformational impact that mutations and inhibitors such as Ibrutinib have on full-length BTK should inform future strategies to effectively target this kinase in the context of disease.The bacterial expression constructs for murine BTK LKD, SH3-SH2-Linker-kinase domain (32LKD) and full-length (FL) have been described previously . All BTK2PO4, pH 8.0, 150 mM NaCl, 20 mM imidazole and 0.5 mg/ml lysozyme) and stored at −80°C. Cells were lysed by thawing and the action of lysozyme, and 3000 U DNAse I (Sigma) and 1 mM PMSF were added to the lysate, incubated at RT for 20 min and then spun at 16,000 rpm for 1 hr at 4°C. Glycerol was added to the supernatant to a final concentration of 10% and was then incubated with Ni-NTA resin (QIAGEN) for 2 hr, washed with Tris pH 8.0, 75 mM NaCl, 40 mM imidazole, and eluted in 20 mM Tris pH 8.0, 150 mM NaCl, 250 mM Imidazole, and 10% glycerol. Eluted protein was flash frozen in liquid nitrogen and stored at −80°C. The proteins were concentrated and dialyzed into the final NMR buffer which consists of 20 mM Tris pH 8.0, 150 mM Sodium chloride, 0.02% Sodium azide, and 10% glycerol. For HDX-MS analysis, the proteins were further purified by size exclusion chromatography . The fractions containing pure protein were pooled, concentrated, snap frozen and stored at −80° C. The final buffer consists of 20 mM Tris pH 8.0, 150 mM Sodium chloride, 0.02% Sodium azide and 10% glycerol. Phosphorylation level on Y551 of all purified BTK proteins used in this study is below western immuno-detection.Full-length, kinase-active BTK, 32LKD, and BTK LKD were produced by co-expressing BTK with YopH in BL21(DE3) (Millipore Sigma) or BL21-Gold(DE3) cells (Agilent Technologies) as described previously . Briefly15N-labeled BTK samples were produced as described earlier by growth in modified M9 minimal media containing 15N ammonium chloride  as the sole source of nitrogen  with a resolution of 0.10 Da and an output mass range of 65,000–90,000 Da.Prior to intact mass analysis, purified BTK full-length wild-type (20 μM) and Ibrutinib (40 μM), both in 20 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, 2% DMSO, were allowed to interact at 21°C for 1 hr. Both the free wild-type BTK, as a control, and Ibrutinib-labeled proteins (70 picomoles) were injected into a Waters nanoACQUITY with HDX technology set-up for intact mass analysis at 20°C. The proteins were injected into the sample loop and desalted for 3 min at 100 μL/min using water (0.1% formic acid) on an in-house packed POROS 20 R-2 trap. Proteins were eluted into the mass spectrometer using a 15–70% ACN (0.1% formic acid) gradient in 10 min at a flow rate of 100 μL/min. Mass spectra were acquired using a Waters Synapt HDMSE mass spectrometer. Peptides generated from online pepsin digestion were identified with Waters Protein Lynx Global Server 3.0 using separate unlabeled protein that was prepared in the same manner as protein labeled with deuterium. Deuterium incorporation was quantified using Waters DynamX 3.0. Deuterium levels for each peptide were calculated by subtracting the average mass of the undeuterated control sample from that of the deuterium-labeled sample; the data were not corrected for back exchange and are therefore reported as relative. Vertical difference maps in General procedures for HDX-MS of BTK have been described in detail previously . Details2, 1 mM DTT, 5% glycerol, 1 mM Pefabloc, and 200 μM ATP) at room temperature for 10 min. The reactions were stopped by the addition of SDS-PAGE loading buffer, and the samples were boiled, separated by SDS−PAGE, and western blotted with the anti-BTK pY551 antibody (BD Pharmingen) or anti-His antibody (EMD Millipore) as described previously .Thank you for submitting your article "BTK regulatory domains escape Ibrutinib inhibition" for consideration by eLife requires that both papers be merged into one (see reviews below). We are providing the same merged review for both papers. Note that only one resubmission link is provided for both papers.Despite the overall favorable response to both papers, the reviewers and the editors are in agreement that further consideration at eLife. The reviewers are strongly supportive of further consideration of a single merged manuscript. We recognize, however, that there may be important factors that prevent you from merging the two papers. Please let us know if that is the case – we will then release the two papers from eLife, allowing you to seek rapid publication of both papers elsewhere. On the other hand, support for a merged publication at eLife is strong, and we anticipate being able to give you a rapid decision after receiving a single merged resubmission.The reviewers agree that the two papers  jointly represent substantial and important advances in the understanding of BTK, a clinically important kinase. Nevertheless, the reviewers have also identified some key weaknesses in each paper that would, on balance, preclude further consideration of the individual papers at Note that new experiments are not required in order to respond to the review – textual justifications in the merged manuscript will suffice. If you do have additional data that support points made in the revised manuscript we would, of course, welcome them.SummaryThe work by Joseph, Amatya et al. "Differential impact of BTK active site inhibitors on the conformational state of full-length BTK" examines the mechanism of inhibition of different Bruton's tyrosine kinase (BTK) active site inhibitor binding on the conformation of the protein.This study addresses the allosteric effects of ATP-competitive inhibitors for Bruton's tyrosine kinase (BTK) and their mechanism. Using NMR and HDX solution measurements, the study compares one covalent inhibitor (ibrutinib) and four non-covalent inhibitors . The study reports three findings: First, NMR shows variable effects on W395 indole NH, which based on NMR and X-ray comparisons of linker-kinase BTK, reports active and inactive conformations of helix aC. These solution conformations differ from those predicted based on X-ray structures of each inhibitor bound to the BTK kinase domain. Second, HDX measurements show variable effects of inhibitors on FL-BTK. The results reveal clear conformational differences between BTK:drug complexes in solution, and an unexpected effect of dasatinib on increased conformational mobility that propagates throughout the kinase domain. Third, the binding interactions of each inhibitor with active site elements are used to construct a structural model which explains why only ibrutinib and dasatinib disrupt the remote SH3-SH2 interactions. The model proposes that L460 and/or T474 relay conformational changes from the active site to the regulatory domains through connections between R spine residues. This is then tested by mutation of L460A, which elevates HDX throughout the SH3-SH2 region as well as within the kinase domain, supporting its likely role in maintaining the allosteric connection between the active site and SH3-SH2 . The T474A mutation does not affect the SH3-SH2 and kinase domain, but increases HDX in the linker region and autoinhibitory PHTH domain. Both mutants interfere with kinase activity partially (T474A) or fully (L460A).The strengths of this manuscript are its analysis of the solution behavior of full length BTK using a powerful combination of NMR and HDX. The results establish evidence for the remote regulation of the SH3-SH2 regulatory domain, variable impact of different inhibitors on this allosteric mechanism, and variable conformational selection by different inhibitors within the active site. The model for allostery involving L460 as a sensor linking the kinase active site to the regulatory domains is exciting, and at least for ibrutinib is supported by HDX evidence. A principal weakness of the study is the limited rigorous mechanistic understanding of the effects of inhibitors on BTK conformation.In Joseph, Wales, et al. "BTK regulatory domains escape Ibrutinib inhibition", the authors examine full length BTK using 1H-15N NMR and HDX-MS to investigate effects of an irreversible covalent inhibitor, ibrutinib, and a T316A drug resistance mutation whose mechanism is unknown. There are three main findings: First, NMR and HDX show that the inhibitor preserves the conformation of the kinase domain predicted from the X-ray structure, but disrupts remote autoinhibitory contacts between SH3-SH2 and the linker-kinase, leading to behavior resembling that of the partially active form. Second, the T316A resistance mutation enhances BTK kinase activity, while increasing HDX in the SH3-SH2 domains, consistent with disengagement to a partially activated state, and shifting a W395 NH resonance towards a productive active site conformation. Third, the effect of ibrutinib on remote conformational changes is nearly the same between WT-BTK and the T316A mutant. The study concludes that ibrutinib has long distance allosteric effects, displacing the SH3 and SH2 domains from their compact autoinhibited conformation and that T316A resistance mutation located outside of the kinase domain works by increasing the catalytic activity of BTK.Strengths of the manuscript are its unique combination of NMR and HDX, and its excellent illustration of the power of this approach in probing regulatory conformational changes, in a way that has been elusive using X-ray crystallography. The description of NMR assignments are rigorous and the data interpretation, particularly with HDX are accurate and not overstated. There are few studies of full-length BTK, and together with the co-submitted article, the manuscript makes a clear case for allostery unique to the full-length form that can be regulated by a clinically important inhibitor and a poorly understood resistance mechanism. Weaknesses are that the manuscript is confusing to understand without extensive reading of previous articles in Structure 2017 and JMB 2016. It would benefit from revisions that clarify a number of ambiguities and confusing terminologies. In addition, the study needs a more convincing explanation for why the results necessarily explain drug resistance by T316A. HDX-MS experiments indicate that despite stabilizing an inactive conformation of the kinase domain, ibrutinib induces disassembly of the inhibitory interactions with the SH3 and SH2 domains. This is a surprising finding that could have important implications for the effects of the drug, which is an FDA-approved therapeutic. Studies with the T316 mutant show that it induces disassembly of the SH3/SH2 interactions and consequently enhances BTK catalytic activity, but the molecular explanation for this is unsatisfactory.Comments to address when revising and merging the manuscripts:A) Specific comments pertaining to Joseph, Amatya, et al. "Differential Effects of BTK active site inhibitors.…"Major points to address:1) There is concern that the L460 and T474 mutations are hard to interpret – they are non-conservative substitutions in key regions of the kinase domain that almost certainly destabilize it to some extent, and can therefore be expected to affect SH3/SH2 regulatory interactions. Indeed the authors see this for the L460A mutation. So it may be difficult to ascribe HDX changes with inhibitors to a role for these residues in allosteric communication. It is possible that similar results with many non-conservative substitutions in this region of the kinase domain. The authors should consider either removing these experiments, or justify their inclusion with additional text.2) "The BTK:CC292 complex shares structural similarity with the ibrutinib, GDC and CGI BTK complex crystal structures but the solution behavior of the CC292 complex is distinct. HDX reveals only limited protection upon CC292 binding localized primarily to the N-lobe surrounding the active site."Detailed analyses of drug-enzyme interactions are needed to support this statement. Superficial examination of Figure 1A suggests that the BTK:CC292 cocrystal structure might have more space for water in the active site.Related to this point, it would be helpful to note in the manuscript which of these structures were generated from co-crystallized proteins and which were generated by soaking.3) T474A does not phenocopy effects of L460A on HDX . Therefore, it was hard to understand how the proposed mechanism explains why dasatinib necessarily phenocopies ibrutinib effects on HDX within the remote SH3-SH2 regions. The conclusions about T474A are not integrated very well with results in Figures 3 and 4 to explain the distinct effects of dasatinib. The authors propose that both ibrutinib and dasatinib perturb L460 in different ways, where ibrutinib interacts directly with L460 to push helix aC out, while dasatinib interacts with T474 to allow helix aC-in, which also perturbs the back pocket. The part of the mechanism involving dasatinib is largely based on the X-ray structure, which seems incongruent with the conclusion from Figure 3 that the Xray structures for CC292 and dasatinib are not representative of their solution conformations. Therefore, it seems possible that perturbation of T474 (the gatekeeper) signals to the remote SH3-SH2 by an alternative route. Perhaps prior findings in JMB 2016 showing that T474M activates the kinase would be useful to integrate into this Discussion. It might also be useful to show the outcomes of Figure 8 experiments using dasatinib instead of ibrutinib.Overall these two large sections read somewhat incoherently, and it was difficult to parse out which aspects were supported by evidence and which were speculation. The sections need to be reconsidered or explained more carefully.4) The authors should place their model in the context of “Conformational Selection” , which is a general mechanism that could explain all their results.B) Specific comments pertaining to Joseph, Wales, et al. (BTK regulatory domains escape.…)Major points to address:1) It would help to clarify that the active mutants used in the prior work as well as T316A in this study only activate BTK by 2-3x. Therefore all mutants cause only a partially active state of BTK, that is intermediate between the autoinhibited and fully active kinase. This was hard to grasp from the manuscript, but explained in a short review of the 2017 Structure paper by Agnew and Jura. Thus the current manuscript is somewhat misleading in using "active conformation" terminology throughout. Is there work comparing HDX in full length BTK vs SH3 and SH2 domains alone? It is possible that the HDX increase would be much greater in fully activated BTK than in the 2-fold activated mutants. It would help to say this, and explain that the mutants increase HDX by only 1-2 Da because the detachment reflects only a partially activated intermediate conformation.Related to this, can any form of FL-BTK yield full activation in vitro? For example, does stoichiometric phosphorylation at Y551 by Lyn and/or autophosphorylation at Y223, with or without PIP3 binding, result in a fully active kinase? If available, it would be useful to know how HDX of SH3-SH2 in the fully activated state compares to that of the mutants.2) Given prior data, including the crystal structure of the BTK kinase domain with ibrutinib and crystal structure of a SH3/Sh2/kinase fragment of BTK in the autoinhibited state, it might be expected that ibrutinib would tend to stabilize the assembled, autoinhibited state of BTK because it stabilizes the inactive conformation of the kinase domain (which is in turn coupled to SH3/SH2 interactions). But the present HDX-MS data clearly suggests otherwise. Is it possible that the destabilization arises from binding of ibrutinib to a solvent exposed cysteine elsewhere in BTK – for example the one in the SH2 domain? The authors present beautiful mass spec data showing 1:1 labeling with ibrutinib, but was this labeling experiment carried out under the same conditions as ibrutinib binding for HDX-MS? Also the mass spec experiment shows not even a trace of unlabeled BTK or double labeled BTK – was some sort of mass selection or filtering applied in this experiment to select the singly-labeled species? Also, Materials and methods should be provided for this mass spec experiment.3) It was unconvincing that T316A should promote resistance by activating FL-BTK.First, the extent of activation by the mutation was only 2-fold. The assay measures autophosphorylation at Y551, which only occurs in vitro and might be irrelevant to cellular activation, therefore might fail to report potentially larger effects of the mutation on activity measured with a true substrate or autophosphorylation at pY223.Related to point #3 above, what is the effect of the T316A mutation on the activity of a fully activated BTK (if it exists), and the degree of its inhibition by ibrutinib? Perhaps T316A increases activity to a greater extent when BTK is fully phosphorylated.Second, as noted by the authors, the irreversible coupling of ibrutinib might mask any effects of the mutant on binding affinity. The latter might be detectable using BTK-C481A to measure ibrutinib binding, or by measuring rates of covalent coupling of ibrutinib to BTK at varying concentrations of a competing inhibitor.Third, elevated activity of unbound BTK-T316A would not be expected to bypass the effects of drug, if drug truly binds the mutant in cells.4) Enzymes exist in ensembles of multiple conformations in solution. This is presented well with the TROSY 1H15N spectra in Figure 2. Crystallography provides high resolution snapshots of only one likely conformation. It is erroneous for the authors to validate their ensemble measurements with crystal structures. The conclusions from the NMR titrations in Figure 2A are correlated with inactive state in Figure 1D. If anything, the authors' results caution against relying on simplistic single snapshot conformational analyses to explain allostery.5) It will greatly help the reader if the authors discuss and reassess their results on the basis of “Conformational Selection”, “Induced Fit” or a combination of both models. It could be argued that the inhibitor favors one conformation and shifts the equilibrium. The authors throughout the manuscript imply Ibrutinib binding allosterically displaces the remote regulatory domain- and then there is a notion of regulatory domains escaping the inhibitor from the title.Supporting the ensemble behavior and conformational selection, the authors conclude from the NMR and HDXMS studies that the T316A mutant shifts the ensemble to a more active kinase conformation consistent with a mutation-induced bias toward the active state.6) An important control would be to compare HDXMS upon Ibrutinib binding with a non-specific covalent modification of BTK at C481 with biotin maleimide for instance. An alternate control would be analysis of the C481M mutant, which might mimic the covalent modification of C481. Have such experiments been done?In this context, the noncovalent interaction of Ibrutinib with C481S is intriguing. Does it give the same equivalent inhibitory effect? Does it compete equivalently with ATP? Are the HDX-MS differences equivalent to wt C481?eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and John Kuriyan as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Natalie G Ahn (Reviewer #1); Ganesh Srinivasan Anand (Reviewer #2); Michael J Eck (Reviewer #3).Thank you for submitting your revised article "Differential impact of BTK active site inhibitors on the conformational state of full-length BTK" for consideration by eLife. Note that one of the reviewers has raised some important points of clarification. Please edit the manuscript to incorporate your response to these comments. We leave this to your discretion, and the revised manuscript will be accepted for publication immediately, without further editorial intervention.Your paper is now suitable for publication in Reviewer #1:The revised manuscript addresses all of my concerns raised in the initial review and is greatly improved in coherency and readability. The current manuscript represents a valuable contribution that illustrates the power of combined NMR and HDX-MS approaches to understand solution conformational changes caused by ligand binding to and mutations of protein kinases that are clinically targeted.Reviewer #2:The revised manuscript is much improved and I support publication of the work.The following points should be addressed by the reviewers:1) In the Abstract the authors state "including unexpected shifts in theconformational equilibria of the regulatory domains." Allosteric propagation from catalytic to regulatory domains are more the norm than the exception. The authors should consider replacing “unexpected” with “propagated” or large-scale etc.2) The authors refer to the comparative magnitude protection in deuterium exchange for one drug molecule compared to another as “more intense”. Protection is either more or less! “Intense” is better suited to describing function.3) I would like to point the authors to a recent publication that addresses integrative allostery by two drugs on kinases by Ghode et al., 2020, Biophysical Journal "Synergistic allostery in multiligand-protein interactions"Reviewer #3:eLife.The combined manuscript is much improved and the authors have addressed my concerns. I support publication of the revised work in  Comments to address when revising and merging the manuscripts:A) Specific comments pertaining to Joseph, Amatya, et al. "Differential Effects of BTK active site inhibitors.…"Major points to address:1) There is concern that the L460 and T474 mutations are hard to interpret – they are non-conservative substitutions in key regions of the kinase domain that almost certainly destabilize it to some extent, and can therefore be expected to affect SH3/SH2 regulatory interactions. Indeed the authors see this for the L460A mutation. So it may be difficult to ascribe HDX changes with inhibitors to a role for these residues in allosteric communication. It is possible that similar results with many non-conservative substitutions in this region of the kinase domain. The authors should consider either removing these experiments, or justify their inclusion with additional text.As per the reviewer’s suggestion, experiments involving BTK L460 and T474 have been removed from the merged manuscript. The potential role of L460 is now described exclusively in the Discussion section.2) "The BTK:CC292 complex shares structural similarity with the ibrutinib, GDC and CGI BTK complex crystal structures but the solution behavior of the CC292 compex is distinct. HDX reveals only limited protection upon CC292 binding localized primarily to the N-lobe surrounding the active site."Detailed analyses of drug-enzyme interactions are needed to support this statement. Superficial examination of Figure 1A suggests that the BTK:CC292 cocrystal structure might have more space for water in the active site.This section of the manuscript has been edited and expanded to discuss the occupancy of CC-292 within the active site as suggested.Related to this point, it would be helpful to note in the manuscript which of these structures were generated from co-crystallized proteins and which were generated by soaking.The figure caption  has been modified to address this point.3) T474A does not phenocopy effects of L460A on HDX . Therefore, it was hard to understand how the proposed mechanism explains why dasatinib necessarily phenocopies ibrutinib effects on HDX within the remote SH3-SH2 regions. The conclusions about T474A are not integrated very well with results in Figures 3 and 4 to explain the distinct effects of dasatinib. The authors propose that both ibrutinib and dasatinib perturb L460 in different ways, where ibrutinib interacts directly with L460 to push helix aC out, while dasatinib interacts with T474 to allow helix aC-in, which also perturbs the back pocket. The part of the mechanism involving dasatinib is largely based on the X-ray structure, which seems incongruent with the conclusion from Figure 3 that the Xray structures for CC292 and dasatinib are not representative of their solution conformations. Therefore, it seems possible that perturbation of T474 (the gatekeeper) signals to the remote SH3-SH2 by an alternative route. Perhaps prior findings in JMB 2016 showing that T474M activates the kinase would be useful to integrate into this Discussion. It might also be useful to show the outcomes of Figure 8 experiments using dasatinib instead of ibrutinib.Overall these two large sections read somewhat incoherently, and it was difficult to parse out which aspects were supported by evidence and which were speculation. The sections need to be reconsidered or explained more carefully.As per the reviewer’s suggestion earlier (in point #1), experiments involving BTK L460 and T474 have been removed from the merged manuscript. Instead we now discuss a potential role for the back pocket residue L460 (part of the regulatory spine) in the Discussion section.4) The authors should place their model in the context of “Conformational Selection” , which is a general mechanism that could explain all their results.We agree that conformational selection is an appealing mechanism to explain the observations in this work. However, unlike the excellent study published very recently on ABL , we have not characterized the distinct conformations populated by full-length apo BTK and so it is not possible to unequivocally ascribe the changes we observe upon drug binding or mutation to a conformational selection model, induced fit model, or a combination of both.B) Specific comments pertaining to Joseph, Wales, et al. (BTK regulatory domains escape.…)Major points to address:1) It would help to clarify that the active mutants used in the prior work as well as T316A in this study only activate BTK by 2-3x. Therefore all mutants cause only a partially active state of BTK, that is intermediate between the autoinhibited and fully active kinase. This was hard to grasp from the manuscript, but explained in a short review of the 2017 Structure paper by Agnew and Jura. Thus the current manuscript is somewhat misleading in using "active conformation" terminology throughout. Is there work comparing HDX in full length BTK vs SH3 and SH2 domains alone? It is possible that the HDX increase would be much greater in fully activated BTK than in the 2-fold activated mutants. It would help to say this, and explain that the mutants increase HDX by only 1-2 Da because the detachment reflects only a partially activated intermediate conformation.This is an excellent point. We have previously compared isolated SH3 and SH2 domains to full-length BTK by HDX-MS and indeed the magnitude of the increase in deuterium uptake is small in the full length T316A mutant when compared to either 1) the isolated domains, 2) the D656K activating mutation or perhaps more relevant, 3) binding of full-length BTK to PIP3, an activating ligand that binds the PH domain . This suggests that the T316A mutation induces a small shift in the conformational ensemble of the regulatory domains of full-length BTK away from the autoinhibited conformation (as compared to the larger shift promoted by PIP3 binding). This is now carefully acknowledged in the text describing the effect of T316A.The manuscript has now been modified throughout to clearly indicate the conformational state probed by the NMR experiment. The “active” versus “inactive” state terminology has been replaced in the NMR experiments to reflect the αC-in versus -out conformation of the kinase domain. A panel has been added to Figure 2 to more clearly show the αC helix and W395 conformations in structures of the active and inactive kinase. The HDX-MS changes are interpreted in the context of the autoinhibited structure since this is the only available multidomain structure for BTK. The data show the location of changes that are consistent with release of the regulatory domains from the kinase domain.Related to this, can any form of FL-BTK yield full activation in vitro? For example, does stoichiometric phosphorylation at Y551 by Lyn and/or autophosphorylation at Y223, with or without PIP3 binding, result in a fully active kinase? If available, it would be useful to know how HDX of SH3-SH2 in the fully activated state compares to that of the mutants.The PIP3 bound, Y551 phosphorylated full-length BTK protein is presumed to be the fully activated state, however we have not characterized this form due to challenges associated with producing sufficient uniformly Y551 phosphorylated sample. For this reason, all NMR and HDX-MS experiments have been carried out using unphosphorylated BTK to ensure sample homogeneity.2) Given prior data, including the crystal structure of the BTK kinase domain with ibrutinib and crystal structure of a SH3/Sh2/kinase fragment of BTK in the autoinhibited state, it might be expected that ibrutinib would tend to stabilize the assembled, autoinhibited state of BTK because it stabilizes the inactive conformation of the kinase domain (which is in turn coupled to SH3/SH2 interactions). But the present HDX-MS data clearly suggests otherwise. Is it possible that the destabilization arises from binding of ibrutinib to a solvent exposed cysteine elsewhere in BTK – for example the one in the SH2 domain? The authors present beautiful mass spec data showing 1:1 labeling with ibrutinib, but was this labeling experiment carried out under the same conditions as ibrutinib binding for HDX-MS? Also the mass spec experiment shows not even a trace of unlabeled BTK or double labeled BTK – was some sort of mass selection or filtering applied in this experiment to select the singly-labeled species? Also, Materials and methods should be provided for this mass spec experiment.In the HDX MS experiments, we were not able to identify any cysteine-containing peptides that were conjugated to Ibrutinib, including those peptides containing C481. However, peptic peptides that contain C481 were absent only in the Ibrutinib labeled BTK samples while all other C containing peptides retained signal in the mass spectrometer suggesting that Ibrutinib conjugated only to C481. We are therefore confident, when combined with intact MS analysis , that there were no substantial sideconjugation reactions to other cysteines in the protein.Thank you for pointing out our error in not describing the methods for the acquisition of the intact mass data. The complete methods for the intact mass analyses are now included in the Materials and methods section of the text. These added methods describe that intact mass analysis was performed under the same conditions as those for the HDX MS experiments, and during mass spectral acquisition there was no mass selection or filtering applied for either the Ibrutinib conjugated or free wild-type BTK. Mass spectra were also acquired after 4 hours of labeling BTK with Ibrutinib and still there was a single mass adduct (data not shown).3) It was unconvincing that T316A should promote resistance by activating FL-BTK.First, the extent of activation by the mutation was only 2-fold. The assay measures autophosphorylation at Y551, which only occurs in vitro and might be irrelevant to cellular activation, therefore might fail to report potentially larger effects of the mutation on activity measured with a true substrate or autophosphorylation at pY223.Related to point #3 above, what is the effect of the T316A mutation on the activity of a fully activated BTK (if it exists), and the degree of its inhibition by ibrutinib? Perhaps T316A increases activity to a greater extent when BTK is fully phosphorylated.We acknowledge that the kinase assay measuring phosphorylation on Y551 might occur only in vitro. However, this assay does report on the intrinsic activity of the kinase and shows a 2-fold increase in activity which is consistent with the population shift measured by NMR. The drug resistance mutation in the ABL SH2 domain, T231R, has been studied extensively and, while this substitution directly stabilizes the active ABL conformation, the fold activation observed using an in vitro kinase assay is also two-fold . In this case drug resistance is also thought to arise from a conformational shift away from a form to which the drug binds. While the T316A mutation could have additional effects in the context of a fully phosphorylated form of BTK, the impact of the mutation on the unphosphorylated protein is likely playing a role in Ibrutinib resistance in vivo given the pharmacokinetics of this drug. Finally, we are aware of data  supporting a resistance causing mutation in a SRC module kinase that does not affect drug binding to the active site.Second, as noted by the authors, the irreversible coupling of ibrutinib might mask any effects of the mutant on binding affinity. The latter might be detectable using BTK-C481A to measure ibrutinib binding, or by measuring rates of covalent coupling of ibrutinib to BTK at varying concentrations of a competing inhibitor.We did consider these approaches to measure binding affinity. Using the C481A mutant to measure binding affinity of Ibrutinib would be an approximation and not reflect the true binding kinetics of a covalent inhibitor. The use of a competing inhibitor (eg. fluorescent tagged ADP) would require that it binds with the same affinity to both the wild-type and mutant proteins. However, previous work has shown that destabilization of the autoinhibited conformation changes the affinity for ADP. Given these complications, we tested binding using HDX-MS and acknowledge the deficiencies in the approach.Third, elevated activity of unbound BTK-T316A would not be expected to bypass the effects of drug, if drug truly binds the mutant in cells.The pharmacokinetics of an inhibitor is a key factor that must be taken into consideration in assessing mutational outcomes. Covalent inhibitors such as Ibrutinib, unlike reversible inhibitors, are rapidly cleared from the serum and Ibrutinib levels drop to basal levels within 4 hours. Newly synthesized BTK T316A could escape this narrow inhibition time window. The unconjugated T316A mutant therefore could promote BTK signaling due to its elevated activity, unlike wild-type BTK which is autoinhibited. In the merged manuscript we address this at the beginning of the Discussion section. In addition, we have acknowledged in this section that the T316A mutation could also adversely affect Ibrutinib binding to the active site thereby contributing to resistance especially in the context of activation loop phosphorylation.4) Enzymes exist in ensembles of multiple conformations in solution. This is presented well with the TROSY 1H15N spectra in Figure 2. Crystallography provides high resolution snapshots of only one likely conformation. It is erroneous for the authors to validate their ensemble measurements with crystal structures. The conclusions from the NMR titrations in Figure 2A are correlated with inactive state in Figure 1D. If anything, the authors' results caution against relying on simplistic single snapshot conformational analyses to explain allostery.We agree that our solution work are ensemble measurements and that crystal structures provide snapshots of a single conformation. Inhibitor and mutational effects are interpreted as shifts in this conformational equilibrium and the crystal structures are used as a guide to interpret these changes.5) It will greatly help the reader if the authors discuss and reassess their results on the basis of “Conformational Selection”, “Induced Fit” or a combination of both models. It could be argued that the inhibitor favors one conformation and shifts the equilibrium. The authors throughout the manuscript imply Ibrutinib binding allosterically displaces the remote regulatory domain- and then there is a notion of regulatory domains escaping the inhibitor from the title.Supporting the ensemble behavior and conformational selection, the authors conclude from the NMR and HDXMS studies that the T316A mutant shifts the ensemble to a more active kinase conformation consistent with a mutation-induced bias toward the active state.It is noteworthy that the recent publication referenced above  is one of the few examples in the kinase field where multiple structures within an ensemble have been detected and structures solved so that conformational shifts induced by mutations could be correlated to structures that exist in the ensemble of the wild type protein – supporting conformational selection. The NMR approach used in these studies (CEST) requires long acquisition times that might prohibit application to full-length BTK.6) An important control would be to compare HDXMS upon Ibrutinib binding with a non-specific covalent modification of BTK at C481 with biotin maleimide for instance. An alternate control would be analysis of the C481M mutant, which might mimic the covalent modification of C481. Have such experiments been done?In this context, the noncovalent interaction of Ibrutinib with C481S is intriguing. Does it give the same equivalent inhibitory effect? Does it compete equivalently with ATP? Are the HDX-MS differences equivalent to wt C481?Ibrutinib and CC-292  are both covalent inhibitors that modify BTK C481. CC292 can therefore be considered as a control for Ibrutinib and in the general sense modification at C481 – the consequences of Ibrutinib and CC-292 on the overall conformational preferences and dynamics of full-length BTK are strikingly different.50 for Ibrutinib in vivo. ATP binding to the BTK C481S mutant has not been tested. Our NMR and HDX-MS analysis of the C481S mutant and wild-type protein show no significant differences between the apo proteins  or when bound to Ibrutinib (data not shown). It is likely that loss of the covalent bond to Ibrutinib results in a faster off rate and subsequent clearance of Ibrutinib from the serum, in other words BTK C481S is not “trapped” by the drug.C481S is an Ibrutinib resistance mutation. The C481S mutant exhibits an increase in IC


Lipid synthesis is the recently found metabolism of cancer cells after their metastasis to lymph nodes (LNs). Carbonic acid is the main byproduct of the lipid metabolism in such cells which resulted in acidification of LN ambient. Hence, calibrated pH sensing could be a diagnostic method to find involved LNs. Here, we designed a simple pH sensing method by a syringe containing sterile PBS and embedded by litmus paper to intraoperatively check the pH of LN fluid. Injected phosphate buffer saline (PBS) would homogenize the LN fluid and litmus needle would detect the pH of the LN. We presented an experimental pathological calibration for the pH values in correlation with cancerous states of the LNs. This system named metabolism based metastatic lymph diagnoser (MMLD) could be a real-time noninvasive tool for precise and fast diagnosis of involved LNs. Lymph nodes are small glands that filter lymphatic fluid (the clear fluid that circulates through the lymphatic system) . The lymCurrently, the only common method for the detection of metastatic cancer cells in the lymph (sentinel and auxiliary) nodes during surgery is the frozen pathology which is highly dependent on the experience and skills of the pathologist. Due to the lack of time in the frozen section to respond to the surgeon, just part of the removed lymph node  is often selected for frozen examination . So, in One of these works is to inject an anti-epidermal growth factor (EGFR) or anti-herceptin2 (HER2) fluorescent substances followed by a 99mTc-phytate radioactive material around the mouse primary tumor ,14. UsinSimilarly, tissue impedance imaging techniques were also used to detect cancer cells in the lymph nodes. The average time to scan an area by this method is approximately 5–10 min .Although above methods made many improvements in intraoperative diagnosis of metastatic SLNs, still we haven’t a real-time noninvasive, simple, and reliable method to detect metastatic lymph nodes.Here, we reported an accurate simple method to diagnose involved LNs based on pH sensing by a litmus paper  embedded into a gauge 18 syringe needle (named as Metabolism based Metastatic Lymph Diagnoser (MMLD)), without any pre-preparation process which declared LN metastasis in less than 30 s. This process has been based on acidification of involved LNs due to fatty acid oxidation metabolism of cancer cells in LNs which was published recently . By caliThe system contains a micro-syringe  filled by 100 µl of injectable sterile PBS and a syringe needle embedded by a thin litmus paper. The mechanism was entering the needle, injection of PBS rubbing the LNs, and checking the color of litmus paper which indicates the acidity or basicity of the lymph node fluid based on lipid synthesis of cancer cells in LNs. This test can be individually repeated by three needles and if even one of the needles showed acidic pH with pH lower than 6 , then the LN would be declared as involved LNs.In vitro fresh lymph node samples prepared from 25 patients’ candidate for breast cancer were recorded from 2018-10-30 to 2019-11-30.Patients provided consent according to an ethically approved protocol (IR.TUMS.VCR.REC.1397.532) at our breast cancer central clinics and assistant hospitals. Hematoxylin and eosin (H&E) staining is the most routine staining procedure in histopathology. This method is a combination of two dyes. H&E staining is used to demonstrate the nucleus and cytoplasmic inclusions in clinical specimens. Hematoxylin, which contains Alum, stains the nucleus in light blue. In the presence of an acid, the dye turns into the red. Therefore, by treating the tissue with an acid solution, the differentiation is achieved. In the bluing step, the initial soluble is converted to red color within the nucleus to an insoluble blue color. By utilizing Eosin, the counterstaining is done, which imparts pink color to the cytoplasm. The initial step of the H&E staining process is deparaffinized a tissue section and flaming the slide on a burner and placing it in the xylene. The process treatment must be repeated afterward, and the hydration process should be done. To hydrate the tissue section, one should be passing it through a decreasing concentration of alcohol baths and water . Then the sample should be stained in hematoxylin for approximately 3–5 min, in the next step, it should be washed in running tap water until sections ‘blue’ for 5 min or less. In the next level, the sample should be placed in 1% acid alcohol  for 5 min. Subsequently, the sample should be washed in running tap water until it turns into blue again by dipping in an alkaline solution  followed by tap water washing process. Furthermore, in 1% Eosin Y for 10 min, the sample is stained and should be washed in tap water for 1–5 min. Finally, to dehydrate the sample, one should dip it in increasing concentration of alcohol and clear in xylene.The metabolism of cancer cells metastasize to LNs is very complicated. Recently, Lee et al.  used com2. Moreover, the acetyl-CoA, formed from reactions, enters the Krebs cycle to oxidize carbon dioxide and water. Finally, the reduced NADH and FADH2 coenzymes are oxidized by giving protons and electrons to the oxygen present in the mitochondria to produce ATP by oxidative phosphorylation in the electron transport cycle [Oxidation of fatty acids occurs in three stages. First, β-oxidation of fatty acids breaks down the two-carbon units (α and β carbons) of the carboxyl end of the fatty acyl-CoA and forms acetyl-CoA. This reaction continues until the entire fatty acyl chain gets broken down to acetyl-CoA molecules. Then, acetyl groups produced from β-fatty acid oxidation participate in the Krebs cycle and form NADH and FADHrt cycle .2 molecules during lipid synthesis of cancer cells would be dissolved in lymphatic fluids and resulted in the production of HCO3− (bicarbonate) and H+ ions through below reversible reaction [Also, released COreaction   was injected to SLNs immediately after dissection and MMLD was entered to the LNs. Then, the pH was read due to the color of litmus paper. After pathological evaluation of the LNs, the pH and diagnosis of each LNs were recorded in Supplementary Table S1. A schematic diagram of MMLD made to detect the acidity or basicity of a lymph node fluid is shown in This method has been tested on 65 lymph nodes immediately after dissection (through standard guidelines) from 25 breast cancer patients. Some of them were normal and some others were metastatic due to pathological evaluations (Supplementary Table S1). Changes in the pH value of the LNs from more than 7.0 in healthy LNs to less than 6.0 in cancerous ones indicate well differentiation ability of the lymphatic fluid (LF) pH as metastasis indicator. The results of the MMLD sensor were compared by frozen section diagnostics based on permanent H&E of the lymph nodes as the gold standard. Among 65 samples, MMLD had 1 false-positive and 1 false-negative. MMLD showed approximately 90% selectivity and 92% sensitivity detecting cancer cells metastasized to lymph nodes while this value in conventional frozen pathology was both 83% (The pH of the SLN fluid secreted in the cancerous lymph was less than 6.0 and for healthy ones were 7.0–10.0, respectively .The H&E images of the normal and cancerous lymph nodes with different percentage of involved cancer cells were shown in In summary, the MMLD was designed to detect metastatic cancer cells in the lymph node during surgery. This tool can also be very effective in the surgical process to prevent the removal of healthy lymph nodes. Also, it can help to make an accurate and real-time diagnosis in the operating room with no need for unnecessary secondary surgery and can reduce recurrences. Another application of this system would be in radiological process to help interventional radiologist for better identification of involved LNs.Click here for additional data file.