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The field of targeted protein degradation (TPD) has grown exponentially over the past decade with the goal of developing therapies that mark proteins for destruction leveraging the ubiquitin-proteasome system. One common approach to achieve TPD is to employ a heterobifunctional molecule, termed as a degrader, to recruit the protein target of interest to the E3 ligase machinery. The resultant generation of an intermediary ternary complex (target-degrader-ligase) is pivotal in the degradation process. Understanding the ternary complex geometry offers valuable insight into selectivity, catalytic efficiency, linker chemistry, and rational degrader design. In this study, we utilize hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify degrader-induced protein-protein interfaces. We then use these data in conjunction with constrained protein docking to build three-dimensional models of the ternary complex. The approach was used to characterize complex formation between the E3 ligase CRBN and the first bromodomain of BRD4, a prominent oncology target. We show marked differences in the ternary complexes formed in solution based on distinct patterns of deuterium uptake for two degraders, CFT-1297 and dBET6. CFT-1297, which exhibited positive cooperativity, altered the deuterium uptake profile revealing the degrader-induced protein-protein interface of the ternary complex. For CFT-1297, the ternary complexes generated by the highest scoring HDX-constrained docking models differ markedly from those observed in the published crystal structures. These results highlight the potential utility of HDX-MS to provide rapidly accessible structural insights into degrader-induced protein-protein interfaces in solution. They further suggest that degrader ternary complexes exhibit significant conformation flexibility and that biologically relevant complexes may well not exhibit the largest interaction surfaces between proteins. Taken together, the results indicate that methods capable of incorporating linker conformation uncertainty may prove an important component in degrader design moving forward. In addition, the development of scoring functions modified to handle interfaces with no evolved complementarity, for example, through consideration of high levels of water infiltration, may prove valuable. Furthermore, the use of crystal structures as validation tools for novel degrader methods needs to be considered with caution.

Recent advances in targeted protein degradation have enabled chemical hijacking of the ubiquitin-proteasome system to treat disease. The catalytic rate of cereblon (CRBN)-dependent bifunctional degradation activating compounds (BiDAC), which recruit CRBN to a chosen target protein, resulting in its ubiquitination and proteasomal degradation, is an important parameter to consider during the drug discovery process. In this work, an in vitro system was developed to measure the kinetics of BRD4 bromodomain 1 (BD1) ubiquitination by fitting an essential activator kinetic model to these data. The affinities between BiDACs, BD1, and CRBN in the binary complex, ternary complex, and full ubiquitination complex were characterized. Together, this work provides a new tool for understanding and optimizing the catalytic and thermodynamic properties of BiDACs.

The BRAF kinase plays a critical role in the MAPK signaling pathway and is mutated in ~8% of all human cancers including melanoma (~60%), thyroid (~60%), and lung adenocarcinoma (~10%). The most common mutation in BRAF is V600E (Class I), which is found in >70% in these cancers. Despite the clinical success of approved small molecule inhibitors of BRAF V600E (vemurafenib, dabrafenib and encorafenib), this remains an area of unmet medical need because nearly all patients progress, due to either primary or acquired resistance. A bifunctional degradation activating compound (or BiDACTM degrader) may address the liabilities of approved drugs by overcoming, or preventing the emergence of, resistance to BRAF inhibitors. Here we describe CFT1946, an orally bioavailable cereblon-based BiDAC degrader of BRAF V600 mutant proteins, and provide an overview of the medicinal chemistry path leading to its discovery. CFT1946 degrades BRAF V600 mutant proteins, while maintaining exquisite selectivity against the proteome, sparing wild type BRAF (BRAF-WT), ARAF, and CRAF. In A375 cells, CFT1946 potently degraded BRAF V600E and inhibited ERK phosphorylation and cell growth while having no effect in the mutant KRAS, BRAF-WT driven cell line HCT116. In the A375 xenograft model, oral delivery of CFT1946 at 10 mg/kg PO BID resulted in deeper and more durable tumor regression compared to a clinically relevant dose of encorafenib. Further evaluation of CFT1946 in an engineered, clinically relevant BRAFi-resistant A375 cell line (endogenous BRAF V600E + engineered expression of NRAS Q61K) demonstrated that CFT1946 both degraded BRAF V600E and caused a loss of viability in these cells, while treatment with encorafenib had no effect. In xenografts derived from this BRAFi-resistant cell line, oral dosing of CFT1946 as a single agent led to tumor growth inhibition, while treatment with a clinically relevant dose of encorafenib had no effect on tumor growth. Furthermore, dosing CFT1946 in combination with the MEK inhibitor, trametinib, resulted in significant tumor regression, whereas combining encorafenib with the same dose of trametinib had no effect. The medicinal chemistry campaign resulting in CFT1946 focused on the improvement of in vivo pharmacokinetics and rational linker design to achieve high oral bioavailability in a beyond Rule of 5 heterobifunctional degrader. The preclinical data presented herein support the planned Phase 1/2 clinical trial of CFT1946 for the treatment of BRAF-V600 mutant solid tumors. Citation Format: Yanke Liang, Mathew E. Sowa, Katrina L. Jackson, Jeffrey R. Simard, Bridget Kreger, Ping Li, Laura Poling, Joelle Baddour, Andrew Good, Hongwei Huang, Scott Eron, Christopher G. Nasveschuk, Robert Yu, Mark Fitzgerald, Victoria Garza, Morgan W. O’Shea, Gesine Veits, Jeremy Y. Yap, Moses Moustakim, Ashley Hart, Roman V. Agafonov, Grace Sarkissian, Joe S. Patel, Richard Deibler, Kyle S. Cole, David Cocozziello, Fazlur Rahman, Andrew J. Phillips, Elizabeth Norton, Adam S. Crystal, Roy M. Pollock, Stewart L. Fisher. The discovery and characterization of CFT1946: A potent, selective, and orally bioavailable degrader of mutant BRAF for the treatment of BRAF-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3425.

Introduction: The chromatin factor BRD9 is a genetic dependency in some cancers, often referred to as SMARCB1-perturbed cancers. Two types of genetic alterations result in SMARCB1 perturbation: SS18-SSX gene fusion and SMARCB1 loss-of-function mutations. In synovial sarcoma, a rare and aggressive soft tissue malignancy comprising approximately 10% of all soft tissue sarcomas, the presence of the SS18-SSX fusion gene drives the disruption of SMARCB1 function and leads to a synthetic lethal dependence on BRD9. In SMARCB1-null solid tumors, for example malignant rhabdoid tumors (MRT), poorly differentiated chordomas, and epithelioid sarcomas, the absence of SMARCB1 protein results in a similar BRD9 dependence. Thus, in SMARCB1-perturbed cancers, including synovial sarcoma and SMARCB1-null cancers, degradation of BRD9 is hypothesized to result in an anticancer effect. CFT8634 is an orally bioavailable heterobifunctional degrader that induces ternary complex formation with BRD9 and an E3 ligase, leading to the ubiquitination of BRD9 and its subsequent degradation by the proteasome. Results: Here we describe the chemical structure of CFT8634 and an overview of the medicinal chemistry path leading to its discovery. In vitro, CFT8634 promotes rapid, potent, deep, and selective degradation of BRD9 with a half-maximal degradation concentration (DC50) of 2 nM in a synovial sarcoma cell line. In long-term growth assays, CFT8634 is effective at impairing cell growth in a concentration-dependent manner specifically in SMARCB1-perturbed contexts. In vivo, oral dosing of CFT8634 in xenograft models of SMARCB1-perturbed cancers leads to robust and dose-dependent degradation of BRD9, which translates to significant and dose-dependent inhibition of tumor growth in preclinical xenograft models. Conclusion: The preclinical data presented herein support the clinical development of CFT8634 for the treatment of synovial sarcoma and SMARCB1-null tumors. Citation Format: Katrina L. Jackson, Roman V. Agafonov, Mark W. Carlson, Prasoon Chaturvedi, David Cocozziello, Kyle Cole, Richard Deibler, Scott J. Eron, Andrew Good, Ashley A. Hart, Minsheng He, Christina S. Henderson, Hongwei Huang, Marta Isasa, R. Jason Kirby, Linda Lee, Michelle Mahler, Moses Moustakim, Christopher G. Nasveschuk, Michael Palmer, Laura L. Poling, Roy M. Pollock, Matthew Schnaderbeck, Stan Spence, Gesine K. Veits, Jeremy L. Yap, Ning Yin, Rhamy Zeid, Adam S. Crystal, Andrew J. Phillips, Stewart L. Fisher. The discovery and characterization of CFT8634: A potent and selective degrader of BRD9 for the treatment of SMARCB1-perturbed cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND09.

Introduction: CFT7455 is a highly potent and novel Ikaros Family Zinc Finger Protein 1/3 (IKZF1/3) degrader. In xenograft models, CFT7455 has more potent IKZF1/3 degradation compared to other degraders. Early observations from the FIH clinical trial (NCT04756726) along with supporting translational studies are presented here. Methods: Pre-clinical studies comparing CFT7455 and CC-92480 in both in vitro and xenograft models were performed. The pre-clinical studies’ results coincided with observations from the on-going clinical trial. The clinical trial is an open-label, Phase 1/2, multi-center, FIH study in heavily pretreated relapsed/refractory (R/R) MM and non-Hodgkin’s lymphoma (NHL) patients evaluating safety, tolerability, and PK of CFT7455. Eligible MM patients are R/R to therapy and are not candidates for regimens known to provide clinical benefit. A starting dose of 50 μg QD 21 days on/7 days off (21/7) in 28-day cycles was administered. Results: CFT7455 and CC-92480 showed similar cereblon binding profiles and in vitro IKZF1/3 degradation kinetics, translating into sub-nanomolar GI50 values in proliferation assays across a panel of MM cell lines. In the NCI-H929 xenograft model, 100 μg/kg/day of CFT7455 resulted in durable tumor regressions, while 1000 μg/kg/day of CC-92480 gave tumor stasis. Similar results were seen in a systemic model of MM, MM1.S. Both compounds achieved >95% IKZF3 degradation in tumors 4h post dose. At 48h post dose, CFT7455 was more effective than CC-92480 in maintaining IKZF3 degradation (65% vs. 6% respectively). When levels of CFT7455 and CC-92480 in plasma and tumor were compared, CFT7455 concentrations were > DC80 in tumor 48h post dose, while CC-92480 levels were undetectable in tumor and plasma, demonstrating CFT7455 has longer exposure resulting in sustained IKZF1/3 degradation in pre-clinical models. In cohort A, 5 heavily pre-treated MM patients (pts) received single agent CFT7455. 4 pts have received at least 3 cycles, with 2 pts receiving 5 cycles. Neutropenia (grade 4) was observed in 3/5 pts without coincident fever or infection. Additionally, a 2-4 fold accumulation in plasma CFT7455 exposure at steady state was observed. Early pharmacodynamic (PD) data demonstrates deep persistent degradation of IKZF3 (~100%) and serum free light chain reduction (up to 72%) in response to treatment. Stable disease has been observed in 34 pts, suggesting clinical benefit. Conclusions: While CFT7455 showed clinical benefit at 50 ug with deep target degradation, neutropenia was dose limiting. PK/PD modeling suggests alternative dosing regimens may result in increased tolerability with preserved efficacy, and evaluation of them is underway. Updated results will be presented at the meeting. Citation Format: Sagar Lonial, Shambavi Richard, Jeffrey V Matous, Andrew J. Yee, Urvi Shah, Neha Mehta-Shah, Thomas Martin, Eli Muchtar, Sikander Ailawadhi, Paul G. Richardson, Manisha Bhutani, Samantha Perino, Jason Kirby, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, Matthew Schnaderbeck, Michael R. Palmer, Cathleen Gorman, Oliver Schoenborn-Kellenberger, Amanda Hoerres, Stewart L. Fisher, Roy M. Pollock, Adam Crystal, Michelle Mahler, Jesus Bardeja. Pharmacokinetic (PK) profile of a novel IKZF1/3 degrader, CFT7455, enables significant potency advantage over other IKZF1/3 degraders in models of multiple myeloma (MM) and the results of the initial treatment cohort from a first-in-human (FIH) phase 1/2 study of CFT7455 in MM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT186.

The BRAF kinase is a critical node in the MAPK signaling pathway and is mutated in approximately 8% of human cancers including melanoma (~60%), thyroid (~60%), and lung adenocarcinoma (~10%). The most common mutation in BRAF is V600E (Class I), occurring in half of malignant melanomas. This mutation hyperactivates ERK and signals as a RAF inhibitor-sensitive monomer. BRAF inhibitors including vemurafenib, dabrafenib and encorafenib have produced impressive responses in V600X patients, however resistance usually emerges within a year, including RAS mutation, BRAFV600E amplification, and BRAFV600E intragenic deletion or splice variants. These inhibitors are also ineffective against non-V600 BRAF mutants (Class II & III). To address some of these limitations we have developed CFT1946, a bifunctional degradation activating compound (BiDAC™) degrader comprising a BRAF kinase domain targeting ligand linked to a cereblon ligand. CFT1946 is capable of degrading BRAF V600E (Class I), G469A (Class II), G466V (Class III) mutations, and the p61-BRAFV600E splice variant while maintaining exquisite selectivity against the proteome including WT BRAF and CRAF. In A375 cells, CFT1946 potently degraded BRAFV600E (Emax = 26%; DC50 = 14nM at 24hr) and, inhibited ERK phosphorylation (IC50 = 11nM at 24hr) and cell growth (GI50 = 94nM at 96hr) while having no effect in the mutant KRAS driven cell line HCT116. In A375 xenografts, oral delivery of CFT1946 resulted in deeper tumor regressions when dosed at 10 mg/kg PO BID and compared favorably to a clinically relevant dose of encorafenib. We further evaluated CFT1946 in an engineered A375-BRAFV600E/NRASQ61K double mutant model of BRAF inhibitor resistance. CFT1946 was able to degrade BRAFV600E in these cells and was much more effective than encorafenib at inhibiting viability in vitro. In this model, in vivo dosing of single agent CFT1946 caused robust tumor growth inhibition and combination with the MEK inhibitor, trametinib, resulted in tumor regressions. The combination of encorafenib and trametinib showed no activity in the same model. Next, we demonstrated that CFT1946 was able to degrade additional BRAF mutant proteins including G469A (Class II), G466V (Class III), and the p61-BRAFV600E splice variant using heterologous expression in HEK293T cells. Additionally, we also showed that CFT1946, but not encorafenib, inhibited proliferation of the BRAFG466V heterozygous lung tumor cell line H1666. Based on its activity in preclinical models, including models of BRAF inhibitor resistance, and its drug-like properties we are progressing CFT1946 as a candidate for clinical development in patients with solid tumors bearing BRAF V600X mutations. Further, given CFT1946’s activity on non-V600 BRAF mutations, we are continuing to explore CFT1946 and related BiDAC degraders as therapeutic options for patients bearing Class II or Class III BRAF mutations. Citation Format: Mathew E. Sowa, Bridget Kreger, Joelle Baddour, Yanke Liang, Jeffrey R. Simard, Laura Poling, Ping Li, Robert Yu, Ashley Hart, Roman V. Agafonov, Grace Sarkissian, Joe Sahil Patel, Richard Deibler, Kyle S. Cole, Scott Eron, David Cocozziello, Fazlur Rahman, Moses Moustakim, Christopher G. Nasveschuk, Katrina L. Jackson, Mark Fitzgerald, Victoria Garza, Morgan O’Shea, Gesine Veits, Jeremy L. Yap, Andrew J. Phillips, Elizabeth Norton, Adam S. Crystal, Stewart L. Fisher, Roy M. Pollock. Preclinical evaluation of CFT1946 as a selective degrader of mutant BRAF for the treatment of BRAF driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2158.

Introduction: Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 inhibits the growth of multiple myeloma (MM) cells, confirming their dependency on IKZF1/3. IMiDs (lenalidomide, pomalidomide) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. However, most patients treated with lenalidomide or pomalidomide eventually develop progressive disease due to acquired resistance, underscoring the unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high binding affinity to cereblon (CRBN), rapid and deep IKZF1/3 degradation, and potent dose-dependent efficacy in vivo. Results: A series of novel benzoimidazolone-based CRBN ligands with potent binding affinity were discovered and their binding modes were informed by CRBN co-crystal structures. Although the benzoimidazolone-based CRBN binders did not exhibit IKZF1/3 degradation activity, structural insights into their unique binding modes and knowledge of the IKZF1/3 degradation pharmacophore were combined to enable identification of a novel benzoisoindolone-based ligand that exhibited a 10-fold potency increase in biochemical CRBN binding and a 30-fold potency increase in H929 MM cell growth inhibition when compared to lenalidomide. Additional rounds of structure-based drug design, degradation and phenotypic profiling led to the discovery of CFT7455, a highly potent, selective and orally bioavailable degrader of IKZF1/3. CFT7455 demonstrated an 800 and 1600-fold improvement in CRBN binding compared to pomalidomide in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced >75% degradation of IKZF1 within 1.5 hrs. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines, as well as H929 cells made resistant to IMiDs. In vivo, CFT7455 catalyzed deep and durable degradation of IKZF3, translating into potent antitumor activity in multiple myeloma xenograft models. CFT7455 also retained its activity in models resistant or insensitive to clinically approved IMiDs as single agent or in combination with standard of care agent dexamethasone. Conclusion: Overall, CFT7455 is a next generation IKZF1/3 degrader, with improved potency and anticancer efficacy in preclinical models compared to existing IMiDs. These features make CFT7455 an exciting drug candidate, as a single agent or for use in combination. CFT7455 is currently being studied in a Ph1 clinical trial. Citation Format: James A. Henderson, Scott J. Eron, Andrew Good, R Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Ashley A. Hart, Christina S. Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Adam S. Crystal, Roy M. Pollock, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. The discovery and characterization of CFT7455: A potent and selective degrader of IKZF1/3 for the treatment of relapsed/refractory multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND13.

In addition to the therapeutic applicability of targeted protein degradation (TPD), the modality also harbors unique properties that enable the development of innovative chemical biology tools to interrogate complex biology. TPD offers an all-chemical strategy capable of the potent, durable, selective, reversible, and time-resolved control of the levels of a given target protein in both in vitro and in vivo contexts. These properties are particularly well-suited for enabling the precise perturbation of a given gene to understand its biology, identify dependencies/vulnerabilities in disease contexts, and as a strategy to control gene therapies. To leverage these elegant properties, we developed the AchillesTag (aTAG) degradation system to serve as a tool in target identification and validation efforts. The aTAG degradation system provides a novel degradation tag based on the MTH1 protein paired with three fully validated bifunctional degraders with both in vitro and in vivo applicability. We catalog the development of the aTAG system from selection and validation of the novel MTH1 aTAG, alongside a comprehensive SAR campaign to identify high performing tool degraders. To demonstrate the utility of the aTAG system to dissect a complex biological system, we apply the technology to the control of Chimeric Antigen Receptor (CAR) activity. Using aTAG, we demonstrate the ability to potently and selectively control CAR protein levels, resulting in the exquisite rheostat control of CAR mediated T-cell activity. Furthermore, we showcase the in vivo application of the system via degradation of the aTAG-fused CAR protein in a human xenograft model. The aTAG degradation system provides a complete chemical biology tool to aid foundational target validation efforts that inspire drug discovery campaigns towards therapeutic applicability.

Introduction Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 in MM cells inhibits growth, confirming their dependency on IKZF1/3. IMiDs (lenalidomide[len], pomalidomide[pom]) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. Most patients treated with len or pom eventually develop progressive disease due to acquired resistance, underscoring an unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high affinity cereblon (CRBN) binding, rapid IKZF1/3 degradation, and potent in vivo efficacy. Results CFT7455 demonstrated an 800 to 1600-fold improvement in CRBN binding compared to pom in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced >75% degradation of IKZF1 within 1.5 hr. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines as well as H929 cells made resistant to IMiDs. In RPMI-8226 MM mice xenografts, CFT7455 (0.1 mg/kg/day) resulted in deep, durable degradation of IKZF3 (21% and 9.5% of vehicle levels at 4 and 24 hr, respectively). Protein levels for IRF4, an essential TF in MM, declined over 7 days with daily CFT7455 treatment to 8% of vehicle levels. Dose dependent efficacy ranged from 0.003-0.1 mg/kg/day, with tumor regression evident at doses ≥0.01 mg/kg/day. Pom was inactive in this model at a human equivalent dose (3 mg/kg/day), with no observed tumor shrinkage in these mice following 17 days of dosing. Switching pom treatment to CFT7455 (0.1 mg/kg/day) on Day 18 led to tumor regression in 67% of animals on Day 28 and 100% tumor regression on Day 35, demonstrating that CFT7455 penetrates large tissues and is efficacious in rapidly growing, IMiD resistant tumors. In the H929 tumor xenograft model, administration of CFT7455 (0.1 mg/kg/day) promoted tumor regression (95% tumor growth inhibition by 7 days); dosing was stopped after 21 days. On Day 63, half the tumors remained below their starting tumor volume. Additionally, CFT7455 demonstrated durable tumor regression in the aggressive MM1.S systemic MM tumor model. In mice bearing RPMI-8226 xenograft tumors, the combination of CFT7455 (QD) and dexamethasone (QW) was more active, and demonstrated a significant survival improvement, compared to either agent alone. Conclusions CFT7455 is a highly potent, catalytic degrader of IKZF1/3, with marked antitumor activity as a single agent and in combination with dexamethasone. Importantly, CFT7455 retains activity in models resistant or insensitive to IMiDs. These results warrant investigation of CFT7455 as a therapeutic approach for MM and a clinical study is planned. Citation Format: James A. Henderson, R. Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Scott J. Eron, Andrew Good, Ashley A. Hart, Christina Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Roy M. Pollock, Adam S. Crystal, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. CFT7455: A novel, IKZF1/3 degrader that demonstrates potent tumor regression in IMiD-resistant multiple myeloma (MM) xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB007.

With early life likely to have existed in a hot environment, enzymes had to cope with an inherent drop in catalytic speed caused by lowered temperature. Here we characterize the molecular mechanisms underlying thermoadaptation of enzyme catalysis in adenylate kinase using ancestral sequence reconstruction spanning 3 billion years of evolution. We show that evolution solved the enzyme’s key kinetic obstacle—how to maintain catalytic speed on a cooler Earth—by exploiting transition-state heat capacity. Tracing the evolution of enzyme activity and stability from the hot-start toward modern hyperthermophilic, mesophilic, and psychrophilic organisms illustrates active pressure versus passive drift in evolution on a molecular level, refutes the debated activity/stability trade-off, and suggests that the catalytic speed of adenylate kinase is an evolutionary driver for organismal fitness.

Targeted protein degradation (TPD) with heterobifunctional small molecules is emerging as a promising new therapeutic modality in oncology. We sought to understand how TPD compares versus targeted protein inhibition. We used BRD4 as a surrogate target given literature showing BET degraders have a global suppressive effect on transcription compared to BET inhibitors that preferentially affect superenhancers. We selected the BET degrader CFT-743 from a library of CRBN-biased degraders based on its specificity, potency and optimum in vivo properties that enable a therapeutic window. While subtle changes in chemistry around CFT-743 had negligible impact on vitro potency, the effect on therapeutic index was significant. In vitro, CFT-743 is more potent than a BET inhibitor (AZD5153) and a CDK9 inhibitor (dinaciclib) in 100% and 97% of 38 leukemia lines tested. In mice bearing HL60 tumors, CFT-743 has similar efficacy as dinaciclib (81% regression versus 99% tumor growth inhibition (TGI)). Pre-dosing mice with the CRBN binding molecule pomalidomide significantly attenuates CFT-743 activity (TGI 40%) rendering the observed efficacy on par with BET inhibitor CPI-203 (TGI 2%) and thus confirming superior efficacy is dependent on BET degradation. In Kasumi1 and THP1 tumor models, CFT-743 efficacy is more like CPI-203 (regressions in Kasumi1 and strong TGI/moderate regressions in THP1) than dinaciclib (moderate regression and 0% TGI). In RS4;11 tumors CFT-743 promotes complete regression whereas dinaciclib and CPI-203 display modest efficacy (80% and 58% TGI). Pharmacokinetic and pharmacodynamic studies in mice bearing MV4;11 tumors demonstrate that a high initial plasma exposure of CFT-743 followed by fast compound clearance is sufficient to drive rapid loss of BRD4 in tumors ( Collectively, these data highlight the in vivo features important for efficacy and tolerability with BET degraders and establish the relationships between degrader pharmacokinetics, target protein degradation and efficacy. Citation Format: Brendon Ladd, David Cocozziello, Mark E. Fitzgerald, Ryan E. Michael, Ellen F. Vieux, Linda Lee, Marta Issa, Jacob D. Feala, Roman V. Agafonov, Eun Sun Park, Christopher G. Nasveschuk, David A. Proia. In vivo profiling of BET degraders establishes optimal pharmacological properties that showcase distinct biological differences from BET inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1949.

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinase Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome., eLife digest Protein kinases are a family of enzymes found in all living organisms. These enzymes help to control many biological processes, including cell division. When particular protein kinases do not work correctly, cells may start to divide uncontrollably, which can lead to cancer. One example is the kinase Aurora A, which is over-active in many common human cancers. As a result, researchers are currently trying to design drugs that reduce the activity of Aurora A in the hope that these could form new anticancer treatments. In general, drugs are designed to be as specific in their action as possible to reduce the risk of harmful side effects to the patient. Designing a drug that affects a single protein kinase, however, is difficult because there are hundreds of different kinases in the body, all with similar structures. Because drugs often work by binding to specific structural features, a drug that targets one protein kinase can often alter the activity of a large number of others too. Gleevec is a successful anti-leukemia drug that specifically works on one target kinase, producing minimal side effects. It was recently discovered that the drug works through a phenomenon called ‘induced fit’. This means that after the drug binds it causes a change in the enzyme’s overall shape that alters the activity of the enzyme. The shape change is complex, and so even small structural differences can change the effect of a particular drug. Do other drugs that target other protein kinases also produce induced fit effects? To find out, Pitsawong, Buosi, Otten, Agafonov et al. studied how three anti-cancer drugs interact with Aurora A: two drugs specifically designed to switch off Aurora A, and Gleevec (which does not target Aurora A). The two drugs that specifically target Aurora A were thought to work by targeting one structural feature of the enzyme. However, the biochemical and biophysical experiments performed by Pitsawong et al. revealed that these drugs instead work through an induced fit effect. By contrast, Gleevec did not trigger an induced fit on Aurora A and so bound less tightly to it. In light of these results, Pitsawong et al. suggest that future efforts to design drugs that target protein kinases should focus on exploiting the induced fit process. This will require more research into the structure of particular kinases.

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.eLife digestThe Ser/Thr kinase Aurora A is an important target for the development of new anticancer therapies. A longstanding question is how to specifically and effectively inhibit only this kinase in a background of over 550 protein kinases with very similar structures. To this end, understanding the inhibition mechanism of Aurora A by different drugs is essential. Here, we characterize the kinetic mechanism of three distinct kinase drugs, Gleevec (Imatinib), Danusertib (PHA739358) and AT9283 (Pyrazol-4-yl Urea) for Aurora A. We show that inhibitor affinities do not rely exclusively on the recognition of a specific conformation of the Asp-Phe-Gly loop of the kinase. Our quantitative kinetics data put forward an opposing mechanism in which a slow conformational change after drug binding (i.e., induced-fit step) dictates drug affinity.

Macromolecular function is rooted in energy landscapes, where sequence determines not a single structure but an ensemble of conformations. Hence, evolution modifies a protein’s function by altering its energy landscape. Here, we recreate the evolutionary pathway between two modern human oncogenes, Src and Abl, by reconstructing their common ancestors. Our evolutionary reconstruction combined with x-ray structures of the common ancestor and pre–steady-state kinetics reveals a detailed atomistic mechanism for selectivity of the successful cancer drug Gleevec. Gleevec affinity is gained during the evolutionary trajectory toward Abl and lost toward Src, primarily by shifting an induced-fit equilibrium that is also disrupted in the clinical T315I resistance mutation. This work reveals the mechanism of Gleevec specificity while offering insights into how energy landscapes evolve.

Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8,000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. Here we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, molecular-dynamics simulations and crystallography of active complexes. We find that the Mg(2+) cofactor activates two distinct molecular events: phosphoryl transfer (>10(5)-fold) and lid opening (10(3)-fold). In contrast, mutation of an essential active site arginine decelerates phosphoryl transfer 10(3)-fold without substantially affecting lid opening. Our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a preorganized active site.

NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by characterizing lowly populated conformations that are in dynamic exchange with the major state. Paramagnetic NMR is a versatile tool for investigating the structures and dynamics of proteins. These two techniques were combined here to measure accurate and precise pseudocontact shifts of a lowly populated conformation. This method delivers valuable long-range structural restraints for higher energy conformations of macromolecules in solution. Another advantage of combining pseudocontact shifts with relaxation dispersion is the increase in the amplitude of dispersion profiles. Lowly populated states are often involved in functional processes, such as enzyme catalysis, signaling, and protein/protein interactions. The presented results also unveil a critical problem with the lanthanide tag used to generate paramagnetic relaxation dispersion effects in proteins, namely that the motions of the tag can interfere severely with the observation of protein dynamics. The two-point attached CLaNP-5 lanthanide tag was linked to adenylate kinase. From the paramagnetic relaxation dispersion only motion of the tag is observed. The data can be described accurately by a two-state model in which the protein-attached tag undergoes a 23° tilting motion on a timescale of milliseconds. The work demonstrates the large potential of paramagnetic relaxation dispersion and the challenge to improve current tags to minimize relaxation dispersion from tag movements.

Protein kinases are obvious drug targets against cancer, owing to their central role in cellular regulation. Since the discovery of Gleevec, a potent and specific inhibitor of Abl kinase, as a highly successful cancer therapeutic, the ability of this drug to distinguish between Abl and other tyrosine kinases such as Src has been intensely investigated but without much success. Using NMR and fast kinetics, we establish a new model that solves this longstanding question of how the two tyrosine kinases adopt almost identical structures when bound to Gleevec but have vastly different affinities. We show that, in contrast to all other proposed models, the origin of Abl's high affinity lies predominantly in a conformational change after binding. An energy landscape providing tight affinity via an induced fit and binding plasticity via a conformational-selection mechanism is likely to be general for many inhibitors.

For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, (TR) 2 FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR) 2 FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond time-resolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by (TR) 2 FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism.

Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X- and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.V(i)-bound biochemical state of myosin, which presumably mimics the S1.ADP.P(i) state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.V(i) biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin.

Sophisticated protein kinase networks, empowering complexity in higher organisms, are also drivers of devastating diseases such as cancer. Accordingly, these enzymes have become major drug targets of the twenty-first century. However, the holy grail of designing specific kinase inhibitors aimed at specific cancers has not been found. Can new approaches in cancer drug design help win the battle with this multi-faced and quickly evolving enemy? In this perspective we discuss new strategies and ideas that were born out of a recent breakthrough in understanding the molecular basis underlying the clinical success of the cancer drug Gleevec. An “old” method, stopped-flow kinetics, combined with old enzymes, the ancestors dating back up to about billion years, provides an unexpected outlook for future intelligent design of drugs.

Protein phosphorylation is an essential regulatory mechanism that affects all aspect of cellular life from division and growth to aging and death. Misregulation of the signaling cascades leads to severe detrimental effects, and in humans often associated with cancer and other diseases. Phosphorylation is performed by a class of protein called kinases. Activation and deactivation of kinases is normally under tight control and is regulated via different mechanisms that are incredibly complex. In this work we combine phylogenetic resurrection techniques with biophysical and chemical approaches to analyze the regulatory mechanisms of modern tyrosine oncokinases Src and Abl, their common ancestor and the common ancestors between several other families of tyrosin kinases. Our results show how the regulatory elements appeared and developed throughout the evolution enabling selective regulation of complex modern cascades.

Proteins populate a landscape of conformations that changes upon binding native ligands or drugs, altering protein function. Different conformations reveal distinct potential drug target sites, including allosteric sites distal to the active site of the protein. It has been historically difficult to identify allosteric modulators early in the screening process since the structural methods required to reveal binding sites are relatively low throughput.

Design of specific kinase inhibitors is an appealing approach for developing new anticancer treatments. However, only a few success stories have been reported to date. Here we demonstrate how the combination of old-fashioned and new biophysical tools together with recent advances in genomics and molecular evolution can aid in overcoming existing limitations.

We are using site-directed spin-labeling (SDSL) and dipolar electron-electron resonance (DEER) to study the structural dynamics of proteins involved in muscle function. Muscle proteins are ideal systems for DEER applications, since these proteins form large dynamic assemblies that are inaccessible to crystallography and NMR. DEER is a pulsed ELDOR technique that detects the spin-spin dipolar interaction as a decay and modulation of a spin echo signal. While the CW EPR spectrum is sensitive only to distances on the order of 1 to 2 nm, DEER is sensitive from 1.5 to 5 nm and more. DEER is sensitive to the width of the distance distribution and can resolve multiple structural populations simultaneously. However, a limitation of DEER is the use of spin probes with flexible linkers, which adds uncertainty to the locations of protein backbone atoms. A bifunctional spin label (BSL), which attaches at position (i, i+4) of a helix, was used to measure the distance between two di-Cys pairs. BSL is rigidly coupled to the protein backbone and is a breakthrough for high resolution measurement of orientation, dynamics, and distance. These distance measurements were compared to conventional labeling of cysteine residues with MSL/MSL pairs.

We have used two complementary time-resolved spectroscopic techniques, dipolar electron–electron resonance and fluorescence resonance energy transfer to determine conformational changes in a single structural element of the myosin motor domain, the relay helix, before and after the recovery stroke. Two double-Cys mutants were labeled with optical probes or spin labels, and interprobe distances were determined. Both methods resolved two distinct structural states of myosin, corresponding to straight and bent conformations of the relay helix. The bent state was occupied only upon nucleotide addition, indicating that relay helix, like the entire myosin head, bends in the recovery stroke. However, saturation of myosin with nucleotide, producing a single biochemical state, did not produce a single structural state. Both straight and bent structural states of the relay helix were occupied when either ATP (ADP.BeF x ) or ADP.P i (ADP.AlF 4 ) analogs were bound at the active site. A greater population was found in the bent structural state when the posthydrolysis analog ADP.AlF 4 was bound. We conclude that the bending of the relay helix in the recovery stroke does not require ATP hydrolysis but is favored by it. A narrower interprobe distance distribution shows ordering of the relay helix, despite its bending, during the recovery stroke, providing further insight into the dynamics of this energy-transducing structural transition.

We have used transient time-resolved FRET (TR2-FRET) to detect structural changes within the relay helix in real time during the myosin recovery stroke. The most intriguing problem in myosin's function is the mechanism of signal transduction between the nucleotide binding site and the force-generating domain of myosin during the recovery stroke. Atomic structures of myosin provide clues, but structural measurements during transient kinetics are needed to reveal the details of this mechanism. Kinetics of nucleotide binding is reliably determined from the fluorescence of mant nucleotides or single Trp myosin mutants (W129, W131). Single Trp mutant W501 has provided insight into structural kinetics in the force-generating domain, but the complex fluorescence signals observed during the recovery stroke do not yield clear structural interpretation. To overcome this problem, we have performed FRET measurements that monitor well-defined structural changes in the relay helix, which is proposed to be a key structural element in myosin's force-generating domain. We engineered two double-Cys myosin mutants in the Cys-lite D. discoideum myosin construct (gift from J. Spudich), with one Cys placed at the C-terminus of the relay helix (K498C) and the other at one of two stable positions in the lower 50K domain (D515C or A639C). We labeled these mutants with a donor-acceptor FRET pair (IAEDANS-DABSYL), then used time-resolved FRET (TR-FRET, detects donor lifetime at nanosecond timescale) to detect interprobe distances in equilibrium states trapped by nucleotide analogs, reflecting pre- and post-powerstroke structures of myosin. We then performed these nanosecond time-resolved measurements repetitively every 0.1 ms, in the transient phase after ATP addition using stopped flow. These transient time-resolved FRET (TR2-FRET) signals showed directly that the relay helix changes from straight to bent conformation in the recovery stroke, from the post-powerstroke to the pre-powerstroke state.

We have engineered a mutant of Dictyostelium discoideum ( Dicty ) myosin II that contains the same fast-reacting “SH1” thiol as in muscle myosin, spin-labeled it, and performed electron paramagnetic resonance (EPR) to compare the structure of the force-generating region of the two myosins. Dicty myosin serves as a model system for muscle myosin because of greater ease of mutagenesis, expression, and crystallization. The catalytic domains of these myosins have nearly identical crystal structures in the apo state, but there are significant differences in ATPase kinetics, and there are no crystal structures of skeletal muscle myosin with bound nucleotides, so another structural technique is needed. Previous EPR studies, with a spin label attached to SH1 in muscle myosin, have resolved the key structural states of this region. Therefore, we have performed identical experiments on both myosins spin-labeled at equivalent sites. Spectra were identical for the two myosins in the apo and ADP-bound states. With bound ADP and phosphate analogs, ( i ) both proteins exhibit two resolved structural states (prepowerstroke, postpowerstroke) in a single biochemical state (defined by the bound nucleotide), and ( ii ) these structural states are essentially identical in the two myosins but ( iii ) are occupied to different extents as a function of the biochemical state. We conclude that ( i ) myosin structural and biochemical states do not have a one-to-one correspondence, and ( ii ) Dicty myosin can serve as a good analog for structural studies of muscle myosin only if differences in the coupling between biochemical and structural states are taken into account.

Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pHin established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pHout=7.8, we found that pHin approximately 5.4-5.7 in the state of photosynthetic control, and pHin approximately 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference DeltapH approximately 1.8-2.1. These values of DeltapH are consistent with a point of view that under steady-state conditions the proton gradient DeltapH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H+/ATP is nor =4-4.7.

We have developed a high-throughput time-resolved fluorescence spectrometer capable of recording a high-resolution time-domain (sub-nanosecond) fluorescence decay, with high S/N every 100 μs. Coupled with a conventional stopped-flow rapid mixer, this technology has allowed us to measure changes in time-resolved fluorescence decays occurring during the course of a millisecond-resolved biochemical transient experiment. Most instruments used in fluorescence-based kinetic studies are limited to detecting a single fluorescence intensity signal on the millisecond time scale. While this type of fluorescence intensity-based measurement is informative, it provides scant information compared to a full sub-nanosecond resolved fluorescence decay- which is exquisitely sensitive to the structure, dynamics, and interactions of molecules in the sample. Current fluorescence lifetime technology is too slow by a factor of at least 1000 to detect this decay accurately within the millisecond timescale of a typical biochemical transient. Our system, based on a direct acquisition data collection approach, records the entire fluorescence decay with S/N > 100 from a single pulse form a 10 kHz microlaser. Using this approach, we can resolve individual fluorescence lifetime components comprising as little as 10% of a complex multi-exponential fluorescence decay. When used to monitor structural transitions by time resolved FRET, we are capable of isolating individual structural states within complex structural ensembles. Using this approach we were able to simultaneously monitor the binding of myosin to different classes of binding sites on actin filaments. This type of analysis is not possible with conventional fluorescence based stopped flow measurements.

We have used pulsed EPR spectroscopy (DEER) to determine the structural effects of point mutations in the force generation region of myosin (I499A, F506A, F692A, D.discoideum sequence). As it was previously reported, these myosin mutants maintain basal ATPase activity, but completely lose their motor function. It was proposed that F506A, located in the relay loop, disrupts communication between the nucleotide binding site and the force generation region in myosin, and F692A and I499A, located on the interface with the converter domain, affect coordination of the converter domain relative to the myosin head. In this study we assayed the structure of the relay helix in these mutants and in wild-type myosin. The mutations were introduced into the A639C:K498C myosin construct, both Cys were labeled with maleimide spin probes, and interprobe distance was measured. Previous studies showed that probes at these sites detect the nucleotide-induced bending of the relay helix. Observed spin echo decays were interpreted in terms of one or two Gaussian distance distributions, corresponding to one or two myosin structural states. Nucleotide analogs were used to trap myosin in several biochemical states. In the wild type, two structural states, corresponding to pre- and post-recovery stroke, are present in a single biochemical state. The mutations affect the equilibrium between these two structural states and increase the conformational disorder, indicating that these mutants destabilize the structure of the force-generation region, particularly in the post-recovery state. These results provide a structural explanation for the functional perturbation, introduced to myosin.

We have used electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) to measure structural transitions of the catalytic domain (CD) in Dictyostelium myosin II. The use of BSL is a critical feature in this work. The bifunctional attachment of BSL eliminates most of the nanosecond motions characteristic of monofunctional labels, making it possible to measure protein structural transitions with a precision not previously achievable. Two double-Cys constructs were engineered with Cys residues at helical locations i and i+4 (494.498 and 639.643). Residues 494.498 are located on the relay helix and residues 639.643 are located on a helix adjacent to the relay helix. After BSL labeling, myosin was bound to actin in oriented muscle fibers, making it possible to measure helix orientation relative to the fiber axis. Spectra were acquired in APO and ADP states. Simulation of the 494.498.BSL spectra demonstrates that in APO and ADP states there are two highly ordered populations of the relay helix. APO and ADP spectra contain the same two spectral components, but the distribution of these two components differs between states. Similarly, spectra from the 639.643 construct reveal an ADP-induced structural transition, but the difference between APO and ADP spectra demonstrates a 3o rotation of the 639.643 helix. Our results demonstrate structural transitions of two helixes within the CD of actin-bound myosin associated with nucleotide binding. Measuring these transitions is essential to understanding the molecular mechanism of force generation. This is particularly true for the relay helix, which plays a key role in the coupling of myosin ATPase and motor function. Additionally, these results demonstrate the utility of BSL for measuring transitions in protein orientation, order, and dynamics.

We have used transient time-resolved FRET (TR2FRET) to monitor the conformation of the relay helix in a myosin II functional mutant during the recovery stroke in real time. Myosin was perturbed with the F506A mutation (Dictyostelium discoideum sequence), located within the relay loop in the force-generating region. F506 is a highly conserved residue in myosin II and is a hypertrophic cardiomyopathy mutation site. Previous studies [Tsiavaliaris, EMBO Rep, 2002, 3(11), 1099] showed a significant effect of the F506A mutation on myosin function. Actin affinity in the presence of ATP was increased, and the mutant did not move actin filaments in in vitro motility assays. A small decrease in intrinsic fluorescence was observed upon addition of excess ATP, but ATP binding and hydrolysis were not affected by the mutation. It was proposed that the F506A disrupts the communication between the active site and the lever arm. We engineered a double-Cys myosin mutant (A639C:K498C) in the Cys-less background with the F506A functional mutation, and labeled the mutant with optical probes. We used TR-FRET to determine the interprobe distance, and TR2FRET measurements after rapid mixing with ATP revealed changes in the relay helix conformation during the recovery stroke in real time. The mutation induced significant disorder of the relay helix in the force-generating region, but myosin still produces a recovery stroke, changing the relay helix conformation from straight to bent. We conclude that (a) the relay helix is disordered in myosin functional mutant F506A, which demonstrates the importance of the relay loop - relay helix interaction in the relay helix stabilization, and (b) the relay helix is the major structural element in the force-generating region of myosin, responsible for communication from the active site to the converter domain and the lever arm.

We have used dipolar electron-electron resonance (DEER) and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the role of the myosin relay helix in coupling between the active site and the force-generating region of myosin II. Two double-Cys Dictyostelium myosin constructs have been engineered, and the structure of the relay helix was monitored by measuring interprobe distances in MSL/MSL or IAEDANS/Dabcyl-labeled myosin. Experiments were performed on WT myosin and on F506A, a functional mutant that has close to normal enzymatic activity but completely lacks motor functions (e.g. unable to move actin filaments or support cell development). We found that the WT myosin relay helix adopts two distinct states (straight and bent), with the bent conformation populated when ATP and ADP.Pi analogs are bound at the active site. In contrast, binding of nucleotide analogs had very little effect on relay helix conformation in the F506A mutant. In addition, the width of the distance distribution was significantly larger in the F506A compared to WT myosin, indicating loss of structural organization. Our results demonstrate that the relay helix plays a key role in coupling of myosin ATPase and motor activities. Loss of functionality observed in F506A myosin can be explained by the disruption of the relay helix-relay loop interactions that normally stabilize well-defined conformations of the myosin force-generating region allowing it to switch between distinct structural states.

We have used three complementary spectroscopic techniques, pulsed EPR (DEER), time-resolved fluorescence resonance energy transfer (TR-FRET) and transient time-resolved FRET [(TR)2FRET], to elucidate the mechanisms of energy transduction in myosin - an important motility protein involved in a variety of processes including muscle contraction. We engineered two double-Cys myosin mutants that were labeled with optical probes or spin labels, and determined structural changes in a single structural element of the myosin motor domain, the relay helix, as affected by nucleotide binding and hydrolysis. In all cases, time-domain detection permitted the reliable determination of the interprobe distance distribution, quantitating both order and disorder, and resolving coexisting structural states. While DEER offered superior distance resolution, (TR)2FRET permitted detection of transient structural changes after rapid mixing (stopped-flow) with ATP. All methods demonstrated two structural states of myosin during the recovery stroke, corresponding to straight and bent conformations of the relay helix, in parallel with straight and bent conformations of the entire myosin head. A narrower interprobe distance distribution in the post-recovery state shows ordering of the relay helix structure during the recovery stroke. These experiments reveal changes in both structure and dynamics of the relay helix and identify it as a key player in the interdomain coupling mechanism. This methodology is applicable to any enzyme in which double Cys mutants can be engineered in a key region of energy transduction, permitting resolution of structural states and dynamics in real time during the transient phase of a biochemical reaction.

We have used DEER (double electron-electron resonance) and TR-FRET (time-resolved fluorescence resonance energy transfer) to study conformational changes within the myosin II relay helix. Major structural changes during the myosin II ATPase cycle take place in the force-generating domain. Crystal structures show that the converter domain, the relay helix, and SH1 helix have different conformations in the proposed pre- and post- powerstroke structural states of myosin. This dramatic structural change is detected in solution studies by intrinsic fluorescence [1, 2] and EPR of a spin label attached to SH1 [3]. In the present study, we focus on the relay helix as a crucial structural element involved in coupling between the force-generating domain and the nucleotide-binding pocket. Cysteine mutations were introduced into a Cys-lite construct of Dictyostelium discoideum (Dicty) myosin in the lower 50k domain (either D515C or A639C) and the C-terminal end of the relay helix (K498C). These constructs were selectively modified with either MSL/MSL or IAEDANS/DABCYL pairs, and the distance between probes was measured in different myosin conformations trapped with nucleotides or nucleotide analogs Two conformations of the relay helix (with distinct probe-to-probe distances, presumably corresponding to the “straight” and “bent“ states of the relay helix) were resolved. Observed distances were in good agreement with existing crystal structures, but at least two distinct structural states were present in certain biochemical states (e.g, with bound ADP.BeF3, ADP.Vi, ADP.AlF4). The mole fraction of the “bent” conformation was higher with post-hydrolysis analogs (ADP.Vi, ADP.AlF4) bound at the active site. Our results reveal structural rearrangements within a single subdomain of myosin and provide insights into the coupling between ATP binding and changes in the force-generating region.

SummaryMolecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here, we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsin kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using nuclear magnetic resonance (NMR) spectroscopy, stopped-flow kinetics, and isothermal titration calorimetry, we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Protein dynamics in free recoverin limits the overall rate of binding.

Catalytic function of many enzymes is comprised of a number of microscopic steps including enzyme structural rearrangements and the chemical steps, in which bonds of the substrate(s) are broken/made to synthesize the product(s). Separation of these processes is a challenging task that hinders our understanding of enzyme catalysis. We have used nuclear magnetic resonance (NMR) and rapid-quenching techniques to independently study these processes in adenylate kinase (ADK) and to characterize their energetic contribution at atomic resolution. Adenylate kinase is an important enzyme that catalyzes a reversible reaction: 2ADP ↔ ATP +AMP, and is involved in maintaining energy homeostasis in a cell. Two binding sites of ADK and three different ligands result in a variety of biochemical states (determined by the bound ligands). Each of these states has different dynamic properties making the overall structural dynamics of ADK complex. We have tackled this problem by engineering a loss-of-function ADK mutant (with greatly reduced rate of turnover), which allowed us to “trap” the enzyme in well-defined biochemical states. Our results lead to a detailed energy profile of ADK, providing insights into the molecular mechanism of its functioning. This work reveals fundamental principles of enzyme catalysis and highlights the role of protein's intrinsic dynamics.

Cancer is a serious illness and the second leading cause of death in the US. It is caused by misregulation of the signaling cascades and uncontrolled cell proliferation. Protein kinases are the key players in the regulatory machinery, and in the last 20 years they have become an increasingly important drug targets. The main requirement for such drugs is selectivity: ideally, only one kinase should be affected by an individual drug. The prominent success story is an FDA-approved chronic myeloid leukemia therapeutic Gleevec. Gleevec is a highly specific inhibitor of the Abl kinase and has very little potency towards a closely related Src subfamily of kinases. This specificity is particularly intriguing considering that the drug-binding pockets look nearly identical in these kinases. We combined NMR and pre-steady-state kinetic experiments to monitor Gleevec binding in real time and with residue-specific precision. Our data establish a novel model that fully and quantitatively explains the observed 3000-fold difference in Gleevec's affinities to Abl and Src, solving a long-standing paradox in the field. These results reveal the general principles of kinase-drug interactions and highlight the molecular mechanisms behind nanomolar affinities found in clinically relevant therapeutics.