Cryo-EM structure of P-glycoprotein bound to triple elacridar inhibitor molecules.

P-glycoprotein (P-gp) is an ATP-binding cassette transporter known for its roles in expelling xenobiotic compounds from cells and contributing to cellular drug resistance through multidrug efflux. This mechanism is particularly problematic in cancer cells, where it diminishes the therapeutic efficacy of anticancer drugs. P-gp inhibitors, such as elacridar, have been developed to circumvent the decrease in drug efficacy due to P-gp efflux. An earlier study reported the cryo-EM structure of human P-gp-Fab (MRK-16) complex bound by two elacridar molecules, at a resolution of 3.6 Å. In this study, we have obtained a higher resolution (2.5 Å) structure of the P-gp- Fab (UIC2) complex bound by three elacridar molecules. This finding, which exposes a larger space for compound-binding sites than previously acknowledged, has significant implications for the development of more selective inhibitors and enhances our understanding of the compound recognition mechanism of P-gp.

Optimizing smartphone psychotherapy for depressive symptoms in patients with cancer: Multiphase optimization strategy using a decentralized multicenter randomized clinical trial (J-SUPPORT 2001 Study).

AIM: Patients with cancer experience various forms of psychological distress, including depressive symptoms, which can impact quality of life, elevate morbidity risk, and increase medical costs. Psychotherapy and pharmacotherapy are effective for reducing depressive symptoms among patients with cancer, but most patients prefer psychotherapy. This study aimed to develop an efficient and effective smartphone psychotherapy component to address depressive symptom. METHODS: This was a decentralized, parallel-group, multicenter, open, individually randomized, fully factorial trial. Patients aged ≥20 years with cancer were randomized by the presence/absence of three cognitive-behavioral therapy (CBT) skills (behavioral activation [BA], assertiveness training [AT], and problem-solving [PS]) on a smartphone app. All participants received psychoeducation (PE). The primary outcome was change in the patient health questionnaire-9 (PHQ-9) total score between baseline and week 8. Secondary outcomes included anxiety. RESULTS: In total, 359 participants were randomized. Primary outcome data at week 8 were obtained for 355 participants (99%). The week 8 PHQ-9 total score was significantly reduced from baseline for all participants by -1.41 points (95% confidence interval [CI] -1.89, -0.92), but between-group differences in change scores were not significant (BA: -0.04, 95% CI -0.75, 0.67; AT: -0.16, 95% CI -0.87, 0.55; PS: -0.19, 95% CI -0.90, 0.52). CONCLUSION: As the presence of any of the three intervention components did not contribute to a significant additive reduction of depressive symptoms, we cannot make evidence-based recommendations regarding the use of specific smartphone psychotherapy.

Design of complicated all-α protein structures.

A wide range of de novo protein structure designs have been achieved, but the complexity of naturally occurring protein structures is still far beyond these designs. Here, to expand the diversity and complexity of de novo designed protein structures, we sought to develop a method for designing 'difficult-to-describe' α-helical protein structures composed of irregularly aligned α-helices like globins. Backbone structure libraries consisting of a myriad of α-helical structures with five or six helices were generated by combining 18 helix-loop-helix motifs and canonical α-helices, and five distinct topologies were selected for de novo design. The designs were found to be monomeric with high thermal stability in solution and fold into the target topologies with atomic accuracy. This study demonstrated that complicated α-helical proteins are created using typical building blocks. The method we developed will enable us to explore the universe of protein structures for designing novel functional proteins.

Prognostic impact of HER2-low positivity in patients with HR-positive, HER2-negative, node-positive early breast cancer.

Adjuvant therapy for patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, node-positive, early breast cancer (EBC) remains challenging. The prognostic significance of HER2-low positivity in these patients is not fully understood. In our retrospective study, we analyzed 647 patients with HR-positive, HER2-negative, node-positive EBC, stratifying them into three cohorts based on axillary lymph node involvement, tumor size, and characteristics. Cohort 1 included patients with either ≥ 4 positive axillary lymph nodes or 1-3 positive nodes with histological grade 3 or tumor size ≥ 5 cm. Cohort 2 consisted of patients with 1-3 positive nodes, histological grade < 3, tumor size < 5 cm, and Ki-67 ≥ 20%. Cohort 3 comprised patients with 1-3 positive nodes, histological grade < 3, tumor size < 5 cm, and Ki-67 < 20%. We compared invasive disease-free survival (IDFS) and distant relapse-free survival (DRFS) between HER2-low (IHC1+ or IHC2+/FISH-) and HER2-zero (IHC0) groups in each cohort. In cohort 1, HER2-low patients exhibited significantly better 5-year IDFS (84.2% vs. 73.6%, p = 0.0213) and DRFS (88.2% vs. 79.8%, p = 0.0154). However, no significant differences were observed in cohorts 2 and 3. Our findings suggest HER2-low positivity as a prognostic factor in HR-positive, HER2-negative, and node-positive EBC.

Discordance in PD-L1 expression using 22C3 and SP142 assays between primary and metastatic triple-negative breast cancer.

AIMS: SP142 and 22C3 assays are approved companion diagnostic assays for anti-PD-1/PD-L1 therapy selection in metastatic triple-negative breast cancer (TNBC). The discordance in PD-L1 status between primary and metastatic tumors in the same patient has been poorly characterized. Here, we examined the concordance of PD-L1 status between the two assays and between primary tumors and metastases for each assay. METHODS: We retrospectively evaluated tumor samples from 160 patients with TNBC, including 45 patients with paired primary and metastatic tumors. PD-L1 status was assessed using SP142 and 22C3 assays, to determine the immune cell (IC) score, tumor cell (TC) score (SP142 and 22C3), and combined proportion score (CPS: 22C3). RESULTS: The concordance of PD-L1 positivity at diagnostic cutoffs for SP142 (IC ≥ 1) and 22C3 (CPS ≥ 10) was substantial (κ = 0.80) in primary tumors and moderate (κ = 0.60) in metastatic tumors. In comparison, between primary and metastatic tumors, the concordance with 22C3 was moderate (κ = 0.50), whereas that with SP142 was poor (κ = -0.03). Among patients who were PD-L1 negative for both assays in primary tumors, 7/30 (23.3%) were PD-L1 positive for both or either 22C3 or SP142 in the metastatic tumors. CONCLUSIONS: The inter-assay concordance of PD-L1 positivity at diagnostic cutoffs was substantial in primary tumors and moderate in metastatic tumors. Discordance between PD-L1 status in primary and metastatic tumors was frequently observed, especially with SP142. Some patients with a PD-L1-negative status in primary tumors may still be candidates for immunotherapy, depending on the PD-L1 status in their metastatic tumors.

Assessment of nuclear grade-based recurrence risk classification in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative, node-positive high-risk early breast cancer.

BACKGROUND: Histological grade (HG) has been used in the MonrachE trial to select patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, node-positive high-risk early breast cancer (EBC). Although nuclear grade (NG) is widely used in Japan, it is still unclear whether replacing HG with NG can appropriately select high-risk patients. METHODS: We retrospectively reviewed 647 patients with HR-positive, HER2-negative, node-positive EBC and classified them into the following four groups: group 1: ≥ 4 positive axillary lymph nodes (pALNs) or 1-3 pALNs and either grade 3 of both grading systems or tumors ≥ 5 cm; group 2: 1-3 pALNs, grade < 3, tumor < 5 cm, and Ki-67 ≥ 20%; group 3: 1-3 pALNs, grade < 3, tumor < 5 cm, and Ki-67 < 20%; and group 4: group 2 or 3 by HG classification but group 1 by NG classification. We compared invasive disease-free survival (IDFS) and distant relapse-free survival (DRFS) among the four groups using the Kaplan-Meier method with the log-rank test. RESULTS: Group 1 had a significantly worse 5-year IDFS and DRFS than groups 2 and 3 (IDFS 80.8% vs. 89.5%, P = 0.0319, 80.8% vs. 95.5%, P = 0.002; DRFS 85.2% vs. 95.3%, P = 0.0025, 85.2% vs. 98.4%, P < 0.001, respectively). Group 4 also had a significantly worse 5-year IDFS (78.0%) and DRFS (83.6%) than groups 2 and 3. CONCLUSIONS: NG was useful for stratifying the risk of recurrence in patients with HR-positive, HER2-negative, node-positive EBC and was the appropriate risk assessment for patient groups not considered high-risk by HG classification.

Nuclear Magnetic Resonance Detection of Hydrogen Bond Network in a Proton Pump Rhodopsin RxR and Its Alteration during the Cyclic Photoreaction.

Hydrogen bond formation and deformation are crucial for the structural construction and functional expression of biomolecules. However, direct observation of exchangeable hydrogens, especially for oxygen-bound hydrogens, relevant to hydrogen bonds is challenging for current structural analysis approaches. Using solution-state NMR spectroscopy, this study detected the functionally important exchangeable hydrogens (i.e., Y49-ηOH and Y178-ηOH) involved in the pentagonal hydrogen bond network in the active site of R. xylanophilus rhodopsin (RxR), which functions as a light-driven proton pump. Moreover, utilization of the original light-irradiation NMR approach allowed us to detect and characterize the late photointermediate state (i.e., O-state) of RxR and revealed that hydrogen bonds relevant to Y49 and Y178 are still maintained during the photointermediate state. In contrast, the hydrogen bond between W75-εNH and D205-γCOO- is strengthened and stabilizes the O-state.

Six states of Enterococcus hirae V-type ATPase reveals non-uniform rotor rotation during turnover.

The vacuolar-type ATPase from Enterococcus hirae (EhV-ATPase) is a thus-far unique adaptation of V-ATPases, as it performs Na+ transport and demonstrates an off-axis rotor assembly. Recent single molecule studies of the isolated V1 domain have indicated that there are subpauses within the three major states of the pseudo three-fold symmetric rotary enzyme. However, there was no structural evidence for these. Herein we activate the EhV-ATPase complex with ATP and identified multiple structures consisting of a total of six states of this complex by using cryo-electron microscopy. The orientations of the rotor complex during turnover, especially in the intermediates, are not as perfectly uniform as expected. The densities in the nucleotide binding pockets in the V1 domain indicate the different catalytic conditions for the six conformations. The off-axis rotor and its' interactions with the stator a-subunit during rotation suggests that this non-uniform rotor rotation is performed through the entire complex.

Anti-nanodisc antibodies specifically capture nanodiscs and facilitate molecular interaction kinetics studies for membrane protein.

Nanodisc technology has dramatically advanced the analysis of molecular interactions for membrane proteins. A nanodisc is designed as a vehicle for membrane proteins that provide a native-like phospholipid environment and better thermostability in a detergent-free buffer. This enables the determination of the thermodynamic and kinetic parameters of small molecule binding by surface plasmon resonance. In this study, we generated a nanodisc specific anti-MSP (membrane scaffold protein) monoclonal antibody biND5 for molecular interaction analysis of nanodiscs. The antibody, biND5 bound to various types of nanodiscs with sub-nanomolar to nanomolar affinity. Epitope mapping analysis revealed specific recognition of 8 amino acid residues in the exposed helix-4 structure of MSP. Further, we performed kinetics binding analysis between adenosine A2a receptor reconstituted nanodiscs and small molecule antagonist ZM241385 using biND5 immobilized sensor chips. These results show that biND5 facilitates the molecular interaction kinetics analysis of membrane proteins substituted in nanodiscs.

Class B1 GPCR activation by an intracellular agonist.

G protein-coupled receptors (GPCRs) generally accommodate specific ligands in the orthosteric-binding pockets. Ligand binding triggers a receptor allosteric conformational change that leads to the activation of intracellular transducers, G proteins and ß-arrestins. Because these signals often induce adverse effects, the selective activation mechanism for each transducer must be elucidated. Thus, many orthosteric-biased agonists have been developed, and intracellular-biased agonists have recently attracted broad interest. These agonists bind within the receptor intracellular cavity and preferentially tune the specific signalling pathway over other signalling pathways, without allosteric rearrangement of the receptor from the extracellular side1-3. However, only antagonist-bound structures are currently available1,4-6, and there is no evidence to support that biased agonist binding occurs within the intracellular cavity. This limits the comprehension of intracellular-biased agonism and potential drug development. Here we report the cryogenic electron microscopy structure of a complex of Gs and the human parathyroid hormone type 1 receptor (PTH1R) bound to a PTH1R agonist, PCO371. PCO371 binds within an intracellular pocket of PTH1R and directly interacts with Gs. The PCO371-binding mode rearranges the intracellular region towards the active conformation without extracellularly induced allosteric signal propagation. PCO371 stabilizes the significantly outward-bent conformation of transmembrane helix 6, which facilitates binding to G proteins rather than ß-arrestins. Furthermore, PCO371 binds within the highly conserved intracellular pocket, activating 7 out of the 15 class B1 GPCRs. Our study identifies a new and conserved intracellular agonist-binding pocket and provides evidence of a biased signalling mechanism that targets the receptor-transducer interface.

Physical pictures of rotation mechanisms of F1- and V1-ATPases: Leading roles of translational, configurational entropy of water.

We aim to develop a theory based on a concept other than the chemo-mechanical coupling (transduction of chemical free energy of ATP to mechanical work) for an ATP-driven protein complex. Experimental results conflicting with the chemo-mechanical coupling have recently emerged. We claim that the system comprises not only the protein complex but also the aqueous solution in which the protein complex is immersed and the system performs essentially no mechanical work. We perform statistical-mechanical analyses on V1-ATPase (the A3B3DF complex) for which crystal structures in more different states are experimentally known than for F1-ATPase (the α3ß3γ complex). Molecular and atomistic models are employed for water and the structure of V1-ATPase, respectively. The entropy originating from the translational displacement of water molecules in the system is treated as a pivotal factor. We find that the packing structure of the catalytic dwell state of V1-ATPase is constructed by the interplay of ATP bindings to two of the A subunits and incorporation of the DF subunit. The packing structure represents the nonuniformity with respect to the closeness of packing of the atoms in constituent proteins and protein interfaces. The physical picture of rotation mechanism of F1-ATPase recently constructed by Kinoshita is examined, and common points and differences between F1- and V1-ATPases are revealed. An ATP hydrolysis cycle comprises binding of ATP to the protein complex, hydrolysis of ATP into ADP and Pi in it, and dissociation of ADP and Pi from it. During each cycle, the chemical compounds bound to the three A or ß subunits and the packing structure of the A3B3 or α3ß3 complex are sequentially changed, which induces the unidirectional rotation of the central shaft for retaining the packing structure of the A3B3DF or α3ß3γ complex stabilized for almost maximizing the water entropy. The torque driving the rotation is generated by water with no input of chemical free energy. The presence of ATP is indispensable as a trigger of the torque generation. The ATP hydrolysis or synthesis reaction is tightly coupled to the rotation of the central shaft in the normal or inverse direction through the water-entropy effect.

A prediction model for distant metastasis after isolated locoregional recurrence of breast cancer.

PURPOSE: The impact of progesterone receptor (PR) status on the prognosis of breast cancer after isolated locoregional recurrence (ILRR) remains unclear. This study evaluated the impact of clinicopathologic factors, including PR status of ILRR, on distant metastasis (DM) after ILRR. METHODS: We retrospectively identified 306 patients with ILRR diagnosed at the National Cancer Center Hospital between 1993 and 2021 from the database. Cox proportional hazards analysis was performed to examine factors associated with DM after ILRR. We developed a risk prediction model based on the number of detected risk factors and estimated survival curves using the Kaplan-Meier method. RESULTS: During a median follow-up time of 4.7 years after ILRR diagnosis, 86 patients developed DM, and 50 died. Multivariate analysis revealed that seven risk factors were associated with poor distant metastasis-free survival (DMFS): estrogen receptor-positive/PR-negative/human epidermal growth factor receptor 2-negative ILRR, short disease-free interval, recurrence site other than ipsilateral breast, no-resection of ILRR tumor, chemotherapy for the primary tumor, nodal stage in the primary tumor, and no endocrine therapy for ILRR. The predictive model classified patients into 4 groups based on the number of risk factors: low-, intermediate-, high-, and the highest-risk groups with 0 to 1, 2, 3 to 4, and 5 to 7 factors, respectively. This revealed significant variation in DMFS among the groups. A higher number of the risk factors was associated with poorer DMFS. CONCLUSION: Our prediction model, which considered the ILRR receptor status, may contribute to the development of a treatment strategy for ILRR.

Clinical significance of tumor cell seeding associated with needle biopsy in patients with breast cancer.

BACKGROUND/OBJECTIVE: The occurrence of iatrogenic tumor cell seeding (seeding) in needle tract scars formed by core needle biopsy (CNB) or vacuum-assisted biopsy (VAB) is well known. Some risk factors for seeding have been reported, but the clinicopathological risk factors and its prognosis have not been fully investigated. We evaluated the clinical features and prognosis of seeding. METHODS: We included 4405 patients who had undergone surgery (lumpectomy or mastectomy) with a diagnosis of breast cancer by preoperative CNB or VAB at our hospital between January 2012 and February 2021. Data of patients with confirmed presence of seeding in resected specimens were collected from pathological records. We analyzed the risk factors of seeding using logistic regression analysis and compared the ipsilateral breast tumor recurrence (IBTR) rate between cases based on the presence or absence of seeding in the lumpectomy group. RESULTS: Of the 4405 patients, 133 (3.0%) had confirmed seeding. Univariate analysis revealed the association of clinicopathological features of seeding with lower nuclear grade (NG1 vs NG2-3; p = 0.043), lower Ki-67 (<30 vs. ≥30; p = 0.049), estrogen receptor (ER) positivity (positive vs negative; p<0.01), and human epidermal growth factor receptor 2 (HER2) negativity (negative vs positive; p = 0.016). Multivariate analysis showed ER positivity (odds ratio, 5.23; p<0.05) as an independent risk factor of seeding. The IBTR rate was not significantly different between the seeding and non-seeding groups. CONCLUSIONS: Seeding was more likely to occur in ER positive, HER2 negative carcinomas with less aggressive features, and may remain subclinical if adequate adjuvant treatments are administered.

Impact of adjuvant chemotherapy and radiotherapy on tumour-infiltrating lymphocytes and PD-L1 expression in metastatic breast cancer.

BACKGROUND: Chemotherapy and radiotherapy were postulated to induce an inflamed tumour microenvironment. We aimed to evaluate the effects of adjuvant chemotherapy/radiotherapy on tumour-infiltrating lymphocytes (TILs) and programmed death-ligand 1 (PD-L1) expression in metastatic breast cancer. METHODS: We identified paired primary and metastatic tumours in 85 patients with breast cancer. Stromal TILs were assessed according to international guidelines. PD-L1 expression was evaluated using the VENTANA SP142 assay. RESULTS: TILs were significantly lower in metastatic tumours than in primary tumours (12.2 vs. 8.3%, p = 0.049). PD-L1 positivity was similar between primary and metastatic tumours (21.2 vs. 14.1%, p = 0.23). TILs were significantly lower in patients who received adjuvant chemotherapy than in those who did not (-9.07 vs. 1.19%, p = 0.01). However, radiotherapy had no significant effect on TILs (p = 0.44). Decreased TILs predicted worse post-recurrence survival (hazard ratio, 2.94; 95% confidence interval [CI]: 1.41-6.13, p = 0.003), while increased TILs was associated with a better prognosis (HR, 0.12; 95% CI: 0.02-0.08, p = 0.04). CONCLUSIONS: TILs decreased in metastatic tumours, particularly in patients who relapsed after adjuvant chemotherapy. Changes in TILs from primary to metastatic sites could be a prognostic factor after recurrence.

Integrative prognostic analysis of tumor-infiltrating lymphocytes, CD8, CD20, programmed cell death-ligand 1, and tertiary lymphoid structures in patients with early-stage triple-negative breast cancer who did not receive adjuvant chemotherapy.

PURPOSE: Stromal tumor-infiltrating lymphocytes (TILs) are independent prognostic factors in systemically untreated early-stage triple-negative breast cancer (TNBC). Other immune biomarkers including CD8, CD20, programmed cell death-ligand 1 (PD-L1), and tertiary lymphoid structures (TLS) are also reported to be associated with prognosis. However, whether combining other immune biomarkers with TILs would allow for further prognostic stratification is unknown. METHODS: We retrospectively analyzed 125 patients with early-stage TNBC not receiving perioperative chemotherapy. Stromal TILs and TLS were evaluated on hematoxylin-eosin slides. PD-L1 expression was evaluated using the SP142 assay. CD8 and CD20 were assessed by immunohistochemistry and counted by digital pathology. RESULTS: Immune biomarker levels were positively correlated (p < 0.001). Adding CD8 and PD-L1 to multivariable analysis including clinicopathological factors (stage and histological grade) and TILs significantly improved the prognostic model (likelihood ratio χ2 = 9.24, p = 0.01). In Cox regression analysis, high CD8 was significantly associated with better prognosis [hazard ratio (HR) 0.69, 95% confidence interval (CI) 0.48-0.98, p = 0.04], and PD-L1 positivity was significantly associated with worse prognosis (HR 4.33, 95%CI 1.57-11.99, p = 0.005). Patients with high CD8/PD-L1 (-) tumors had the most favorable prognosis [5 year invasive disease-free survival (iDFS), 100%], while patients with low CD8/PD-L1( +) tumors had the worst prognosis (5 year iDFS, 33.3%). CONCLUSION: CD8 and PD-L1 levels add prognostic information beyond TILs for early-stage TNBC not receiving perioperative chemotherapy. CD8-positive T cells and PD-L1 may be useful for prognostic stratification and in designing future clinical trials of TNBC.

Clinical significance of discordances in sentinel lymph node reactivity between radioisotope and indocyanine green fluorescence in patients with cN0 breast cancer.

BACKGROUND: /Objective: To evaluate the usefulness of combining radioisotopes (RI) and indocyanine green (ICG) and investigate discordances in sentinel lymph node (SN) reactivity using each tracer in cN0 breast cancer patients. METHODS: In total, 338 cN0 primary breast cancer patients who underwent SN biopsy with RI and ICG and axillary lymph node (ALN) dissection were included. SN positivity with RI, ICG, and a combination of RI and ICG was denoted as SN(RI), SN(ICG), and SN(RI+ICG), respectively. We retrospectively estimated metastatic SN detection rates, each method's discordance rate, and the correlation of discordances in SN reactivity with postoperative N staging. RESULTS: The combination of RI and ICG had higher metastatic SN detection rates (99.7%) than RI or ICG alone (91.7% and 96.4%, respectively; p < 0.01). The discordance rate between SN(RI) and SN(ICG) in detecting metastatic SNs was 11.3% (38/337 cases). The absence of SN(RI), cT stage (cT2-3), higher histological grade (G3), and histological type (special type) were identified as risk factors of pN2-3 disease (odds ratios: 8.64, 2.56, 1.92, and 3.28, respectively; p < 0.01). CONCLUSION: Discordances in SN reactivity between RI and ICG helps in identifying SN metastasis. Although the absence of SN(RI) is rare, it is a significant sign of advanced ALN metastases. The findings of our study indicate that ALN dissection should be considered for accurate nodal staging in such cases.

Identifying antibiotics based on structural differences in the conserved allostery from mitochondrial heme-copper oxidases.

Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.