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Nature 正刊· Máté Pénzes·· 25 天前AI 评分34

抑制肌球蛋白实现毛细血管选择性舒张，阻断缺血级联反应

Capillary-selective vasodilation through myosin inhibition halts ischaemic cascades

AI 摘要

Nature 刊发研究显示，通过抑制肌球蛋白（myosin）可选择性舒张毛细血管，从而阻断缺血后的无复流级联反应。研究基于毛细血管周细胞收缩机制，在缺血模型中验证了该策略对微循环灌注的改善作用。该工作为缺血性损伤的微血管靶点干预提供了新的药理学路径。 brief: yes

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Data availability

The data supporting the findings of this study have been deposited in the figshare repository at https://doi.org/10.6084/m9.figshare.32956559. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) under accession codes 29ZY (SMM–R(+)2501) and 29ZZ (SMM–S(−)2501). Any extra data supporting the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We would like to thank Mihály Ruppert (Semmelweis University) for the help in LADO operations; András Szabó (Motorpharma) for his contributions to the synthesis of CLN-derivatives; and Nikolett Takács, Gábor Glatz and Maryam Gazeran (Motorpharma) for the professional technical assistance; and beam line scientists of PX2A (SOLEIL synchrotron) for excellent support during data collection.

We thank Louise Canon for her help in the beginning of this work. We also thank Prof. Mark Fishman (Harvard University), Prof. Mihály Kovács (Eötvös University), Prof.

Ádám Miklósi (Eötvös University), Eszter Nagy (Cebina) and Balázs Jelinek (Motorpharma) for detailed discussions and helpful comments on the manuscript.

Funding

This study has been supported by the Hungarian National Research Development and Innovation Office grant No. 2021-1.1.4-GYORSÍTÓSÁV-2022-00017 and 2025-1.1.4-LIFE_SCIENCES_II-2025-00002.

2-photon imaging methodology was developed with the support of the HUN-REN Hungarian Research Network (HUN-REN). Crystallography by the A.H. team was supported by grants from CNRS and ANR 21-CE11-0022-01. The A.H.

team is part of the Labex Cell(n)Scale (ANR-11-LBX-0038) and IDEX PSL (ANR-10-IDEX-0001-02-PSL). C.G. acknowledges doctoral funding from FRM (PBR202306017868). S.M. is supported by the British Heart Foundation (FS/IBSRF/21/25060). Pig model development by T.R.

and A.A.S. was supported by a grant from the National Research, Development and Innovation Office (NKFIH) of Hungary (K134939), and project no. RRF-2.3.1-21-2022-00003 has been implemented with the support provided by the European Union.

Author information

Author notes

  1. These authors contributed equally: Máté Pénzes, Máté Winternitz, Ádám I. Horváth

Authors and Affiliations

  1. Motorpharma, Budapest, Hungary

    Máté Pénzes, Máté Winternitz, Ádám I. Horváth, Usman Awan, Tamás Szimler, Anna Á. Rauscher, Máté Gyimesi & András Málnási-Csizmadia

  2. HUN-REN-ELTE Motor Pharmacology Research Group, Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary

    Máté Pénzes, Máté Winternitz, Ádám I. Horváth & András Málnási-Csizmadia

  3. Department of Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK

    Svetlana Mastitskaya

  4. Structural Motility, Institut Curie, CNRS, UMR144, PSL Research University, Paris, France

    Camille Glaser, Carlos Kikuti, Marie Juillé, Leila Heidsieck & Anne Houdusse

  5. Clinch Biosciences, Boston, MA, USA

    Anna Á. Rauscher, Máté Gyimesi & András Málnási-Csizmadia

  6. Heart and Vascular Center, Department of Experimental Cardiology and Surgical Techniques, Semmelweis University, Budapest, Hungary

    Tímea Bálint, Dávid Nagy, Tamás Radovits & Alex Ali Sayour

  7. Baim Institute for Clinical Research, Boston, MA, USA

    C. Michael Gibson

Authors

  1. Máté Pénzes
  2. Máté Winternitz
  3. Ádám I. Horváth
  4. Svetlana Mastitskaya
  5. Usman Awan
  6. Tamás Szimler
  7. Camille Glaser
  8. Carlos Kikuti
  9. Marie Juillé
  10. Leila Heidsieck
  11. Anna Á. Rauscher
  12. Tímea Bálint
  13. Dávid Nagy
  14. Tamás Radovits
  15. Alex Ali Sayour
  16. C. Michael Gibson
  17. Anne Houdusse
  18. Máté Gyimesi
  19. András Málnási-Csizmadia

Contributions

M.P., M.W. and Á.I.H. contributed equally to this work and share first authorship.

Conceptualization: M.P., M.G., A.M.-C.; development of methodology: M.P., M.W., Á.I.H., S.M., C.G., C.K., L.H., A.H., T.R., A.A.S., M.G., A.M.-C.; investigation: M.P., M.W., Á.I.H., S.M., U.A., T.S., C.G., C.K., M.J., L.H., A.Á.R., T.B., D.N., T.R., A.A.S., A.H., M.G., A.M.-C.; acquisition of data: M.P., M.W., Á.I.H., S.M., U.A., T.S., C.G., C.K., L.H., A.A.S., T.B., D.N., T.R., M.G., A.M.-C.; analysis and interpretation of data: M.P., M.W., Á.I.H., S.M., A.Á.R., C.G., C.K., M.J., L.H., A.H., T.B., D.N., A.A.S., M.G., A.M.-C.; supervision: M.G., A.M.-C.; visualization: M.P., C.G., M.G.; Writing: M.P., M.W., Á.I.H., S.M., C.K., A.H., M.G., A.M.-C.; writing – review and editing, all authors.

Corresponding authors

Correspondence to Máté Pénzes or András Málnási-Csizmadia.

Ethics declarations

Competing interests

The authors declare the following competing interests: M.G. and A.M.-C. are owners of Clinch Biosciences; M.G., A.Á.R. and A.M.-C. are owners of Motorpharma. Several authors are inventors on patents relevant to this work.

WO2019202346A3 (‘Pharmaceutically effective compounds inhibiting selectively the myosin 2 isoforms’) covers selective myosin-2 isoform inhibitors and has been granted in several jurisdictions, including US11845758B2, EP3781263B1, JP7463344B2 and IL278105B1; it is held by Printnet Kereskedelmi és Szolgáltató and Eötvös Loránd Tudományegyetem.

PCT/US25/38451 (published as WO2026020167A1, ‘Inhibitors of smooth muscle myosins and uses thereof’) covers SMM inhibitors and their therapeutic uses, and is a pending international (PCT) application held by Clinch Biosciences under assignment by Motorpharma.

Peer review

Peer review information

Nature thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 R(+)-selective inhibition of SMM over CM, with crystallographic validation.

a, Screening of matched R(+) and S(–) enantiomer pairs across SMM and CM revealed that R(+) derivatives consistently showed selective inhibition of SMM, as demonstrated by representative examples in (Extended Data Fig. 1f–h) (nR(+) = 57, nS(-) = 52 compounds). b-e, Validation of ligand positioning in the crystallographic structures. b, Two views of the 2Fo-Fc electron density map of R(+)2501-bound SMM contoured at 1.0 σ (blue), and the corresponding Fo-Fc difference map at 3.5 σ. c, Two views of the R(+)2501 polder map52 at 3.5 σ. (d) and (e) The same analysis performed with S(-)2501-bound SMM, with the polder map contoured at 5 σ for S(-)2501. For both drugs, the maps show well-defined density for the entire compounds, including their chiral hydroxyls, without ambiguity. Protein carbons are colored as in Fig. 1e. f-h, Actin-activated ATPase inhibition of SMM (red) and cardiac muscle myosin-S1 (CM, gray) by CLN-family derivatives. Derivatives containing an OH-group at the bridgehead (chiral) position and a methyl-group on the A-ring (f): (left-to-right) CLN-222 (nR(+),S(-) = 3, n is individual experiment/dose), CLN-2501 (nR(+) = 6, nS(-) = 3, n is individual experiment/dose), CLN-2503 (nR(+),S(-) = 3, n is individual experiment/dose), CLN-2531 (nR(+),S(-) = 3, n is individual experiment/dose), CLN-2532-I (nR(+),S(-) = 3, n is individual experiment/dose), CLN-2557 (nR(+),S(-) = 3, n is individual experiment/dose); an NH2-group at the bridgehead (chiral) position (g): (left-to-right) CLN-3400 (nR(+),S(-) = 3, n is individual experiment/dose), CLN-3403 (nR(+),S(-) = 3, n is individual experiment/dose) and CLN-3407 (nR(+),S(-) = 3, n is individual experiment/dose); an CONH2-group at the bridgehead (chiral) position (h): CLN-4401 (nR(+),S(-) = 3, n is individual experiment/dose). All plots show the measured relative ATPase activities (solid circles) and the fitted curves of a quadratic function used to determine the IC50 and maximal inhibition (MaxInh) parameters using SMM (red) and CM (gray). Structures of all derivatives, and the fitted parameters are shown in Supplementary Table 1.

Source data

Extended Data Fig. 2 Synthesis, quality control, and structure–activity analysis of R(+)2501 binding to smooth muscle myosin.

a, Flow Diagram for the synthesis of R(+)2501, b-f, Quality Check (QC) analysis of R(+)2501. b, QC by HPLC detected at 240 nm, purity: 99.47% (area%). c, QC by MS (APCI) confirming exact mass of 342 [MH]+ m/z. d, QC by chiral HPLC detected at 240 nm, R(+) enantiomer purity: 100% (area%). e, QC of racemate mixture by chiral HPLC detected at 240 nm, enantiomer distribution: 47.46% (area%) S(-)2501, 52.54% (area%) R(+)2501. f, QC by 1H-NMR confirms structure and purity. g-h, SAR analysis of SMM inhibitors. g, Structure of R(+)2501 with the four rings marked by letters A-D and functional groups. h, Structure of CK-571. Chemical groups are marked by letters A-D according to their position relative to R(+)2501. W1 and W2 denote structurally important water molecules involved in stabilizing the ligand–protein interaction network. i, R(+)2501-bound SMM structure (purple) superimposed with SMM in complex with CK-57134 (green). Cartoon radius is set by B-factor. j, Structural overlay of R(+)2501 and CK-571. k, Sequence alignment of the R(+)2501/CK-571 pocket in human SMM (MYH11), non-muscle myosin-2B (NM2B, MYH10), β-cardiac myosin heavy chain (Car, MYH7) and fast skeletal muscle myosin-2x/d (Sk2, MYH1). Residues involved in R(+)2501 binding are red. Conserved residues for binding of R(+)2501/CK-571 are indicated with a star. Colored boxes denote interaction differences between CK-571 and R(+)2501: blue, different; pink, exclusive to R(+)2501 binding; green, exclusive to CK-571 binding (Extended Data Fig. 1g,h, Extended Data Table 2). l-m, Structural mobility of R(+)2501– and S(−)2501–bound SMM. l, R(+)2501–bound SMM structure colored by B-factor (temperature factor), where warmer colors indicate higher atomic displacement and greater local structural flexibility, and cooler colors indicate more ordered regions of the protein. m, S(−)-2501–bound SMM structure colored by B-factor using the same scale. The coloring highlights differences in local flexibility and structural stabilization around the ligand-binding regions.

Extended Data Fig. 3 Tissue distribution, vessel-size-dependent relaxation, and cardiac tolerability of R(+)2501.

a, Tissue distribution of R(+)2501. Mean tissue concentrations of R(+)2501 two hours after intravenous injection, shown for each dose. Concentrations were below approximately 0.3 μM across all examined organs, including at the 40 mg/kg dose (n5mg/kg = 5, n10mg/kg = 2, n40mg/kg = 2 animals). b, Diameter-based relaxation half-lives. Capillary relaxation half-life determined by 2-PM and logarithmic x axis. BA: basilar artery, FA: femoral artery, CCA: common carotid artery (nBA = 28, nFA = 1, nCCA = 1, mean value is presented for capillary (0.4). c, Ex vivo Langendorff heart perfusion. Ventricular capillary dilation with 25 μM R(+)2501 and full washout (Wo) recovery (nBaseline = 60, nR(+)2501 = 75, nAfter Wo = 65 capillaries from the same animal, ****p(Baseline-R(+)2501) < 0.0001, ****p(R(+)2501-After Wo) < 0.0001). d-j, Effects of R(+)2501 on cardiac function and hemodynamics in healthy rats. Intravenous administration of R(+)2501 at 5, 10 and 20 mg/kg did not induce marked changes in left ventricular (LV) hemodynamics as shown by stroke work (d), stroke volume (e), heart rate (f), maximum LV pressure (g), together with indices of cardiac contractility (dP/dt_max) and diastolic function (dP/dt_min and Tau; h-j). Values are expressed as a percentage change relative to the corresponding time-matched vehicle condition (0% = vehicle). The vehicle (Veh) time point represents the reference condition and is therefore defined as 0% (n = 3 animals/dose). Data are mean ± SEM for (a,d-j) and mean ± SD for (c,d). Statistical analysis was performed using one-way ANOVA.

Source data

Extended Data Fig. 4 Occlusion-time dependence of renal capillary no-reflow and its reversal by R(+)2501 with recovery of kidney function.

a-b, Kidney can tolerate only ~10 min of occlusion before significant CNR develops. Beyond this threshold, reperfusion did not restore RBC velocity or flux to pre-ischemic levels (green shadow lane) when RBC velocity and flux were measured 30 min after reperfusion start (n10,R = 60, n10,T = 90, n30,R = 40, n30,T = 27, n60,R = 84, n60,T = 34, n120,R = 60, n120,T = 79 capillaries, R means reperfusion, T means R(+)2501 treatment). c, Renal artery occlusion time dependence of RBC velocity (orange) and flux (purple) values after reperfusion normalized to the healthy blood flow parameters, and representation of the non-moving RBC fractions (pink) as a function of occlusion-time (pink). Steep occlusion time dependence suggests that regenerative potentials are maintained only for a very limited time (cca. 10 min of occlusion) and beyond a threshold of 20–30 min the capillary flow deficit becomes refractory to reperfusion alone. Complete blockage of capillaries without R(+)2501 treatment becomes dominant after 60 min of occlusion, indicating that pericytes remain completely constricted and refractory to reperfusion alone (i.e. to restoration of large-artery flow). Kinetics of RBC velocity (d) and flux (e) in intravenous bolus 5 mg/kg R(+)2501 treated renal capillaries following 10, 30, 60 and 120 min of occlusion. Gray line indicates the reperfusion level of 60-minute non-treated capillaries as a reference. In all cases, both RBC velocity and flux increased back to the healthy range (green shadow lane) within 6 min of treatment. Both velocity and flux parameters increased above the healthy range in an occlusion time-dependent manner, suggesting that R(+)2501 is capable of opening the capillaries to a greater extent than the healthy regulated range (n10min = 3 animals, n30min = 2 animals, n60min = 3 animals, n120min = 3 animals). Thus, we also tested the effect of intravenous bolus 5 mg/kg R(+)2501 on the RBC velocity (f) and flux (g) of healthy animals and confirmed that R(+)2501 is capable of full relaxation of capillaries, increasing capillary blood flow to a higher than baseline level (nHealthy = 2, n5 = 2 animals). This confirms that this effect is not RAO-dependent but an inherent feature of the system, confirming that our effect is on the capillary pericytes maintaining regulated baseline capillary blood flow level by SMM-mediated contraction. h, Survival rate of animals after AKI of bilateral-90 min-RAO-operated rats (nV = 32, n5 = 33, n10 = 33, n20 = 3 animals). i, Addition of 100 mg/kg Vitamin C (n = 3 animals), 100 mg/kg allopurinol (n = 3 animals) or 10 mg/kg carprofen (Rimadyl) (n = 3 animals) into the 10 mg/kg R(+)2501 (n = 33 animals) treatment solution did not increase the survival rate of the bilateral-90 min-RAO-operated rats over the 10 mg/kg R(+)2501 treatment baseline. Concentration of creatinine and carbamide in blood plasma of RAO operated rats 24 h after occlusion (j), and calculated kidney function (eGFR) (k) as a function of renal artery occlusion time in RAO operated rats 24 h after occlusion (n0,15,30,120min = 3, n60min = 9, n90min = 6, n240min = 2 animals). Both carbamide and creatinine levels showed large variance among the animals resulting in high standard error of the calculated eGFR between 60-90-minute occlusion times. eGFR levels showed similar behavior as RBC velocity and flux (c.f. Extended Data Fig. 4c) suggesting that capillary blood flow and kidney functions are two closely interrelated parameters. This close correlation between capillary blood flow level and kidney function demonstrates that efficient capillary relaxation can directly influence long-term kidney functions.

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Extended Data Fig. 5 Experimental setup and effects of R(+)2501 on myocardial capillary flow, ECG, and survival after acute myocardial infarction.

a, Schematic of optimized 2-PM experimental setup to quantify capillary changes, RBC velocity and flux after LADO-induced AMI. b-c, Relative RBC velocity and flux in each phase (healthy, reperfusion, vehicle, bolus 20 mg/kg R(+)2501) (nReperfusion = 23, nVehicle = 39, nB(20) = 66 capillaries from 2 hearts, ****p(Reperfusion-B(20)) < 0.0001, ****p(Vehicle-B(20)) < 0.0001). d, ECG traces from different phases of experiments are shown, demonstrating ST-segment elevation during ligation (purple shading). Vehicle-treated animals developed pathological Q-waves and progressive cardiac decline, while R(+)2501-treated animals maintained near-normal ECG patterns at 120 min reperfusion. e, Survival outcomes after the reperfusion phase (120 min) (nB(V) = 10, nB(5) = 7, nB(10) = 7, nB(20) = 5 animals). f, Schematic of optimized 2-PM experimental setup and design to quantify RBC velocity and flux in AMI-induced CRS-I. g, Experimental setup: perivascular flow probe placed on the LAD and intracoronary catheter advanced via the femoral artery into the left main coronary artery for bolus injections. LAD flow, arterial pressure, and ECG were recorded continuously to generate pressure–flow loops. Data are mean ± SEM. Statistical analysis was performed using one-way ANOVA. Components in a, f and g created using BioRender; Winternitz, M. https://BioRender.com/bcb1th9 (2026).

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Extended Data Fig. 6 Genotoxicity assessment of R(+)2501 by in vivo micronucleus and Ames tests.

In vivo micronucleus test using intravenous bolus 10 and 100 mg/kg doses showed that R(+)2501 does not induce chromosome aberrations neither in blood (a) nor in the bone marrow (b) even at extremely high doses (nVehicle = 6, nPos = 6, n10mg/kg = 6, n100mg/kg = 6 animals). c, Hierarchy of gates applied during the flow cytometric analysis of blood and bone marrow samples collected during the in vivo micronucleus test (Extended Data Fig. 6a,b) is demonstrated on the example of a bone marrow sample. Gates are shown as rectangles or ellipses draw around the gated subpopulation of events. Arrows mark hierarchical relationships between the subpopulations. Ames reverse mutagenicity test using TA98 (d) (n2501-SO4 Xtreme 10% = 2, n2501-SO4 Xtreme 30% = 1, n2501 humS9 = 2, n2501-SO4 humS9 = 2, n2501 Xtreme = 1, n2501-SO4-S9 = 1 sample(s)) and TA100 (e) (n2501-SO4 Xtreme 10% = 1, n2501-SO4 Xtreme 30% = 1, n2501-SO4-S9 = 1 sample) S.typhimurium strains shows that R(+)2501 does not induce frameshift and base-substitution mutations, respectively, in the absence and presence of human liver S9 fractions from different sources including the 200-donor, mixed-gender extreme-pool liver S9 fraction.

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Extended Data Table 1 Data collection and refinement statistics for R(+)2501-bound SMM and S(-)2501-bound SMM

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Extended Data Table 2 Interaction table for comparison of R(+)2501 and CK-571 interaction with SMM

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Extended Data Table 3 Cage-side observation of rats following intravenous R(+)2501 at 100 and 200 mg/kg

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Supplementary information

Supplementary Information (download PDF )

This file contains further methodological details, including structural analysis of the R(+)2501 and S(−)2501 crystal structures, characterization of the AKI rat model, and safety pharmacology studies supporting the findings of the main manuscript. This file also contains Supplementary Table 1.

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Supplementary Video 1 (download MP4 )

The R(+)2501 binding site undergoes significant conformational changes during the myosin cycle. Trajectories were obtained using ChimeraX ‘morph’ command with different Myo2 structures as starting points: (1) Rigor (PDB 6BIH); (2) Smm2 actin-bound Strong-ADP (PDB 8SYF); (3) Post-rigor (PDB 3I5F); and (4) PPS (S(−)2501-bound, PDB 29ZZ). In all sequences, the R(+)2501-bound recovery stroke intermediate (PDB 29ZY) was used as the end-point structure. For simplification, the side chains of only a few key residues of the interface are shown.

Supplementary Video 2 (download MP4 )

Representative time-lapse fluorescence video of a capillary segment ensheathed by a pericyte. Capillaries were labelled with Alexa Fluor 488-conjugated WGA, and pericytes were visualized in transgenic NG2-DsRed BAC mice, in which they exhibit red fluorescence enabling unambiguous identification. Application of ET-1 elicits a rapid and marked pericyte contraction, accompanied by constriction of the capillary lumen. Subsequent addition of compound R(+)2501 induces an immediate relaxation of the pericyte, restoring capillary diameter and flow. These observations also support a direct, pericyte-specific action of R(+)2501.

Supplementary Video 3 (download MP4 )

Acute effect of R(+)2501 after 60 min of renal ischaemia. Within 10 s of intravenous injection, R(+)2501 dilates the peritubular renal capillaries and restores healthy perfusion to the pre-ischemic state (video speed 7 fps). The apparent streaks observed under normal flow conditions arise from the interaction between rapid RBC movement and raster scanning during image acquisition, which can elongate fluorescent signals within a single frame. As RBC velocity decreases during ischaemia and early reperfusion, individual cells become clearly resolved within the vessel lumen, eliminating this apparent streaking artefact.

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Pénzes, M., Winternitz, M., Horváth, Á.I. et al. Capillary-selective vasodilation through myosin inhibition halts ischaemic cascades. Nature (2026). https://doi.org/10.1038/s41586-026-11171-6

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  • Received: 17 November 2025

  • Accepted: 15 September 2026

  • Published: 14 October 2026

  • Version of record: 14 October 2026

  • DOI: https://doi.org/10.1038/s41586-026-11171-6

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