Molecular recognition
is fundamental to biological signaling. A
central question is how individual interactions between molecular
moieties affect the thermodynamics of ligand binding to proteins and
...how these effects might propagate beyond the immediate neighborhood
of the binding site. Here, we investigate this question by introducing
minor changes in ligand structure and characterizing the effects of
these on ligand affinity to the carbohydrate recognition domain of
galectin-3, using a combination of isothermal titration calorimetry,
X-ray crystallography, NMR relaxation, and computational approaches
including molecular dynamics (MD) simulations and grid inhomogeneous
solvation theory (GIST). We studied a congeneric series of ligands
with a fluorophenyl-triazole moiety, where the fluorine substituent
varies between the
ortho
,
meta
,
and
para
positions (denoted O, M, and P). The M and
P ligands have similar affinities, whereas the O ligand has 3-fold
lower affinity, reflecting differences in binding enthalpy and entropy.
The results reveal surprising differences in conformational and solvation
entropy among the three complexes. NMR backbone order parameters show
that the O-bound protein has reduced conformational entropy compared
to the M and P complexes. By contrast, the bound ligand is more flexible
in the O complex, as determined by
19
F NMR relaxation,
ensemble-refined X-ray diffraction data, and MD simulations. Furthermore,
GIST calculations indicate that the O
-
bound complex
has less unfavorable solvation entropy compared to the other two complexes.
Thus, the results indicate compensatory effects from ligand conformational
entropy and water entropy, on the one hand, and protein conformational
entropy, on the other hand. Taken together, these different contributions
amount to entropy–entropy compensation among the system components
involved in ligand binding to a target protein.
The beta-galactoside-binding mammalian lectin galectin-1 can bind, via its carbohydrate recognition domain (CRD), to various cell surface glycoproteins and has been implicated in a range of cancers. ...As a consequence of binding to sugar residues on cell surface receptors, it has been shown to have a pleiotropic effect across many cell types and mechanisms, resulting in immune system modulation and cancer progression. As a result, it has started to become a therapeutic target for both small and large molecules. In previous studies, we used fluorescence polarization (FP) assays to determine KD values to screen and triage small molecule glycomimetics that bind to the galectin-1 CRD. In this study, surface plasmon resonance (SPR) was used to compare human and mouse galectin-1 affinity measures with FP, as SPR has not been applied for compound screening against this galectin. Binding affinities for a selection of mono- and di-saccharides covering a 1000-fold range correlated well between FP and SPR assay formats for both human and mouse galectin-1. It was shown that slower dissociation drove the increased affinity at human galectin-1, whilst faster association was responsible for the effects in mouse galectin-1. This study demonstrates that SPR is a sound alternative to FP for early drug discovery screening and determining affinity estimates. Consequently, it also allows association and dissociation constants to be measured in a high-throughput manner for small molecule galectin-1 inhibitors.
Galectin-3 is a carbohydrate-binding protein central to regulating mechanisms of diseases such as fibrosis, cancer, metabolic, inflammatory, and heart disease. We recently found a high affinity (nM) ...thiodigalactoside GB0139 which currently is in clinical development (PhIIb) as an inhaled treatment of idiopathic pulmonary fibrosis. To enable treatment of systemically galectin-3 driven disease, we here present the first series of selective galectin-3 inhibitors combining high affinity (nM) with oral bioavailability. This was achieved by optimizing galectin-3 specificity and physical chemical parameters for a series of disubstituted monogalactosides. Further characterization showed that this class of compounds reduced profibrotic gene expression in liver myofibroblasts and displayed antifibrotic activity in CCl4-induced liver fibrosis and bleomycin-induced lung fibrosis mouse models. On the basis of the overall pharmacokinetic, pharmacodynamic, and safety profile, GB1211 was selected as the clinical candidate and is currently in phase IIa clinical trials as a potential therapy for liver cirrhosis and cancer.
Symmetrical and asymmetrical fluorinated phenyltriazolyl-thiodigalactoside derivatives have been synthesized and evaluated as inhibitors of galectin-1 and galectin-3. Systematic tuning of the ...phenyltriazolyl-thiodigalactosides’ fluoro-interactions with galectin-3 led to the discovery of inhibitors with exceptional affinities (K d down to 1–2 nM) in symmetrically substituted thiodigalactosides as well as unsurpassed combination of high affinity (K d 7.5 nM) and selectivity (46-fold) over galectin-1 for asymmetrical thiodigalactosides by carrying one trifluorphenyltriazole and one coumaryl moiety. Studies of the inhibitor–galectin complexes with isothermal titration calorimetry and X-ray crystallography revealed the importance of fluoro-amide interaction for affinity and for selectivity. Finally, the high affinity of the discovered inhibitors required two competitive titration assay tools to be developed: a new high affinity fluorescent probe for competitive fluorescent polarization and a competitive ligand optimal for analyzing high affinity galectin-3 inhibitors with competitive isothermal titration calorimetry.
A new series of orally available α-d-galactopyranosides with high affinity and specificity toward galectin-1 have been discovered. High affinity and specificity were achieved by changing six-membered ...aryl-triazolyl substituents in a series of recently published galectin-3-selective α-d-thiogalactosides (e.g., GB1107 K d galectin-1/3 3.7/0.037 μM) for five-membered heterocycles such as thiazoles. The in vitro pharmacokinetic properties were optimized, resulting in several galectin-1 inhibitors with favorable properties. One compound, GB1490 (K d galectin-1/3 0.4/2.7 μM), was selected for further characterization toward a panel of galectins showing a selectivity of 6- to 320-fold dependent on galectin. The X-ray structure of GB1490 bound to galectin-1 reveals the compound bound in a single conformation in the carbohydrate binding site. GB1490 was shown to reverse galectin-1-induced apoptosis of Jurkat cells at low μM concentrations. No cell cytotoxicity was observed for GB1490 up to 90 μM in the A549 cells. In pharmacokinetic studies in mice, GB1490 showed high oral bioavailability (F% > 99%).
The design of small and high‐affinity lectin inhibitors remains a major challenge because the natural ligand binding sites of lectin are often shallow and have polar character. Herein we report that ...derivatizing galactose with un‐natural structural elements that form multiple non‐natural lectin–ligand interactions (orthogonal multipolar fluorine–amide, phenyl–arginine, sulfur–π, and halogen bond) can provide inhibitors with extraordinary affinity (low nanomolar) for the model lectin, galectin‐3, which is more than five orders of magnitude higher than the parent galactose; moreover, is selective over other galectins.
Specific interactions working in concert! Derivatizing a low‐affinity monosaccharide with functionalities that form a combination of orthogonal multipolar fluorine–amide, phenyl–arginine, sulfur–π, and halogen bond interactions results in lectin ligands with affinities that far surpass those of common natural ligand fragments. These compounds are the smallest high‐affinity galectin‐3 inhibitors described and thus constitute a new class of promising drug lead structures.
Background
During recent years FLASH radiotherapy (FLASH‐RT) has shown promising results in radiation oncology, with the potential to spare normal tissue while maintaining the antitumor effects. The ...high speed of the FLASH‐RT delivery increases the need for fast and precise motion monitoring to avoid underdosing the target. Surface guided radiotherapy (SGRT) uses surface imaging (SI) to render a 3D surface of the patient. SI provides real‐time motion monitoring and has a large scanning field of view, covering off‐isocentric positions. However, SI has so far only been used for human patients with conventional setup and treatment.
Purpose
The aim of this study was to investigate the performance of SI as a motion management tool during electron FLASH‐RT of canine cancer patients.
Methods
To evaluate the SI system's ability to render surfaces of fur, three fur‐like blankets in white, grey, and black were used to imitate the surface of canine patients and the camera settings were optimized for each blanket. Phantom measurements using the fur blankets were carried out, simulating respiratory motion and sudden shift. Respiratory motion was simulated using the QUASAR Respiratory Motion Phantom with the fur blankets placed on the phantom platform, which moved 10 mm vertically with a simulated respiratory period of 4 s. Sudden motion was simulated with an in‐house developed phantom, consisting of a platform which was moved vertically in a stepwise motion at a chosen frequency. For sudden measurements, 1, 2, 3, 4, 5, 6, 7, and 10 Hz were measured. All measurements were both carried out at the conventional source‐to‐surface distance (SSD) of 100 cm, and in the locally used FLASH‐RT setup at SSD = 70 cm. The capability of the SI system to reproduce the simulated motion and the sampling time were evaluated. As an initial step towards clinical implementation, the feasibility of SI for surface guided FLASH‐RT was evaluated for 11 canine cancer patients.
Results
The SI camera was capable of rendering surfaces for all blankets. The deviation between simulated and measured mean peak‐to‐peak breathing amplitude was within 0.6 mm for all blankets. The sampling time was generally higher for the black fur than for the white and grey fur, for the measurement of both respiratory and sudden motion. The SI system could measure sudden motion within 62.5 ms and detect motion with a frequency of 10 Hz. The feasibility study of the canine patients showed that the SI system could be an important tool to ensure patient safety. By using this system we could ensure and document that 10 out of 11 canine patients had a total vector offset from the reference setup position <2 mm immediately before and after irradiation.
Conclusions
We have shown that SI can be used for surface guided FLASH‐RT of canine patients. The SI system is currently not fast enough to interrupt a FLASH‐RT beam while irradiating but with the short sampling time sudden motion can be detected. The beam can therefore be held just prior to irradiation, preventing treatment errors such as underdosing the target.
Aberrations in glycan and lectin expression and function represent one of the earliest hallmarks of cancer. Among galectins, a conserved family of β-galactoside-binding lectins, the role of ...Galectin-9 in immune–tumor interactions is well-established, although its effect on cancer cell behavior remains unclear. In this study, we assayed for, and observed, an association between Galectin-9 expression and invasiveness of breast cancer cells in vitro and in vivo. Genetic perturbation and pharmacological inhibition using novel cognate inhibitors confirmed a positive correlation between Galectin-9 levels and the adhesion of invasive cancer cells toand their invasion throughconstituted organomimetic extracellular matrix microenvironments. Signaling experiments and unbiased quantitative proteomics revealed Galectin-9 induction of Focal Adhesion Kinase activity and S100A4 expression, respectively. FAK inhibition decreased S100A4 mRNA levels. Our results provide crucial insights into how elevated Galectin-9 expression potentiates the invasiveness of breast cancer cells during early steps of invasion.
We have previously described a new series of selective and orally available galectin-1 inhibitors resulting in the thiazole-containing glycomimetic GB1490. Here, we show that the introduction of ...polar substituents to the thiazole ring results in galectin-1-specific compounds with low nM affinities. X-ray structural analysis of a new ligand-galectin-1 complex shows changes in the binding mode and ligand-protein hydrogen bond interactions compared to the GB1490-galectin-1 complex. These new high affinity ligands were further optimized with respect to affinity and ADME properties resulting in the galectin-1-selective GB1908 (K d galectin-1/3 0.057/6.0 μM). In vitro GB1908 inhibited galectin-1-induced apoptosis in Jurkat cells (IC50 = 850 nM). Pharmacokinetic experiments in mice revealed that a dose of 30 mg/kg b.i.d. results in free levels of GB1908 in plasma over galectin-1 K d for 24 h. GB1908 dosed with this regimen reduced the growth of primary lung tumor LL/2 in a syngeneic mouse model.
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