Galectin-3 N-terminal tail prolines modulate cell activity and glycan-mediated oligomerization/phase separation.

Galectin-3 (Gal-3) has a long, aperiodic, and dynamic proline-rich N-terminal tail (NT). The functional role of the NT with its numerous prolines has remained enigmatic since its discovery. To provide some resolution to this puzzle, we individually mutated all 14 NT prolines over the first 68 residues and assessed their effects on various Gal-3-mediated functions. Our findings show that mutation of any single proline (especially P37A, P55A, P60A, P64A/H, and P67A) dramatically and differentially inhibits Gal-3-mediated cellular activities (i.e., cell migration, activation, endocytosis, and hemagglutination). For mechanistic insight, we investigated the role of prolines in mediating Gal-3 oligomerization, a fundamental process required for these cell activities. We showed that Gal-3 oligomerization triggered by binding to glycoproteins is a dynamic process analogous to liquid-liquid phase separation (LLPS). The composition of these heterooligomers is dependent on the concentration of Gal-3 as well as on the concentration and type of glycoprotein. LLPS-like Gal-3 oligomerization/condensation was also observed on the plasma membrane and disrupted endomembranes. Molecular- and cell-based assays indicate that glycan binding-triggered Gal-3 LLPS (or LLPS-like) is driven mainly by dynamic intermolecular interactions between the Gal-3 NT and the carbohydrate recognition domain (CRD) F-face, although NT-NT interactions appear to contribute to a lesser extent. Mutation of each proline within the NT differentially controls NT-CRD interactions, consequently affecting glycan binding, LLPS, and cellular activities. Our results unveil the role of proline polymorphisms (e.g., at P64) associated with many diseases and suggest that the function of glycosylated cell surface receptors is dynamically regulated by Gal-3.

Chemokines and galectins form heterodimers to modulate inflammation.

Chemokines and galectins are simultaneously upregulated and mediate leukocyte recruitment during inflammation. Until now, these effector molecules have been considered to function independently. Here, we tested the hypothesis that they form molecular hybrids. By systematically screening chemokines for their ability to bind galectin-1 and galectin-3, we identified several interacting pairs, such as CXCL12 and galectin-3. Based on NMR and MD studies of the CXCL12/galectin-3 heterodimer, we identified contact sites between CXCL12 ß-strand 1 and Gal-3 F-face residues. Mutagenesis of galectin-3 residues involved in heterodimer formation resulted in reduced binding to CXCL12, enabling testing of functional activity comparatively. Galectin-3, but not its mutants, inhibited CXCL12-induced chemotaxis of leukocytes and their recruitment into the mouse peritoneum. Moreover, galectin-3 attenuated CXCL12-stimulated signaling via its receptor CXCR4 in a ternary complex with the chemokine and receptor, consistent with our structural model. This first report of heterodimerization between chemokines and galectins reveals a new type of interaction between inflammatory mediators that can underlie a novel immunoregulatory mechanism in inflammation. Thus, further exploration of the chemokine/galectin interactome is warranted.

PLG-007 and Its Active Component Galactomannan-α Competitively Inhibit Enzymes That Hydrolyze Glucose Polymers.

PLG-007 is a developmental therapeutic compound that has been clinically shown to reduce the magnitude of postprandial glucose excursions and has the potential to be an adjunct treatment for diabetes and inflammatory-related diseases. The present investigation is aimed at understanding the molecular mechanism of action of PLG-007 and its galactomannan (GM) components GMα and GMß (in a 1:4 mass ratio, respectively) on enzyme (i.e., α-amylase, maltase, and lactase) hydrolysis of glucose polymers using colorimetric assays and 13C HSQC NMR spectroscopy. The starch-iodine colorimetric assay indicated that GMα strongly inhibits α-amylase activity (~16-fold more potent than GMß) and thus is the primary active component in PLG-007. 13C HSQC experiments, used to follow the α-amylase-mediated hydrolysis of starch and amylopectin, further demonstrate the α-amylase inhibitory effect of GMα via α-amylase-mediated hydrolysis of starch and amylopectin. Maltohexaose (MT6) was used to circumvent the relative kinetic complexity of starch/amylopectin degradation in Michaelis-Menten analyses. The Vmax, KM, and Ki parameters were determined using peak volume integrals from 13C HSQC NMR spectra. In the presence of PLG-007 with α-amylase and MT6, the increase in KM from 7.5 ± 0.6 × 10-3 M (control) to 21 ± 1.4 × 10-3 M, with no significant change in Vmax, indicates that PLG-007 is a competitive inhibitor of α-amylase. Using KM values, Ki was estimated to be 2.1 ± 0.9 × 10-6 M; however, the microscopic Ki value of GMα is expected to be larger as the binding stoichiometry is likely to be greater than 1:1. Colorimetric assays also demonstrated that GMα is a competitive inhibitor of the enzymes maltase and lactase. Overall, this study provides insight as to how PLG-007 (GMα) is likely to function in vivo.

Topsy-turvy binding of negatively charged homogalacturonan oligosaccharides to galectin-3.

Galectin-3 is crucial to many physiological and pathological processes. The generally accepted dogma is that galectins function extracellularly by binding specifically to ß(1→4)-galactoside epitopes on cell surface glycoconjugates. Here, we used crystallography and NMR spectroscopy to demonstrate that negatively charged homogalacturonans (HG, linear polysaccharides of α(1→4)-linked-D-galacturonate (GalA)) bind to the galectin-3 carbohydrate recognition domain. The HG carboxylates at the C6 positions in GalA rings mandate that this saccharide bind galectin-3 in an unconventional, "topsy-turvy" orientation that is flipped by about 180o relative to that of the canonical ß-galactoside lactose. In this binding mode, the reducing end GalA ß-anomer of HGs takes the position of the nonreducing end galactose residue in lactose. This novel orientation maintains interactions with the conserved tryptophan and seven of the most crucial lactose-binding residues, albeit with different H-bonding interactions. Nevertheless, the HG molecular orientation and new interactions have essentially the same thermodynamic binding parameters as lactose. Overall, our study provides structural details for a new type of galectin-sugar interaction that broadens glycospace for ligand binding to Gal-3 and suggests how the lectin may recognize other negatively charged polysaccharides like glycoaminoglycans (e.g. heparan sulfate) on the cell surface. This discovery impacts on our understanding of galectin-mediated biological function.

Pro4 prolyl peptide bond isomerization in human galectin-7 modulates the monomer-dimer equilibrum to affect function.

Human galectin-7 (Gal-7; also termed p53-induced gene 1 product) is a multifunctional effector by productive pairing with distinct glycoconjugates and protein counter-receptors in the cytoplasm and nucleus, as well as on the cell surface. Its structural analysis by NMR spectroscopy detected doubling of a set of particular resonances, an indicator of Gal-7 existing in two conformational states in slow exchange on the chemical shift time scale. Structural positioning of this set of amino acids around the P4 residue and loss of this phenomenon in the bioactive P4L mutant indicated cis-trans isomerization at this site. Respective resonance assignments confirmed our proposal of two Gal-7 conformers. Mapping hydrogen bonds and considering van der Waals interactions in molecular dynamics simulations revealed a structural difference for the N-terminal peptide, with the trans-state being more exposed to solvent and more mobile than the cis-state. Affinity for lactose or glycan-inhibitable neuroblastoma cell surface contact formation was not affected, because both conformers associated with an overall increase in order parameters (S2). At low µM concentrations, homodimer dissociation is more favored for the cis-state of the protein than its trans-state. These findings give direction to mapping binding sites for protein counter-receptors of Gal-7, such as Bcl-2, JNK1, p53 or Smad3, and to run functional assays at low concentration to test the hypothesis that this isomerization process provides a (patho)physiologically important molecular switch for Gal-7.

Galectin-3 binds selectively to the terminal, non-reducing end of ß(1→4)-galactans, with overall affinity increasing with chain length.

Galactans are linear polysaccharides of ß(1→4)-linked galactose residues. Although they can antagonize galectin function, the nature of their binding to galectins needs to be better defined to develop them as drugs. Here, we investigated interactions between galectin-3 (Gal-3) and a series of galactans ranging in weight average molecular weight from 670 to 7550 Da. 15N-1H HSQC NMR studies with 15N-labeled Gal-3 carbohydrate recognition domain (CRD) indicate that each of these galactans interacts primarily with residues in ß-strands 4, 5 and 6 on the canonical, ß-galactoside sugar binding S-face. Although these galactans also bind to full length Gal-3 (CRD plus N-terminal tail) to the same extent, it appears that binding to the S-face attenuates interactions between the CRD F-face and N-terminal tail, making interpretation of site-specific binding unclear. Following assignment of galactan 13C and 1H resonances using HSQC, HMBC and TOCSY experiments, we used 13C-1H HSQC data to demonstrate that the Gal-3 CRD binds to the terminal, non-reducing end of these galactans, regardless of their size, but with binding affinity increasing as the galactan chain length increases. Overall, our findings increase understanding as to how galactans interact with Gal-3 at the non-reducing, terminal end of galactose-containing polysaccharides as found on the cell surface.

NMR-based insight into galectin-3 binding to endothelial cell adhesion molecule CD146: Evidence for noncanonical interactions with the lectin's CRD ß-sandwich F-face.

Galectin-3 (Gal-3) binds to cell adhesion glycoprotein CD146 to promote cytokine secretion and mediate endothelial cell migration. Here, we used Nuclear Magnetic Resonance (NMR) 15N-Heteronuclear Single Quantum Coherence (HSQC) spectroscopy to investigate binding between 15N-labeled Gal-3 and the extracellular domain (eFL) of purified CD146 (five Ig-like ectodomains D1-D5) and a shorter, D5-deleted version of CD146 (D1-D4). Binding of Gal-3 and its carbohydrate recognition domain (CRD) to CD146 D1-D4 is greatly reduced vis-à-vis CD146 eFL, supporting the proposal of a larger number of glycosylation sites on D5. Even though the canonical sugar-binding ß-sheet S-face (ß-strands 1, 10, 3, 4, 5, 6) of the Gal-3 ß-sandwich is involved in interactions with CD146 (e.g. N-linked glycosylation sites), equivalent HSQC spectral perturbations at residues on the opposing Gal-3 F-face ß-sheet (ß-strands 11, 2, 7, 8, 9) indicate involvement of the Gal-3 F-face in binding CD146. This is supported by the observation that addition of lactose, while significantly attenuating Gal-3 binding (primarily with the S-face) to CD146 eFL, does not abolish it. Bio-Layer Interferometry studies with Gal-3 F-face mutants yield KD values to demonstrate a significant decrease (L203A) or increase (V204A, L218A, T243A) in net binding to CD146 eFL compared to wild type Gal-3. However, HSQC lactose titrations show no highly significant effects on sugar binding to the Gal-3 CRD S-face. Overall, our findings indicate that Gal-3 binding to CD146 is more involved than simple interactions with ß-galactoside epitopes on the cell receptor, and that there is a direct role for the lectin's CRD F-face in the CD146 binding process.

Adhesion/growth-regulatory galectins tested in combination: evidence for formation of hybrids as heterodimers.

The delineation of the physiological significance of protein (lectin)-glycan recognition and the structural analysis of individual lectins have directed our attention to studying them in combination. In this report, we tested the hypothesis of hybrid formation by using binary mixtures of homodimeric galectin-1 and -7 as well as a proteolytically truncated version of chimera-type galectin-3. Initial supportive evidence is provided by affinity chromatography using resin-presented galectin-7. Intriguingly, the extent of cell binding by cross-linking of surface counter-receptor increased significantly for monomeric galectin-3 form by the presence of galectin-1 or -7. Pulsed-field gradient NMR (nuclear magnetic resonance) diffusion measurements on these galectin mixtures indicated formation of heterodimers as opposed to larger oligomers. 15N-1H heteronuclear single quantum coherence NMR spectroscopy and molecular dynamics simulations allowed us to delineate how different galectins interact in the heterodimer. The possibility of domain exchange between galectins introduces a new concept for understanding the spectrum of their functionality, particularly when these effector molecules are spatially and temporally co-expressed as found in vivo.

Macromolecular assemblies of complex polysaccharides with galectin-3 and their synergistic effects on function.

Although pectin-derived polysaccharides can antagonize galectin function in various pathological disorders, the nature of their binding interactions needs to be better defined for developing them as drugs. Moreover, given their relatively large size and complexity, pectin-derived polysaccharides are also useful as model systems to assess inter-polysaccharide and protein-polysaccharide interactions. Here, we investigated interactions between galectin-3 (Gal-3) and pectin-derived polysaccharides: a rhamnogalacturonan (RG) and two homogalacturonans (HGs). BioLayer Interferometry and fluorescence-linked immunosorbent assays indicate that these polysaccharides bind Gal-3 with macroscopic or apparent KD values of 49 nM, 46 µM, and 138 µM, respectively. 15N-1H heteronuclear single quantum coherence (HSQC) NMR studies reveal that these polysaccharides interact primarily with the F-face of the Gal-3 carbohydrate recognition domain. Even though their binding to Gal-3 does not inhibit Gal-3-mediated T-cell apoptosis and only weakly attenuates hemagglutination, their combination in specific proportions increases activity synergistically along with avidity for Gal-3. This suggests that RG and HG polysaccharides act in concert, a proposal supported by polysaccharide particle size measurements and 13C-1H HSQC data. Our model has HG interacting with RG to promote increased avidity of RG for Gal-3, likely by exposing additional lectin-binding sites on the RG. Overall, the present study contributes to our understanding of how complex HG and RG polysaccharides interact with Gal-3.

Galectins as Molecular Targets for Therapeutic Intervention.

Galectins are a family of small, highly conserved, molecular effectors that mediate various biological processes, including chemotaxis and angiogenesis, and that function by interacting with various cell surface glycoconjugates, usually targeting ß-galactoside epitopes. Because of their significant involvement in various biological functions and pathologies, galectins have become a focus of therapeutic discovery for clinical intervention against cancer, among other pathological disorders. In this review, we focus on understanding galectin structure-function relationships, their mechanisms of action on the molecular level, and targeting them for therapeutic intervention against cancer.

Novel polysaccharide binding to the N-terminal tail of galectin-3 is likely modulated by proline isomerization.

Interactions between galectins and polysaccharides are crucial to many biological processes, and yet these are some of the least understood, usually being limited to studies with small saccharides and short oligosaccharides. The present study is focused on human galectin-3 (Gal-3) interactions with a 60 kDa rhamnogalacturonan RG-I-4 that we use as a model to garner information as to how galectins interact with large polysaccharides, as well as to develop this agent as a therapeutic against human disease. Gal-3 is unique among galectins, because as the only chimera-type, it has a long N-terminal tail (NT) that has long puzzled investigators due to its dynamic, disordered nature and presence of numerous prolines. Here, we use 15N-1H heteronuclear single quantum coherence NMR spectroscopy to demonstrate that multiple sites on RG-I-4 provide epitopes for binding to three sites on 15N-labeled Gal-3, two within its carbohydrate recognition domain (CRD) and one at a novel site within the NT encompassing the first 40 residues that are highly conserved among all species of Gal-3. Moreover, strong binding of RG-I-4 to the Gal-3 NT occurs on a very slow time scale, suggesting that it may be mediated by cis-trans proline isomerization, a well-recognized modulator of many biological activities. The NT binding epitope within RG-I-4 appears to reside primarily in the side chains of the polysaccharide, some of which are galactans. Our results provide new insight into the role of the NT in Gal-3 function.

Chemokines from a Structural Perspective.

Chemokines are a family of small, highly conserved cytokines that mediate various biological processes, including chemotaxis, hematopoiesis, and angiogenesis, and that function by interacting with cell surface G-Protein Coupled Receptors (GPCRs). Because of their significant involvement in various biological functions and pathologies, chemokines and their receptors have been the focus of therapeutic discovery for clinical intervention. There are several sub-families of chemokines (e.g., CXC, CC, C, and CX3C) defined by the positions of sequentially conserved cysteine residues. Even though all chemokines also have a highly conserved, three-stranded ß-sheet/α-helix tertiary structural fold, their quarternary structures vary significantly with their sub-family. Moreover, their conserved tertiary structures allow for subunit swapping within and between sub-family members, thus promoting the concept of a "chemokine interactome". This review is focused on structural aspects of CXC and CC chemokines, their functional synergy and ability to form heterodimers within the chemokine interactome, and some recent developments in structure-based chemokine-targeted drug discovery.

Binding of polysaccharides to human galectin-3 at a noncanonical site in its carbohydrate recognition domain.

Galectin-3 (Gal-3) is a multifunctional lectin, unique to galectins by the presence of a long N-terminal tail (NT) off of its carbohydrate recognition domain (CRD). Many previous studies have investigated binding of small carbohydrates to its CRD. Here, we used nuclear magnetic resonance spectroscopy ((15)N-(1)H heteronuclear single quantum coherence data) to assess binding of (15)N-Gal-3 (and truncated (15)N-Gal-3 CRD) to several, relatively large polysaccharides, including eight varieties of galactomannans (GMs), as well as a ß(1 → 4)-polymannan and an α-branched mannan. Overall, we found that these polysaccharides with a larger carbohydrate footprint interact primarily with a noncanonical carbohydrate-binding site on the F-face of the Gal-3 CRD ß-sandwich, and to a less extent, if at all, with the canonical carbohydrate-binding site on the S-face. While there is no evidence for interaction with the NT itself, it does appear that the NT somehow mediates stronger interactions between the Gal-3 CRD and the GMs. Significant Gal-3 resonance broadening observed during polysaccharide titrations indicates that interactions occur in the intermediate exchange regime, and analysis of these data allows estimation of affinities and stoichiometries that range from 4 × 10(4) to 12 × 10(4) M(-1) per site and multiple sites per polysaccharide, respectively. We also found that lactose can still bind to the CRD S-face of GM-bound Gal-3, with the binding of one ligand attenuating affinity of the other. These data are compared with previous results on Gal-1, revealing differences and similarities. They also provide research direction to the development of these polysaccharides as galectin-targeting therapeutics in the clinic.

Intra- and intermolecular interactions of human galectin-3: assessment by full-assignment-based NMR.

Galectin-3 is an adhesion/growth-regulatory protein with a modular design comprising an N-terminal tail (NT, residues 1-111) and the conserved carbohydrate recognition domain (CRD, residues 112-250). The chimera-type galectin interacts with both glycan and peptide motifs. Complete (13)C/(15)N-assignment of the human protein makes NMR-based analysis of its structure beyond the CRD possible. Using two synthetic NT polypeptides covering residues 1-50 and 51-107, evidence for transient secondary structure was found with helical conformation from residues 5 to 15 as well as proline-mediated, multi-turn structure from residues 18 to 32 and around PGAYP repeats. Intramolecular interactions occur between the CRD F-face (the 5-stranded ß-sheet behind the canonical carbohydrate-binding 6-stranded ß-sheet of the S-face) and NT in full-length galectin-3, with the sequence P(23)GAW(26)…P(37)GASYPGAY(45) defining the primary binding epitope within the NT. Work with designed peptides indicates that the PGAX motif is crucial for self-interactions between NT/CRD. Phosphorylation at position Ser6 (and Ser12) (a physiological modification) and the influence of ligand binding have minimal effect on this interaction. Finally, galectin-3 molecules can interact weakly with each other via the F-faces of their CRDs, an interaction that appears to be assisted by their NTs. Overall, our results add insight to defining binding sites on galectin-3 beyond the canonical contact area for ß-galactosides.

Defining the potential of aglycone modifications for affinity/selectivity enhancement against medically relevant lectins: synthesis, activity screening, and HSQC-based NMR analysis.

The emerging significance of lectins for pathophysiological processes provides incentive for the design of potent inhibitors. To this end, systematic assessment of contributions to affinity and selectivity by distinct types of synthetic tailoring of glycosides is a salient step, here taken for the aglyconic modifications of two disaccharide core structures. Firstly we report the synthesis of seven N-linked-lactosides and of eight O-linked N-acetyllactosamines, each substituted with a 1,2,3-triazole unit, prepared by copper-catalyzed azide-alkyne cycloaddition (CuAAC). The totally regioselective ß-D-(1 → 4) galactosylation of a 6-O-TBDPSi-protected N-acetylglucosamine acceptor provided efficient access to the N-acetyllactosamine precursor. The resulting compounds were then systematically tested for lectin reactivity in two binding assays of increasing biorelevance (inhibition of lectin binding to a surface-presented glycoprotein and to cell surfaces). As well as a plant toxin, we also screened the relative inhibitory potential with adhesion/growth-regulatory galectins (total of eight proteins). This type of modification yielded up to 2.5-fold enhancement for prototype proteins, with further increases for galectins-3 and -4. Moreover, the availability of (15)N-labeled proteins and full assignments enabled (1)H, (15)N HSQC-based measurements for hu- man galectins-1, -3, and -7 against p-nitrophenyl lactopyranoside, a frequently tested standard inhibitor containing an aromatic aglycone. The measurements confirmed the highest affinity against galectin-3 and detected chemical shift differences in its hydrophobic core upon ligand binding, besides common alterations around the canonical contact site for the lactoside residue. What can be accomplished in terms of affinity/selectivity by this type of core extension having been determined, the applied combined strategy should be instrumental for proceeding with defining structure-activity correlations at other bioinspired sites in glycans and beyond the tested lectin types.

Isomerization of proline-46 in the N-terminal tail of galectin-3 enhances T cell apoptosis via the ROS-ERK pathway.

Galectin-3 (Gal-3) is unique in the galectin family, due to the presence of a long N-terminal tail (NT) arising from its conserved carbohydrate recognition domain (CRD). Although functional significance of the NT has remained elusive, our previous studies demonstrated the importance of NT prolines to Gal-3 function. Here, we show that during the time Gal-3 stands in solution for three or more days, Gal-3 NT undergoes a slow, intra-molecular, time-dependent conformational/dynamical change associated with proline cis-trans isomerization. From initial dissolution of Gal-3 in buffer to three days in solution, Gal-3-mediated T cell apoptosis is enhanced from 23 % to 37 %. Western blotting and flow cytometry show that the enhancement occurs via the ROS-ERK pathway, and not by the PKC-ERK pathway. To assess which proline(s) is (are) responsible for this effect, we individually mutated all 14 NT prolines within the first 68 residues to alanines, and assessed their effect on ROS production. Our study shows that isomerization of P46 alone is responsible for the upregulation of ROS and T cell apoptosis. NMR studies show that this unique effect is mediated by a change in dynamic interactions between the NT and CRD F-face, which in turn leads to this change in Gal-3 function.

Lactose binding to human galectin-7 (p53-induced gene 1) induces long-range effects through the protein resulting in increased dimer stability and evidence for positive cooperativity.

The product of p53-induced gene 1 is a member of the galectin family, i.e., galectin-7 (Gal-7). To move beyond structural data by X-ray diffraction, we initiated the study of the lectin by nuclear magnetic resonance (NMR) and circular dichroism spectroscopies, and molecular dynamics (MD) simulations. In concert, our results indicate that lactose binding to human Gal-7 induces long-range effects (minor conformational shifts and changes in structural dynamics) throughout the protein that result in stabilization of the dimer state, with evidence for positive cooperativity. Monte Carlo fits of (15)N-Gal-7 HSQC titrations with lactose using a two-site model yield K1 = 0.9 ± 0.6 × 10(3) M(-1) and K2 = 3.4 ± 0.8 × 10(3) M(-1). Ligand binding-induced stabilization of the Gal-7 dimer was supported by several lines of evidence: MD-based calculations of interaction energies between ligand-loaded and ligand-free states, gel filtration data and hetero-FRET spectroscopy that indicate a highly reduced tendency for dimer dissociation in the presence of lactose, CD-based thermal denaturation showing that the transition temperature of the lectin is significantly increased in the presence of lactose, and saturation transfer difference (STD) NMR using a molecular probe of the monomer state whose presence is diminished in the presence of lactose. MD simulations with the half-loaded ligand-bound state also provided insight into how allosteric signaling may occur. Overall, our results reveal long-range effects on Gal-7 structure and dynamics, which factor into entropic contributions to ligand binding and allow further comparisons with other members of the galectin family.

Structure-based optimization of angiostatic agent 6DBF7, an allosteric antagonist of galectin-1.

Galectin-1 (gal-1), which binds ß-galactoside groups on various cell surface receptors, is crucial to cell adhesion and migration, and is found to be elevated in several cancers. Previously, we reported on 6DBF7, a dibenzofuran (DBF)-based peptidomimetic of the gal-1 antagonist anginex. In the present study, we used a structure-based approach to optimize 6DBF7. Initial NMR studies showed that 6DBF7 binds to gal-1 on one side of the ß-sandwich away from the lectin's carbohydrate binding site. Although an alanine scan of 6DBF7 showed that the two cationic groups (lysines) in the partial peptide are crucial to its angiostatic activity, it is the hydrophobic face of the amphipath that appears to interact directly with the surface of gal-1. Based on this structural information, we designed and tested additional DBF analogs. In particular, substitution of the C-terminal Asp for alanine and branched alkyl side chains (Val, Leu, Ile) for linear ones (Nle, Nva) rendered the greatest improvements in activity. Flow cytometry with gal-1(-/-) splenocytes showed that 6DBF7 and two of its more potent analogs (DB16 and DB21) can fully inhibit fluorescein isothiocyanate-gal-1 binding. Moreover, heteronuclear single-quantum coherence NMR titrations showed that the presence of DB16 decreases gal-1 affinity for lactose, indicating that the peptidomimetic targets gal-1 as a noncompetitive, allosteric inhibitor of glycan binding. Using tumor mouse models (B16F10 melanoma, LS174 lung, and MA148 ovarian), we found that DB21 inhibits tumor angiogenesis and tumor growth significantly better than 6DBF7, DB16, or anginex. DB21 is currently being developed further and holds promise for the management of human cancer in the clinic.

An Oral Galectin Inhibitor in COVID-19-A Phase II Randomized Controlled Trial.

BACKGROUND: SARS-CoV-2 vaccines play an important role in reducing disease severity, hospitalization, and death, although they failed to prevent the transmission of SARS-CoV-2 variants. Therefore, an effective inhibitor of galectin-3 (Gal-3) could be used to treat and prevent the transmission of COVID-19. ProLectin-M (PL-M), a Gal-3 antagonist, was shown to interact with Gal-3 and thereby prevent cellular entry of SARS-CoV-2 in previous studies. AIM: The present study aimed to further evaluate the therapeutic effect of PL-M tablets in 34 subjects with COVID-19. METHODS: The efficacy of PL-M was evaluated in a randomized, double-blind, placebo-controlled clinical study in patients with mild to moderately severe COVID-19. Primary endpoints included changes in the absolute RT-PCR Ct values of the nucleocapsid and open reading frame (ORF) genes from baseline to days 3 and 7. The incidence of adverse events, changes in blood biochemistry, inflammatory biomarkers, and levels of antibodies against COVID-19 were also evaluated as part of the safety evaluation. RESULTS: PL-M treatment significantly (p = 0.001) increased RT-PCR cycle counts for N and ORF genes on days 3 (Ct values 32.09 ± 2.39 and 30.69 ± 3.38, respectively) and 7 (Ct values 34.91 ± 0.39 and 34.85 ± 0.61, respectively) compared to a placebo treatment. On day 3, 14 subjects in the PL-M group had cycle counts for the N gene above the cut-off value of 29 (target cycle count 29), whereas on day 7, all subjects had cycle counts above the cut-off value. Ct values in placebo subjects were consistently less than 29, and no placebo subjects were RT-PCR-negative until day 7. Most of the symptoms disappeared completely after receiving PL-M treatment for 7 days in more patients compared to the placebo group. CONCLUSION: PL-M is safe and effective for clinical use in reducing viral loads and promoting rapid viral clearance in COVID-19 patients by inhibiting SARS-CoV-2 entry into cells through the inhibition of Gal-3.

Molecular basis for cellular retinoic acid-binding protein 1 in modulating CaMKII activation.

Introduction: Cellular retinoic acid (RA)-binding protein 1 (CRABP1) is a highly conserved protein comprised of an anti-parallel, beta-barrel, and a helix-turn-helix segment outside this barrel. Functionally, CRABP1 is thought to bind and sequester cytosolic RA. Recently, CRABP1 has been established as a major mediator of rapid, non-genomic activity of RA in the cytosol, referred to as "non-canonical" activity. Previously, we have reported that CRABP1 interacts with and dampens the activation of calcium-calmodulin (Ca2+-CaM)-dependent kinase 2 (CaMKII), a major effector of Ca2+ signaling. Through biophysical, molecular, and cellular assays, we, herein, elucidate the molecular and structural mechanisms underlying the action of CRABP1 in dampening CaMKII activation. Results: We identify an interaction surface on CRABP1 for CaMKII binding, located on the beta-sheet surface of the barrel, and an allosteric region within the helix segment outside the barrel, where both are important for interacting with CaMKII. Molecular studies reveal that CRABP1 preferentially associates with the inactive form of CaMKII, thereby dampening CaMKII activation. Alanine mutagenesis of residues implicated in the CaMKII interaction results in either a loss of this preference or a shift of CRABP1 from associating with the inactive CaMKII to associating with the active CaMKII, which corresponds to changes in CRABP1's effect in modulating CaMKII activation. Conclusions: This is the first study to elucidate the molecular and structural basis for CRABP1's function in modulating CaMKII activation. These results further shed insights into CRABP1's functional involvement in multiple signaling pathways, as well as its extremely high sequence conservation across species and over evolution.