Novel Selective Galectin-3 Antagonists Are Cytotoxic to Acute Lymphoblastic Leukemia.

Galectin-3 is a ß-galactoside-specific, carbohydrate-recognizing protein (lectin) that is strongly implicated in cancer development, metastasis, and drug resistance. Galectin-3 promotes migration and ability to withstand drug treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. Due to high amino acid conservation among galectins and the shallow nature of their glycan-binding site, the design of selective potent antagonists targeting galectin-3 is challenging. Herein, we report the design and synthesis of novel taloside-based antagonists of galectin-3 with enhanced affinity and selectivity. The molecules were optimized by in silico docking, selectivity was established against four galectins, and the binding modes were confirmed by elucidation of X-ray crystal structures. Critically, the specific inhibition of galectin-3-induced BCP-ALL cell agglutination was demonstrated. The compounds decreased the viability of ALL cells even when grown in the presence of protective stromal cells. We conclude that these compounds are promising leads for therapeutics, targeting the tumor-supportive activities of galectin-3 in cancer.

Re-amputation and survival following toe amputation: outcome data from a tertiary referral centre.

PURPOSE: Toe amputation is a commonly performed procedure for irreversible foot sepsis. However, outcome and predictors of outcome are poorly understood. Our aim was to determine survival and rate of progression to further amputation following index toe amputation. METHODS: Consecutive patients between 2010 and 2015 were included. Progression to further minor amputation, major amputation or death was recorded. Multivariable Cox regression analyses were undertaken to determine independent predictors of outcome and survival. RESULTS: One hundred forty-six patients were included, with mean age of 65 years. Fifty-five (37.7%) patients underwent hallux amputation, while 91 (62.3%) underwent amputation of non-hallux digit(s). Following index toe amputation, 63 (43.2%) patients progressed to further minor or major ipsilateral amputation, median time to which was 36 months. Twenty-one patients (14.4%) progressed to major ipsilateral amputation. Patients undergoing index non-hallux amputation were significantly more likely to require further minor amputation (P = 0.050); however, the rate of major amputation between hallux (14.5%) and non-hallux (14.3%) groups was similar. Overall, 5-year ipsilateral amputation-free (iAFS) was 39.6 ± 4.1%, ipsilateral major amputation-free (iMAFS) was 55.9 ± 4.1% and overall survival (OS) was 64.3 ± 4.0% and did not differ between index amputation sites. CONCLUSION: Almost half of patients undergoing toe amputation required further digital amputation. However, limb preservation rates are high, and a majority of patients are alive at 5-year follow-up. There was no significant difference in outcome between patients undergoing hallux and non-hallux primary procedures. Overall, increasing age remains the only independent predictor of iMAFS and OS.

Urological service provision during the COVID-19 period: the experience from an Irish tertiary centre.

PURPOSE: Urological service provision has changed dramatically with the advent of the SARS-CoV-2, necessitating restructuring and reorganization. The aim of this study was to review the reorganization of our unit, map the change in volume of departmental activities and discuss potential solutions. METHODS: Departmental activities over the months of April and May 2020 and 2019 were analysed. Details of admissions, operations, diagnostic procedures, outpatient reviews, morbidities and mortalities were recorded. Operations were performed on two sites, with elective operation transferred to an offsite, COVID-free hospital. RESULTS: Seventy-four emergency operations were performed onsite, with 85 elective operations outsourced. A total of 159 operations were performed, compared with 280 in the same period in 2019. Five (5.0%) of 101 admitted patients to the COVID hospital contracted COVID-19. No patients outsourced to the COVID-free hospital were infected there. Outpatient referrals to urology service decreased from 928 to 481. There was a 66% decrease in new cancer diagnoses. A virtual review clinic was established, with remaining outpatients reviewed through a telephone clinic platform. CONCLUSION: Compared with 2019, we performed fewer operations and outpatient procedures, had fewer admissions and diagnosed fewer patients with new cancers. However, outsourcing elective operation to designated non-COVID hospitals prevented the infection of any patient with COVID-19 in the post-operative period. The use of virtual clinic and telephone clinic has had some success in replacing traditional outpatient visits. The overall significant decrease in operative volume will likely precipitate a mismatch between demand and service provision in the coming months, unless capacity is increased.

Neoadjuvant chemotherapy for luminal a breast cancer: Factors predictive of histopathologic response and oncologic outcome.

BACKGROUND: The benefit of chemotherapy (NAC) for patients with ER/PR positive, HER2 negative breast cancer is unclear. Our aim was to determine factors associated with histopathologic response and oncologic outcome following NAC in this group. METHODS: Consecutive female patients undergoing neoadjuvant therapy and surgery for locally advanced Luminal A breast cancer between 2010 and 2015 were studied. Multivariable linear, logistic, and Cox regression analysis was undertaken. RESULTS: 114 patients were studied. Pathological complete response (pCR) was achieved in 7.9% of patients, ypN0 in 25.5%, and downstaging in 33.6%. However, 43.9% exhibited a Sataloff C-D response. Tumor grade independently predicted pCR (P = 0.039), while PR score predicted ypN0 (P = 0.017) and downstaging (P=0.029). 5-year invasive disease-free (iDFS) and overall survival (OS) were 68.5 ± 4.7% and 77.7 ± 4.3%, respectively. CONCLUSION: After NAC for Luminal A breast cancer, pCR rates are low. Patients with high grade tumors with weak PR expression exhibit the most promising response rates.

Construction of a Shape-Diverse Fragment Set: Design, Synthesis and Screen against Aurora-A Kinase.

Historically, chemists have explored chemical space in a highly uneven and unsystematic manner. As an example, the shape diversity of existing fragment sets does not generally reflect that of all theoretically possible fragments. To assess experimentally the added value of increased three dimensionality, a shape-diverse fragment set was designed and collated. The set was assembled by both using commercially available fragments and harnessing unified synthetic approaches to sp3 -rich molecular scaffolds. The resulting set of 80 fragments was highly three-dimensional, and its shape diversity was significantly enriched by twenty synthesised fragments. The fragment set was screened by high-throughput protein crystallography against Aurora-A kinase, revealing four hits that targeted the binding site of allosteric regulators. In the longer term, it is envisaged that the fragment set could be screened against a range of functionally diverse proteins, allowing the added value of more shape-diverse screening collections to be more fully assessed.

Characterization of Three Druggable Hot-Spots in the Aurora-A/TPX2 Interaction Using Biochemical, Biophysical, and Fragment-Based Approaches.

The mitotic kinase Aurora-A and its partner protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2) are overexpressed in cancers, and it has been proposed that they work together as an oncogenic holoenzyme. TPX2 is responsible for activating Aurora-A during mitosis, ensuring proper cell division. Disruption of the interface with TPX2 is therefore a potential target for novel anticancer drugs that exploit the increased sensitivity of cancer cells to mitotic stress. Here, we investigate the interface using coprecipitation assays and isothermal titration calorimetry to quantify the energetic contribution of individual residues of TPX2. Residues Tyr8, Tyr10, Phe16, and Trp34 of TPX2 are shown to be crucial for robust complex formation, suggesting that the interaction could be abrogated through blocking any of the three pockets on Aurora-A that complement these residues. Phosphorylation of Aurora-A on Thr288 is also necessary for high-affinity binding, and here we identify arginine residues that communicate the phosphorylation of Thr288 to the TPX2 binding site. With these findings in mind, we conducted a high-throughput X-ray crystallography-based screen of 1255 fragments against Aurora-A and identified 59 hits. Over three-quarters of these hits bound to the pockets described above, both validating our identification of hotspots and demonstrating the druggability of this protein-protein interaction. Our study exemplifies the potential of high-throughput crystallography facilities such as XChem to aid drug discovery. These results will accelerate the development of chemical inhibitors of the Aurora-A/TPX2 interaction.

Gentle, fast and effective crystal soaking by acoustic dispensing.

The steady expansion in the capacity of modern beamlines for high-throughput data collection, enabled by increasing X-ray brightness, capacity of robotics and detector speeds, has pushed the bottleneck upstream towards sample preparation. Even in ligand-binding studies using crystal soaking, the experiment best able to exploit beamline capacity, a primary limitation is the need for gentle and nontrivial soaking regimens such as stepwise concentration increases, even for robust and well characterized crystals. Here, the use of acoustic droplet ejection for the soaking of protein crystals with small molecules is described, and it is shown that it is both gentle on crystals and allows very high throughput, with 1000 unique soaks easily performed in under 10 min. In addition to having very low compound consumption (tens of nanolitres per sample), the positional precision of acoustic droplet ejection enables the targeted placement of the compound/solvent away from crystals and towards drop edges, allowing gradual diffusion of solvent across the drop. This ensures both an improvement in the reproducibility of X-ray diffraction and increased solvent tolerance of the crystals, thus enabling higher effective compound-soaking concentrations. The technique is detailed here with examples from the protein target JMJD2D, a histone lysine demethylase with roles in cancer and the focus of active structure-based drug-design efforts.

Galectin-3 interactions with glycosphingolipids.

Galectins have essential roles in pathological states including cancer, inflammation, angiogenesis and microbial infections. Endogenous receptors include members of the lacto- and neolacto-series glycosphingolipids present on mammalian cells and contain the tetrasaccharides lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) that form their core structural components and also ganglio-series glycosphingolipids. We present crystallographic structures of the carbohydrate recognition domain of human galectin-3, both wild type and a mutant (K176L) that influenced ligand affinity, in complex with LNT, LNnT and acetamido ganglioside a-GM3 (α2,3-sialyllactose). Key structural features revealed include galectin-3's demonstration of a binding mode towards gangliosides distinct from that to the lacto/neolacto-glycosphingolipids, with its capacity for recognising the core ß-galactoside region being challenged when the core oligosaccharide epitope of ganglio-series glycosphingolipids (GM3) is embedded within particular higher-molecular-weight glycans. The lacto- and neolacto- glycosphingolipids revealed different orientations of their terminal galactose in the galectin-3-bound LNT and LNnT structures that has significant ramifications for the capacity of galectin-3 to interact with higher-order lacto/neolacto-series glycosphingolipids such as ABH blood group antigens and the HNK-1 antigen that is common on leukocytes. LNnT also presents an important model for poly-N-acetyllactosamine-containing glycans and provides insight into galectin-3's accommodation of extended oligosaccharides such as the poly-N-acetyllactosamine-modified N- and O-glycans that, via galectin-3 interaction, facilitate progression of lung and bladder cancers, respectively. These findings provide the first atomic detail of galectin-3's interactions with the core structures of mammalian glycosphingolipids, providing information important in understanding the capacity of galectin-3 to engage with receptors identified as facilitators of major disease.

Investigation into the feasibility of thioditaloside as a novel scaffold for galectin-3-specific inhibitors.

Galectin-3 is extensively involved in metabolic and disease processes, such as cancer metastasis, thus giving impetus for the design of specific inhibitors targeting this ß-galactose-binding protein. Thiodigalactoside (TDG) presents a scaffold for construction of galectin inhibitors, and its inhibition of galectin-1 has already demonstrated beneficial effects as an adjuvant with vaccine immunotherapy, thereby improving the survival outcome of tumour-challenged mice. A novel approach--replacing galactose with its C2 epimer, talose--offers an alternative framework, as extensions at C2 permit exploitation of a galectin-3-specific binding groove, thereby facilitating the design of selective inhibitors. We report the synthesis of thioditaloside (TDT) and crystal structures of the galectin-3 carbohydrate recognition domain in complexes with TDT and TDG. The different abilities of galactose and talose to anchor to the protein correlate with molecular dynamics studies, likely explaining the relative disaccharide binding affinities. The feasibility of a TDT scaffold to enable access to a particular galectin-3 binding groove and the need for modifications to optimise such a scaffold for use in the design of potent and selective inhibitors are assessed.

Taloside inhibitors of galectin-1 and galectin-3.

Galectin-1 and galectin-3 have roles in cancer and inflammation. Galectin-1 has recently emerged as a significant protein produced by tumour cells to promote tumour development, angiogenesis and metastasis and consequently represents an important target to inhibit. The design of inhibitors targeting the carbohydrate recognition domain that is known to recognize galactose is an important approach in the fight against cancer. Based on the analysis of crystal structures, we pursued the concept that if the galactose was replaced with talose (the C2 epimer of galactose) as a scaffold, then O2 substituents would be directed closer to the protein surface and provide opportunity to design inhibitors that are more specific towards particular galectins. Our elucidation of X-ray crystal structures of two of our synthesized talosides in complex with galectin-1 and galectin-3 provides the first atomic information on the interactions of galectins, and indeed any protein, with talosides. These results have enabled a structure-based rationale for the specificity differences shown by galectin-1 and galectin-3 towards these talosides and demonstrate new opportunities for further exploitation as specific inhibitors of galectins.

Galectin inhibitory disaccharides promote tumour immunity in a breast cancer model.

High level galectin-1 expression results in cancer cell evasion of the immune response, increased tumour survival and aggressive metastases. Using a galectin-1 polyclonal antibody, high levels of galectin-1 protein were shown to be expressed by breast cancer cells established from FVB/N MMTV-c-neu mice as well as by the B16F10 melanoma cell line. In mixed lymphocyte cultures using tumour cells as antigenic stimulators, addition of recombinant galectin-1 dose-dependently inhibited lymphocyte production. Disaccharides were identified that inhibited galectin-1 function and increased growth and activation of CD8(+) CTL's killing cancer cells. X-ray crystallographic structures of human galectin-1 in complex with inhibitory disaccharides revealed their mode of binding. Combining galectin-blocking carbohydrates as adjuvants with vaccine immunotherapy in vivo to promote immune responses significantly decreased tumour progression and improved the outcomes for tumour challenged mice. This is the first report showing that suitably selected galectin-1 blocking disaccharides will act as adjuvants promoting vaccine stimulated immune responses against tumours in vivo.

Slow diffusion of lactose out of galectin-3 crystals monitored by X-ray crystallography: possible implications for ligand-exchange protocols.

Galectin-3 is a multifunctional carbohydrate-binding protein that has roles in cancer progression. In addition to carbohydrate-dependent extracellular functions, galectin-3 participates in carbohydrate-independent intracellular signalling pathways, including apoptosis, via protein-protein interactions, some of which engage the carbohydrate-binding groove. When ligands bind within this site, conformational rearrangements are induced and information on unliganded galectin-3 is therefore valuable for structure-based drug design. Removal of cocrystallized lactose from the human galectin-3 carbohydrate-recognition domain was achieved via crystal soaking, but took weeks despite low affinity. Pre-soaking to remove lactose enabled the subsequent binding of cryoprotectant glycerol, whereas when the lactose was not removed a priori the glycerol could not displace it in the short cryosoaking time frame. This slow diffusion of lactose out of the crystals contrasts with the entrance of glycerol, which takes place within minutes. The importance of the removal of incumbent ligands prior to attempts to introduce alternative ligands is indicated, even for proteins exhibiting low affinity for ligands, and has significance for ligand exchange in structure-based drug design.