Synthesis of branched and linear 1,4-linked galactan oligosaccharides

Strategic naphthylidine protection allows for the rapid assembly of linear and branched 1,4-galactans.</p

Structural and Functional Studies of the Wilms' Tumour 1 Protein (WT1) in Interaction with Nucleic acids

The WT1 protein, a product of the Wilms’ tumour 1 gene (WT1) is a zinc finger transcription factor implicated in a number of cellular processes particularly involved in the development of the urogenital system. Mutations in this gene have been implicated in abnormal development of the urogenital system resulting in syndromes such as the Denys-Drash, WAGR and Frasier and in the childhood kidney malignancy, Wilms’ tumour. Although WT1 was originally cloned as a tumour suppressor, it has most recently been described as an oncogene in some adult cancers. This dichotomous activity in cancer is cell and isoform specific though the exact mechanism responsible is yet unknown. In order to perform its regulatory activity WT1 interacts with DNA, RNA and proteins. The DNA and RNA binding activities of WT1 are restricted to the C-terminal zinc finger domain, which is made up of 4 C2H2 zinc fingers. Two of these isoforms are distinguished by the presence or absence of a 3 amino acid insert, Lysine-Threonine-Serine (KTS) in the linker between zinc-fingers 3 and 4. The –KTS and +KTS isoforms of WT1 differentially recognize DNA and RNA. Sequence examination of the zinc fingers of WT1 reveal significant differences between zinc finger 1 and the other 3 zinc fingers. Attempts at identifying DNA binding specificity for these zinc fingers have failed to successfully identify any specificity for zinc finger 1. We seek to elucidate the molecular mechanism that governs nucleic acid binding by WT1. Our biochemical and structural data enable the detailed description of the molecular interactions involved in the binding activity of the two major isoforms of WT1. These interactions reveal that the +KTS isoform binds DNA in a similar manner to the –KTS isoform. The +KTS isoform has until recently only been assigned a posttranscriptional role. These results provide the necessary detail to conclude that the +KTS isoform also possesses transcriptional capabilities similar to those of its counterpart, the -KTS isoform. We also show that the first zinc finger in this domain does not have DNA binding specificity and contributes very little to the overall affinity. The other 3 zinc fingers are specific DNA binding zinc fingers that define the transcriptional targets of WT1. Our studies with RNA reveal a synergistic effect between the KTS insert and the different zinc fingers but do not reveal any significant role for zinc finger 1. This study however suggests that the binding of WT1 to RNA depend strongly on the sequence and 3-dimensional structure of the RNA

New insights into DNA-binding behavior of Wilms tumor protein (WT1)--a dual study.

Wilms Tumor suppressor protein (WT1) is a transcription factor that is involved in a variety of developmental functions during organ development. It is also implicated in the pathology of several different cancer forms. The protein contains four C(2)H(2)-type zinc fingers and it specifically binds GC-rich sequences in the promoter regions of its target genes, which are either up or down regulated. Two properties make WT1 a more unusual transcription factor - an unconventional amino acid composition for zinc finger 1, and the insertion of a tri-peptide KTS in some of the splice isoforms of WT1. Using six WT1 constructs in which zinc fingers are systematically deleted, a dual study based on a bacterial 1-hybrid system and surface plasmon resonance measurements is performed. The experiments show that the effect of zinc finger 1 is not significant in terms of overall DNA-binding kinetics, however it influences both the specificity of target recognition and stability of interaction in presence of KTS. The KTS insertion, however, only mildly retards binding affinity, mainly by affecting the on-rate. We suggest that the insertion disturbs zinc finger 4 from its binding frame, thus weakening the rate of target recognition. Finally, for the construct in which both zinc fingers 1 and 4 were deleted, the two middle fingers 2-3 still could function as a 'minimal DNA-recognition domain' for WT1, however the formation of a stable protein-DNA complex is impaired since the overall affinity was dramatically reduced mainly since the off-rate was severely affected

Porphyrin Binding and Distortion and Substrate Specificity in the Ferrochelatase Reaction: The Role of Active Site Residues.

The specific insertion of a divalent metal ion into tetrapyrrole macrocycles is catalyzed by a group of enzymes called chelatases. Distortion of the tetrapyrrole has been proposed to be an important component of the mechanism of metallation. We present the structures of two different inhibitor complexes: (1) N-methylmesoporphyrin (N-MeMP) with the His183Ala variant of Bacillus subtilis ferrochelatase; (2) the wild-type form of the same enzyme with deuteroporphyrin IX 2,4-disulfonic acid dihydrochloride (dSDP). Analysis of the structures showed that only one N-MeMP isomer out of the eight possible was bound to the protein and it was different from the isomer that was earlier found to bind to the wild-type enzyme. A comparison of the distortion of this porphyrin with other porphyrin complexes of ferrochelatase and a catalytic antibody with ferrochelatase activity using normal-coordinate structural decomposition reveals that certain types of distortion are predominant in all these complexes. On the other hand, dSDP, which binds closer to the protein surface compared to N-MeMP, does not undergo any distortion upon binding to the protein, underscoring that the position of the porphyrin within the active site pocket is crucial for generating the distortion required for metal insertion. In addition, in contrast to the wild-type enzyme, Cu(2+)-soaking of the His183Ala variant complex did not show any traces of porphyrin metallation. Collectively, these results provide new insights into the role of the active site residues of ferrochelatase in controlling stereospecificity, distortion and metallation

Efficacy of the combination of monoclonal antibodies against the SARS-CoV-2 Beta and Delta variants.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the biggest healthcare issue worldwide. This study aimed to develop a monoclonal antibody against SARS-CoV-2 from B cells of recovered COVID-19 patients, which might have beneficial therapeutic purposes for COVID-19 patients. We successfully generated human monoclonal antibodies (hmAbs) against the receptor binding domain (RBD) protein of SARS-CoV-2 using developed hybridoma technology. The isolated hmAbs against the RBD protein (wild-type) showed high binding activity and neutralized the interaction between the RBD and the cellular receptor angiotensin-converting enzyme 2 (ACE2) protein. Epitope binning and crystallography results displayed target epitopes of these antibodies in distinct regions beneficial in the mix as a cocktail. The 3D2 binds to conserved epitopes among multi-variants. Pseudovirion-based neutralization results revealed that the antibody cocktail, 1D1 and 3D2, showed high potency in multiple variants of SARS-CoV-2 infection. In vivo studies showed the ability of the antibody cocktail treatment (intraperitoneal (i.p.) administration) to reduce viral load (Beta variant) in blood and various tissues. While the antibody cocktail treatment (intranasal (i.n.) administration) could not significantly reduce the viral load in nasal turbinate and lung tissue, it could reduce the viral load in blood, kidney, and brain tissue. These findings revealed that the efficacy of the antibody cocktail, 1D1 and 3D2, should be further studied in animal models in terms of timing of administration, optimal dose, and efficacy to mitigate inflammation in targeted tissue such as nasal turbinate and lung

Rational design of balanced dual-targeting antibiotics with limited resistance

Antibiotics that inhibit multiple bacterial targets offer a promising therapeutic strategy against resistance evolution, but developing such antibiotics is challenging. Here we demonstrate that a rational design of balanced multitargeting antibiotics is feasible by using a medicinal chemistry workflow. The resultant lead compounds, ULD1 and ULD2, belonging to a novel chemical class, almost equipotently inhibit bacterial DNA gyrase and topoisomerase IV complexes and interact with multiple evolutionary conserved amino acids in the ATP-binding pockets of their target proteins. ULD1 and ULD2 are excellently potent against a broad range of gram-positive bacteria. Notably, the efficacy of these compounds was tested against a broad panel of multidrug-resistant Staphylococcus aureus clinical strains. Antibiotics with clinical relevance against staphylococcal infections fail to inhibit a significant fraction of these isolates, whereas both ULD1 and ULD2 inhibit all of them (minimum inhibitory concentration [MIC] ≤1 μg/mL). Resistance mutations against these compounds are rare, have limited impact on compound susceptibility, and substantially reduce bacterial growth. Based on their efficacy and lack of toxicity demonstrated in murine infection models, these compounds could translate into new therapies against multidrug-resistant bacterial infections