Rational Design, Synthesis, and Anti-Proliferative Evaluation of Novel 4-Aryl-3,4-Dihydro-2H-1,4-Benzoxazines.

A synthetic pathway to a novel 4-aryl-3,4-dihydro-2H-1,4-benzoxazine scaffold was developed and a series of compounds based on the scaffold were synthesised as potential anticancer agents. The 4-aryl-substituted compounds were prepared via Buchwald-Hartwig cross-coupling between substituted bromobenzenes and various 1,4-benzoxazines, which in turn were generated from a cascade hydrogenation and reductive amination one-pot reaction. These analogues exhibited moderate to good potency against various cancer cell lines. Structure-activity relationship analysis indicated that the inclusion of hydroxyl groups on ring A and ring B was beneficial to biological activity, while having a para-amino group on ring C significantly enhanced potency. Molecule 14f displayed the most potent anticancer activity (IC50 = 7.84-16.2 µM against PC-3, NHDF, MDA-MB-231, MIA PaCa-2, and U-87 MG cancer cell lines), indicating its potential as a lead compound for further structural optimisation. All the synthesised compounds were fully characterised with NMR, HMRS, and IR. The novel benzoxazine scaffold described in this study holds promise and deserves further in-depth studies.

Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site.

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.

Development of Inhibitors against Mycobacterium abscessus tRNA (m1G37) Methyltransferase (TrmD) Using Fragment-Based Approaches.

Mycobacterium abscessus (Mab) is a rapidly growing species of multidrug-resistant nontuberculous mycobacteria that has emerged as a growing threat to individuals with cystic fibrosis and other pre-existing chronic lung diseases. Mab pulmonary infections are difficult, or sometimes impossible, to treat and result in accelerated lung function decline and premature death. There is therefore an urgent need to develop novel antibiotics with improved efficacy. tRNA (m1G37) methyltransferase (TrmD) is a promising target for novel antibiotics. It is essential in Mab and other mycobacteria, improving reading frame maintenance on the ribosome to prevent frameshift errors. In this work, a fragment-based approach was employed with the merging of two fragments bound to the active site, followed by structure-guided elaboration to design potent nanomolar inhibitors against Mab TrmD. Several of these compounds exhibit promising activity against mycobacterial species, including Mycobacterium tuberculosis and Mycobacterium leprae in addition to Mab, supporting the use of TrmD as a target for the development of antimycobacterial compounds.

Dihydropyrrolones as bacterial quorum sensing inhibitors.

Bacteria regulate their pathogenicity and biofilm formation through quorum sensing (QS), which is an intercellular communication system mediated by the binding of signaling molecules to QS receptors such as LasR. In this study, a range of dihydropyrrolone (DHP) analogues were synthesized via the lactone-lactam conversion of lactone intermediates. The synthesized compounds were tested for their ability to inhibit QS, biofilm formation and bacterial growth of Pseudomonas aeruginosa. The compounds were also docked into a LasR crystal structure to rationalize the observed structure-activity relationships. The most active compound identified in this study was compound 9i, which showed 63.1% QS inhibition of at 31.25 µM and 60% biofilm reduction at 250 µM with only moderate toxicity towards bacterial cell growth.

Fragment-based approaches to TB drugs.

Tuberculosis is an infectious disease associated with significant mortality and morbidity worldwide, particularly in developing countries. The rise of antibiotic resistance in Mycobacterium tuberculosis (Mtb) urgently demands the development of new drug leads to tackle resistant strains. Fragment-based methods have recently emerged at the forefront of pharmaceutical development as a means to generate more effective lead structures, via the identification of fragment molecules that form weak but high quality interactions with the target biomolecule and subsequent fragment optimization. This review highlights a number of novel inhibitors of Mtb targets that have been developed through fragment-based approaches in recent years.

Effect of DMSO on Protein Structure and Interactions Assessed by Collision-Induced Dissociation and Unfolding.

Given the frequent use of DMSO in biochemical and biophysical assays, it is desirable to understand the influence of DMSO concentration on the dissociation or unfolding behavior of proteins. In this study, the effects of DMSO on the structure and interactions of avidin and Mycobacterium tuberculosis (Mtb) CYP142A1 were assessed through collision-induced dissociation (CID) and collision-induced unfolding (CIU) as monitored by nanoelectrospray ionization-ion mobility-mass spectrometry (nESI-IM-MS). DMSO concentrations higher than 4% (v/v) destabilize the avidin tetramer toward dissociation and unfolding, via both its effects on charge state distribution (CSD) as well as at the level of individual charge states. In contrast, DMSO both protects against heme loss and increases the stability of CYP142A1 toward unfolding even up to 40% DMSO. Tandem MS/MS experiments showed that DMSO could modify the dissociation pathway of CYP142A1, while CIU revealed the protective effect of the heme group on the structure of CYP142A1.

Fragment-Based Approaches to the Development of Mycobacterium tuberculosis CYP121 Inhibitors.

The essential enzyme CYP121 is a target for drug development against antibiotic resistant strains of Mycobacterium tuberculosis. A triazol-1-yl phenol fragment 1 was identified to bind to CYP121 using a cascade of biophysical assays. Synthetic merging and optimization of 1 produced a 100-fold improvement in binding affinity, yielding lead compound 2 (KD = 15 µM). Deconstruction of 2 into its component retrofragments allowed the group efficiency of structural motifs to be assessed, the identification of more LE scaffolds for optimization and highlighted binding affinity hotspots. Structure-guided addition of a metal-binding pharmacophore onto LE retrofragment scaffolds produced low nanomolar (KD = 15 nM) CYP121 ligands. Elaboration of these compounds to target binding hotspots in the distal active site afforded compounds with excellent selectivity against human drug-metabolizing P450s. Analysis of the factors governing ligand potency and selectivity using X-ray crystallography, UV-vis spectroscopy, and native mass spectrometry provides insight for subsequent drug development.

Synthesis, biological evaluation and structure-activity relationship studies of isoflavene based Mannich bases with potent anti-cancer activity.

Phenoxodiol, an analogue of the isoflavone natural product daidzein, is a potent anti-cancer agent that has been investigated for the treatment of hormone dependent cancers. This molecular scaffold was reacted with different primary amines and secondary amines under different Mannich conditions to yield either benzoxazine or aminomethyl substituted analogues. These processes enabled the generation of a diverse range of analogues that were required for structure-activity relationship (SAR) studies. The resulting Mannich bases exhibited prominent anti-proliferative effects against SHEP neuroblastoma and MDA-MB-231 breast adenocarcinoma cell lines. Further cytotoxicity studies against MRC-5 normal lung fibroblast cells showed that the isoflavene analogues were selective towards cancer cells.

A highly selective, label-free, homogenous luminescent switch-on probe for the detection of nanomolar transcription factor NF-kappaB.

Transcription factors are involved in a number of important cellular processes. The transcription factor NF-κB has been linked with a number of cancers, autoimmune and inflammatory diseases. As a result, monitoring transcription factors potentially represents a means for the early detection and prevention of diseases. Most methods for transcription factor detection tend to be tedious and laborious and involve complicated sample preparation, and are not practical for routine detection. We describe herein the first label-free luminescence switch-on detection method for transcription factor activity using Exonuclease III and a luminescent ruthenium complex, [Ru(phen)(2)(dppz)](2+). As a proof of concept for this novel assay, we have designed a double-stranded DNA sequence bearing two NF-κB binding sites. The results show that the luminescence response was proportional to the concentration of the NF-κB subunit p50 present in the sample within a wide concentration range, with a nanomolar detection limit. In the presence of a known NF-κB inhibitor, oridonin, a reduction in the luminescence response of the ruthenium complex was observed. The reduced luminescence response of the ruthenium complex in the presence of small molecule inhibitors allows the assay to be applied to the high-throughput screening of chemical libraries to identify new antagonists of transcription factor DNA binding activity. This will allow the rapid and low cost identification and development of novel scaffolds for the treatment of diseases caused by the deregulation of transcription factor activity.