Resistance to β-lactam antibacterials, importantly via production of β-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both ...serine- (SBL) and metallo- (MBL) β-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C β‑lactamase from
, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied β-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D β‑lactamases, our data support the proposal that bicyclic boronates are broad-spectrum β‑lactamase inhibitors that work by mimicking a high energy 'tetrahedral' intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful β-lactamase inhibition.
Background
Because of the global spread of coronavirus disease 2019 (COVID‐19), oncology departments across the world have rapidly adapted their cancer care protocols to balance the risk of delaying ...cancer treatments and the risk of COVID‐19 exposure. COVID‐19 and associated changes may have an impact on the psychosocial functioning of patients with cancer and survivors. This study was designed to determine the impact of the COVID‐19 pandemic on young people living with and beyond cancer.
Methods
In this cross‐sectional study, 177 individuals, aged 18 to 39 years, were surveyed about the impact of COVID‐19 on their cancer care and psychological well‐being. Participants also reported their information needs with respect to COVID‐19. Responses were summarized with a content analysis approach.
Results
This was the first study to examine the psychological functioning of young patients and survivors during the first weeks of the COVID‐19 pandemic. A third of the respondents reported increased levels of psychological distress, and as many as 60% reported feeling more anxious than they did before COVID‐19. More than half also wanted more information tailored to them as young patients with cancer.
Conclusions
The COVID‐19 pandemic is rapidly evolving and changing the landscape of cancer care. Young people living with cancer are a unique population and might be more vulnerable during this time in comparison with their healthy peers. There is a need to screen for psychological distress and attend to young people whose cancer care has been delayed. As the lockdown begins to ease, the guidelines about cancer care should be updated according to this population's needs.
This study examines the psychological functioning of young people living with and beyond cancer during the first weeks of the coronavirus disease 2019 (COVID‐19) pandemic. A third have reported increased levels of psychological distress, and as many as 60% have reported feeling more anxious than they did before COVID‐19.
The β-lactams remain the most important antibacterials, but their use is increasingly compromised by resistance, importantly by β-lactamases. Although β-lactam and non-β-lactam inhibitors forming ...stable acyl–enzyme complexes with nucleophilic serine β-lactamases (SBLs) are widely used, these are increasingly susceptible to evolved SBLs and do not inhibit metallo-β-lactamases (MBLs). Boronic acids and boronate esters, especially cyclic ones, can potently inhibit both SBLs and MBLs. Vaborbactam, a monocyclic boronate, is approved for clinical use, but its β-lactamase coverage is limited. Bicyclic boronates rapidly react with SBLs and MBLs forming stable enzyme–inhibitor complexes that mimic the common anionic high-energy tetrahedral intermediates in SBL/MBL catalysis, as revealed by crystallography. The ability of boronic acids to ‘morph’ between sp2 and sp3 hybridisation states may help enable potent inhibition. There is limited structure–activity relationship information on the (bi)cyclic boronate inhibitors compared to β-lactams, hence scope for creativity towards new boron-based β-lactamase inhibitors/antibacterials.
The bicyclic boronate VNRX-5133 (taniborbactam) is a new type of β-lactamase inhibitor in clinical development. We report that VNRX-5133 inhibits serine-β-lactamases (SBLs) and some clinically ...important metallo-β-lactamases (MBLs), including NDM-1 and VIM-1/2. VNRX-5133 activity against IMP-1 and tested B2/B3 MBLs was lower/not observed. Crystallography reveals how VNRX-5133 binds to the class D SBL OXA-10 and MBL NDM-1. The crystallographic results highlight the ability of bicyclic boronates to inhibit SBLs and MBLs via binding of a tetrahedral (sp3) boron species. The structures imply conserved binding of the bicyclic core with SBLs/MBLs. With NDM-1, by crystallography, we observed an unanticipated VNRX-5133 binding mode involving cyclization of its acylamino oxygen onto the boron of the bicyclic core. Different side-chain binding modes for bicyclic boronates for SBLs and MBLs imply scope for side-chain optimization. The results further support the “high-energy-intermediate” analogue approach for broad-spectrum β-lactamase inhibitor development and highlight the ability of boron inhibitors to interchange between different hybridization states/binding modes.
The work described in this thesis focused on exploration of the versatile chemistry of boron to access functionalised bicyclic and acyclic boron-containing structures for use as enzyme inhibitors. ...Cyclic boronic acids/esters are emerging as promising scaffolds for broad-spectrum inhibition of β-lactamases, which are the most clinically relevant determinant of antimicrobial resistance to β-lactam antibiotics in Gram-negative organisms. The work included the development of novel stereoselective synthetic strategies, mechanistic studies, as well as method development for the bicyclic boronic acid pharmacophore. Development of novel methodology to access highly functionalised α-acylamido bicyclic boronate taniborbactam (TAN) is described in Chapter 2. Bicyclic boronates are very broad-spectrum β-lactamase inhibitors and have considerable clinical potential, having inhibitory activity against both serine- and metallo-β-lactamases (SBLs and MBLs, respectively). TAN was synthesised in eleven steps in 3% overall yield, which represents up to 750-fold yield improvement over previously reported syntheses of structurally related compounds. Subsequent biochemical and structural observations obtained through collaborations are described. An unexpected intramolecular cyclisation of the C-3 acylamido oxygen atom of TAN onto the boron of the bicyclic core was observed crystallographically in the complex of TAN with the subclass B1 MBL New Delhi metallo-β-lactamase-1 (NDM-1). The mechanistic proposal for the formation of this previously unobserved tricyclic inhibitor form and its biological significance are discussed. Building on this discovery, comparative analyses of binding modes of TAN and its analogues revealed a striking conservation in the conformations adopted by the fused bicyclic boronate core across SBLs and MBLs. Substantial variations in the acylamino side chain orientations were observed in the complex crystal structures. Studies on the synthesis and inhibitory properties of other fused 6,6-bicyclic boronate analogues with non-α-acylamido side chains are also described in Chapter 2. The synthetic results include an unexpected intramolecular Grignard reaction between an N-tert-butoxycarbonyl group and adjacent carboxylic ester giving rise to a novel borolactone structure. The SBL/MBL inhibition results suggest that there remains scope for side chain optimisation. Structure-based drug design and fragment-based drug design approaches were used to investigate the boronate pharmacophore in Chapters 3 and 4. Routes to bicyclic boronates and acyclic boronic acids were developed. The effects of ring size of the boronate core (6,6- versus 6,5-systems) and the presence of a carboxylate group were examined in the context of MBL inhibition in Chapter 3. Based on biochemical, structural and computational analyses, a pharmacophore fragment was selected for further optimisation studies, yielding potent MBL inhibitors comprising a benzoxaborole with a C-3 sulfonamide side chain (Chapter 4). Overall, the results described in this thesis enable the stereoselective synthesis of α-acylamido bicyclic boronates in improved yields and highlight the therapeutic potential of bicyclic boronates for β-lactamase inhibition. They also showcase the chameleon-like behaviour of organoboron compounds, including in their interactions with proteins.
The effectiveness of β-lactam antibiotics is increasingly compromised by β-lactamases. Boron-containing inhibitors are potent serine-β-lactamase inhibitors, but the interactions of boron-based ...compounds with the penicillin-binding protein (PBP) β-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the Pseudomonas aeruginosa PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by β-lactamase-driven resistance.
Human prolyl‐hydroxylases (PHDs) are hypoxia‐sensing 2‐oxoglutarate (2OG) oxygenases, catalysis by which suppresses the transcription of hypoxia‐inducible factor target genes. PHD inhibition enables ...the treatment of anaemia/ischaemia‐related disease. The PHD inhibitor Molidustat is approved for the treatment of renal anaemia; it differs from other approved/late‐stage PHD inhibitors in lacking a glycinamide side chain. The first reported crystal structures of Molidustat and IOX4 (a brain‐penetrating derivative) complexed with PHD2 reveal how their contiguous triazole, pyrazolone and pyrimidine/pyridine rings bind at the active site. The inhibitors bind to the active‐site metal in a bidentate manner through their pyrazolone and pyrimidine nitrogens, with the triazole π‐π‐stacking with Tyr303 in the 2OG binding pocket. Comparison of the new structures with other PHD inhibitor complexes reveals differences in the conformations of Tyr303, Tyr310, and a mobile loop linking β2–β3, which are involved in dynamic substrate binding/product release.
Ring binder: Co‐crystal structures of human PHD2 with Molidustat and a related inhibitor provide insight into their mode of inhibition. The pyrazolone‐pyrimidine rings of Molidustat chelate the active‐site metal ion, and its triazole ring makes a π‐π‐stacking interaction with Tyr303. The results reveal altered conformations of PHD2 residues, including Tyr303 and Tyr310 on binding of Fe‐chelating PHD inhibitors.
Klebsiella pneumoniae
carbapenemase-2 (KPC-2) is a serine-β-lactamase (SBL) capable of hydrolysing almost all β-lactam antibiotics. We compare KPC-2 inhibition by vaborbactam, a clinically-approved ...monocyclic boronate, and VNRX-5133 (taniborbactam), a bicyclic boronate in late-stage clinical development. Vaborbactam inhibition is slowly reversible, whereas taniborbactam has an off-rate indicating essentially irreversible complex formation and a 15-fold higher on-rate, although both potentiate β-lactam activity against KPC-2-expressing
K. pneumoniae
. High resolution X-ray crystal structures reveal closely related binding modes for both inhibitors to KPC-2, with differences apparent only in positioning of the endocyclic boronate ester oxygen. The results indicate the bicyclic boronate scaffold as both an efficient, long-lasting, KPC-2 inhibitor and capable of supporting further iterations that may improve potency against specific enzyme targets and pre-empt the emergence of inhibitor resistant KPC-2 variants.
Mono- (vaborbactam) and bi-cyclic (taniborbactam) boronate inhibitors bind KPC-2 similarly, but the latter is essentially irreversible.
β-Lactamases comprise the most widely used mode of resistance to β-lactam antibiotics. Cyclic boronates have shown promise as a new class of β-lactamase inhibitor, with pioneering potential to ...potently inhibit both metallo- and serine-β-lactamases. We report studies concerning a bicyclic boronate ester with a thioether rather than the more typical β-lactam antibiotic "C-6/C-7" acylamino type side chain, which is present in the penicillin/cephalosporin antibiotics. The thioether bicyclic boronate ester was tested for activity against representative serine- and metallo-β-lactamases. The results support the broad inhibition potential of bicyclic boronate based inhibitors with different side chains, including against metallo-β-lactamases from B1, B2, and B3 subclasses. Combined with previous crystallographic studies, analysis of a crystal structure of the thioether inhibitor with the clinically relevant VIM-2 metallo-β-lactamase implies that further SAR work will expand the already broad scope of β-lactamase inhibition by bicyclic boronates.
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