Influence of N- methylation and conformation on almiramide anti-leishmanial activity

The almiramide N-methylated lipopeptides exhibit promising activity against trypanosomatid parasites. A structure–activity relationship study has been performed to examine the influences of N-methylation and conformation on activity against various strains of leishmaniasis protozoan and on cytotoxicity. The synthesis and biological analysis of twenty-five analogs demonstrated that derivatives with a single methyl group on either the first or fifth residue amide nitrogen exhibited greater activity than the permethylated peptides and relatively high potency against resistant strains. Replacement of amino amide residues in the peptide, by turn inducing α‑amino γ‑lactam (Agl) and N-aminoimidazalone (Nai) counterparts, reduced typically anti-parasitic activity; however, peptide amides possessing Agl residues at the second residue retained significant potency in the unmethylated and permethylated series. Systematic study of the effects of methylation and turn geometry on anti-parasitic activity indicated the relevance of an extended conformer about the central residues, and conformational mobility by tertiary amide isomerization and turn geometry at the extremities of the active peptides

Synthesis, structure and antileishmanial evaluation of endoperoxide–pyrazole hybrids

Leishmaniases are among the most impacting neglected tropical diseases. In attempts to repurpose antimalarial drugs or candidates, it was found that selected 1,2,4-trioxanes, 1,2,4,5-tetraoxanes, and pyrazole-containing chemotypes demonstrated activity against Leishmania parasites. This study reports the synthesis and structure of trioxolane–pyrazole (OZ1, OZ2) and tetraoxane–pyrazole (T1, T2) hybrids obtained from the reaction of 3(5)-aminopyrazole with endoperoxide-containing building blocks. Interestingly, only the endocyclic amine of 3(5)-aminopyrazole was found to act as nucleophile for amide coupling. However, the fate of the reaction was influenced by prototropic tautomerism of the pyrazole heterocycle, yielding 3- and 5-aminopyrazole containing hybrids which were characterized by different techniques, including X-ray crystallography. The compounds were evaluated for in vitro antileishmanial activity against promastigotes of L. tropica and L. infantum, and for cytotoxicity against THP-1 cells. Selected compounds were also evaluated against intramacrophage amastigote forms of L. infantum. Trioxolane–pyrazole hybrids OZ1 and OZ2 exhibited some activity against Leishmania promastigotes, while tetraoxane–pyrazole hybrids proved inactive, most likely due to solubility issues. Eight salt forms, specifically tosylate, mesylate, and hydrochloride salts, were then prepared to improve the solubility of the corresponding peroxide hybrids and were uniformly tested. Biological evaluations in promastigotes showed that the compound OZ1•HCl was the most active against both strains of Leishmania. Such finding was corroborated by the results obtained in assessments of the L. infantum amastigote susceptibility. It is noteworthy that the salt forms of the endoperoxide–pyrazole hybrids displayed a broader spectrum of action, showing activity in both strains of Leishmania. Our preliminary biological findings encourage further optimization of peroxide–pyrazole hybrids to identify a promising antileishmanial lead.info:eu-repo/semantics/publishedVersio

Characterization and Development of Occidiofungin

Fungal infections caused by opportunistic pathogens tend to be particularly severe and systemic in the case of immunocompromised patients. The current treatment options fall under classes such as azoles, polyenes, echinocandins and nucleoside analogs, to which resistance has been widely reported. Occidiofungin is a novel non-ribosomal peptide with a base mass of 1,200 Da that has sub-micromolar activity against a wide spectrum of fungi. Occidiofungin does not have a similar mechanism of action as the other classes of antifungals. Preliminary toxicological analyses suggested that occidiofungin was well tolerated in mice at high doses. This dissertation is aimed at characterizing the structural, functional and pharmacological aspects of occidiofungin. We describe the structural and functional characteristics of occidiofungin without the xylose group. Loss of the xylose group affected the secretion of occidiofungin by the bacterium but did not affect activity of the purified compound. We analyze a variant that is produced when a free standing thioesterase in the biosynthetic pathway is mutated. We observed that a distinct diastereomer of occidiofungin cyclized by the mutated thioesterase contributed to the activity of occidiofungin. Microscopy assays indicated that the wild type compound rapidly triggered apoptosis. Time course analysis showed immediate concentration of occidiofungin at the bud tips of S. cerevisiae; after an hour of exposure it distributed throughout the parent cells. In S. pombe, localization was seen at the poles and division septum. In vivo and in vitro affinity purification assays indicated binding of occidiofungin to actin. Pharmacokinetic evaluation of occidiofungin indicated that highest peak plasma concentration could be achieved in a murine model via the intravenous route. Lipoformulation of occidiofungin led to a marked increase in the peak plasma concentration. Histopathology performed on mice that were exposed to long duration treatment indicated that changes in all organ tissues were within normal limits. Efficacy of occidiofungin in reducing the fungal load in a murine model of systemic candidiasis could not be demonstrated due to the possibility of high levels of binding of occidiofungin to serum proteins. Future studies will be aimed at the chemical modification of occidiofungin to reduce the binding of serum proteins

The Development of the Bengamides as New Antibiotics against Drug-Resistant Bacteria

The bengamides comprise an interesting family of natural products isolated from sponges belonging to the prolific Jaspidae family. Their outstanding antitumor properties, coupled with their unique mechanism of action and unprecedented molecular structures, have prompted an intense research activity directed towards their total syntheses, analogue design, and biological evaluations for their development as new anticancer agents. Together with these biological studies in cancer research, in recent years, the bengamides have been identified as potential antibiotics by their impressive biological activities against various drug-resistant bacteria such as Mycobacterium tuberculosis and Staphylococcus aureus. This review reports on the new advances in the chemistry and biology of the bengamides during the last years, paying special attention to their development as promising new antibiotics. Thus, the evolution of the bengamides from their initial exploration as antitumor agents up to their current status as antibiotics is described in detail, highlighting the manifold value of these marine natural products as valid hits in medicinal chemistry.Supported by grants RTI2018-098296-BI00 (Ministerio de Ciencia e Innovación), PI19/01478 from Instituto de Salud Carlos III (ISCIII) (FEDER), P20_00540 (Andalusian Government and FEDER), K99GM138758 and R35GM136286 (National Institute of General Medical Sciences of the National Institutes of Health), A-CTS-666-UGR20 (University of Granada) (FEDER), CTS-107 (Andalusian Government) and 2021-GRIN-30998 (University of Castilla-La Mancha). Partial funding for open access charge: Universidad de Málag

Less toxic yet still resistance evasive amphotericins and atomistic probing of the amphotericin B ion channel

Amphotericin B (AmB) is a polyene macrolide natural product with two major functions: antifungal activity and ion channel formation. For over 50 years AmB has remained the last line of defense against systemic fungal infections. Remarkably, AmB has evaded the development of drug resistance during this extensive clinical lifetime. Unfortunately, AmB’s utility is impaired by severe toxicities. A less toxic AmB derivative thus stands to have a major impact on global human health. Similar to most small molecule medicines, AmB exerts its antifungal activity by binding and disabling a specific molecular target. In contrast, some diseases are caused by the malfunctioning of proteins, and thus lie outside this traditional paradigm. For example, treating the underlying cause of cystic fibrosis requires a small molecule capable of replicating the function of the CFTR chloride ion channel. AmB is the prototypical small molecule capable of ion channel formation. Although it has been known for over 40 years that AmB forms ion channels, the structure of these channels, as well as the functional groups responsible for ion conductance and selectivity remain unknown. Understanding these basic tenants of AmB mediated ion channel activity is the first step towards harnessing AmB to replace a missing or malfunctioning protein ion channel in a living system. There is thus a rich opportunity to harness synthesis to understand and optimize both the ion channel and antifungal functions of AmB. The polyol region of AmB has been predicted line channel interior, creating a hyrophillic environment for ions and water. Furthermore, the channel is proposed to be funnel shaped, with the narrowest region near the C3 hydroxyl. Based on this model, we hypothesized that removal of the C3 hydroxyl would impact ion channel conductance. The C3 alcohol was promptly excised, synthesizing C3deoxyAmB in only 9 steps from AmB. NMR characterization revealed that there were no significant alterations in the overall conformation of the of the AmB macrocycle upon removal of the C3 alcohol. Single ion channels of C3deoxyAmB in planar lipid bilayers revealed that C3deoxyAmB is capable of ion channel formation, however its conductance is significantly reduced relative to AmB. This is consistent with models that place the C-3 hydroxyl group at a critical point for ion conductance. During efforts to understand the molecular basis of ion selectivity, an efficient 3-step synthesis of a series of AmB urea derivatives was discovered. Although not useful for ion channel study, they were important probes to test an emerging allosteric modification model for non-toxic AmB derivatization. These derivatives selectively bound ergosterol (the primary sterol in yeast), but not cholesterol (the primary sterol in human cells), and maintained antifungal activity but were significantly less toxic to human cells. Additionally, these derivatives were more efficacious than AmB in a mouse model of disseminated candidiasis, and drastically less toxic in acute toxicity studies in mice. More selective pharmacological action is generally associated with decreased toxicity, but also with increased vulnerability to resistance. This creates an important question. Would a less toxic AmB derivative still evade drug resistance? The AmB urea derivatives were ideal candidates to evaluate this question. AmB urea resistant yeast were generated using mutagenesis and evaluated with a suite a fitness and genomic tests. Despite increased sterol selectivity and decreased toxicity, the development resistance to the AmB ureas was accompanied by significant fitness trade-offs, resulting in completely avirulent yeast. Therefore, it is possible to have a less-toxic yet resistance evasive antifungals. Based on their more selective, non-toxic, and resistance evasive profile, two of these compounds, AmBMU and AmBAU, are exceptionally exciting prospects as a clinical replacement for AmB. Degradative synthesis was vital in accessing both C3deoxyAmB and the AmB urea derivatives leveraged to probe AmB’s ion channel and antifungal functions. However, not all desired derivatives are accessible from this platform. For example, installing 13C labels into the backbone of the AmB framework would enable solid-state NMR studies capable of mapping the interaction between AmB and sterols at the atomistic level. An efficient and flexible total synthesis of AmB was designed grounded in the iterative cross coupling (ICC) strategy for small molecule synthesis. The synthesis was divided into three main phases, building block construction, ICC, and protecting group removal. Advances were made in all three phases. A scalable synthesis of building block one, and its cross coupling with building block two was developed. The planned final deprotection was additionally optimized. These advances contributed to the completion of the total synthesis of a fully protected doubly 13C-labeled AmB

Structural and functional characterisation of Uric Acid Permease, a fungal nucleobase transporter, with novel substrates

The development of novel antifungals capable of eliminating Aspergillus fungal infections and improving patient outcomes in Aspergillosis is essential. This work has focussed on Uric Acid Permease, a nucleobase-ascorbate transporter from Aspergillus nidulans, as a potential antifungal carrier. The existing crystal structure of UapA-G411VΔ1-11 in complex with xanthine (PDB 5I6C)1, has provided invaluable information about the substrate binding site, and allowed for the structure-guided design of derivatised xanthine analogues. Competitive uptake assays were used to obtain the binding affinity of each of the analogues for UapA in Aspergillus nidulans. 3-benzylxanthine (5) was identified as a novel ligand of UapA, binding with 35x higher affinity (KI = 0.2 μM) than the native substrates xanthine (KM = 7 μM) and uric acid (KI = 7 μM). This work describes the functional characterisation of 3-benzylxanthine (5) using fungal growth assays, and fluorescence microscopy. The effect of this substrate on UapA-WT and the thermostabilised mutants UapA-G411VΔ1-11 and UapA-Q408EΔ1-11 was assessed using cellular thermal shift assays (CETSA), fluorescence size-exclusion chromatography (FSEC), CPM-based thermostability assays and nano-differential scanning fluorimetry (nano-DSF). While these approaches had their challenges, they all demonstrated that 3-benzylxanthine (5) did not significantly destabilise UapA-WT or the thermostabilised mutants UapA-G411VΔ1-11 and UapA-Q408EΔ1-11. The later part of this work describes attempts to structurally characterize the binding interaction between 3-benzylxanthine (5) and UapA using X-ray crystallography, hydrogen-deuterium mass spectrometry (HDX-MS) and cryo-electron microscopy (cryo-EM). While the structure of UapA-WT or its mutants with 3-benzylxanthine (5) has not yet been resolved, preliminary results from hydrogen-deuterium mass spectrometry (HDX-MS) suggest that 3-benzylxanthine (5) may bind an outward or occluded conformation of UapA-Q408EΔ1-11. Structural characterisation of UapA with 3-benzylxanthine (5) using cryo-EM is ongoing. Once resolved, this will form the basis of future structure-guided xanthine analogue design initiatives.Open Acces

Amphotericin B as a mycolic acid specific targeting agent in tuberculosis

The serious threat of tuberculosis, especially XDR-TB, is a reality in Southern Africa particularly in individuals with HIV/AIDS. Therefore the importance of development of new or improved anti-TB treatment must now be emphasized more than ever. In this study, a model was created to target isoniazid (toxophore) specifically to a cholesterol rich environment where mycobacteria reside in macrophages, by making use of a sterol binding drug, Amphotericin B (haptophore). Isoniazid was covalently linked to Amphotericin B via a Schiff base to a linker molecule, terephthalaldehyde. Although this molecule showed a loss of biological activity, a discovery was made by serendipity that could have great impact in understanding how Mycobacterium tuberculosis enters and survives in the host macrophage. During the testing of the compound, it was discovered that Amphotericin B bound to mycolic acids at least as well as it binds to cholesterol, its natural ligand. This could provide proof of the structural similarity between mycolic acids and cholesterol but many more controls need to be investigated. As cholesterol was previously shown in literature to be critical for entry and survival of Mycobacterium tuberculosis in macrophages, the indication of a structural mimicry between the cell wall mycolic acids and cholesterol and the attraction of these two chemical entities to one another seems to be highly relevant. This characteristic can now be further explored to improve the understanding of the process of entry and survival of Mycobacterium tuberculosis in the macrophage host. Copyright 2006, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Benadie, Y 2006, Amphotericin B as a mycolic acid specific targeting agent in tuberculosis, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd Dissertation (MSc (Biochemistry))--University of Pretoria, 2008.Biochemistryunrestricte

Advances in Polymeric Materials for Biomedical Applications

Significant research efforts are currently being undertaken in the field of natural and synthetic polymers for a range of biomedical applications. (Co)polymer molecular structure, topology, self-assemblies, biodegradation, and hydrophobicity are of biomaterial importance for intrinsically biocompatible polymer systems. This book is comprised of nine chapters, published previously as original research contributions of the Special Issue focused on advances in polymeric materials for biomedical applications. The authors of these contributions are predominantly from central European countries, Italy and the United Kingdom. The content of this book will be of interest to scientists, scholars and students working in this area of knowledge, reflecting the progress in the development of advanced natural and synthetic polymer biomaterials

In Silico Binding of 2-Aminocyclobutanones to SARS-CoV-2 Nsp13 Helicase and Demonstration of Antiviral Activity

The landscape of viral strains and lineages of SARS-CoV-2 keeps changing and is currently dominated by Delta and Omicron variants. Members of the latest Omicron variants, including BA.1, are showing a high level of immune evasion, and Omicron has become a prominent variant circulating globally. In our search for versatile medicinal chemistry scaffolds, we prepared a library of substituted α-aminocyclobutanones from an α-aminocyclobutanone synthon (11). We performed an in silico screen of this actual chemical library as well as other virtual 2-aminocyclobutanone analogs against seven SARS-CoV-2 nonstructural proteins to identify potential drug leads against SARS-CoV-2, and more broadly against coronavirus antiviral targets. Several of these analogs were initially identified as in silico hits against SARS-CoV-2 nonstructural protein 13 (Nsp13) helicase through molecular docking and dynamics simulations. Antiviral activity of the original hits as well as α-aminocyclobutanone analogs that were predicted to bind more tightly to SARS-CoV-2 Nsp13 helicase are reported. We now report cyclobutanone derivatives that exhibit anti-SARS-CoV-2 activity. Furthermore, the Nsp13 helicase enzyme has been the target of relatively few target-based drug discovery efforts, in part due to a very late release of a high-resolution structure accompanied by a limited understanding of its protein biochemistry. In general, antiviral agents initially efficacious against wild-type SARS-CoV-2 strains have lower activities against variants due to heavy viral loads and greater turnover rates, but the inhibitors we are reporting have higher activities against the later variants than the wild-type (10–20X). We speculate this could be due to Nsp13 helicase being a critical bottleneck in faster replication rates of the new variants, so targeting this enzyme affects these variants to an even greater extent. This work calls attention to cyclobutanones as a useful medicinal chemistry scaffold, and the need for additional focus on the discovery of Nsp13 helicase inhibitors to combat the aggressive and immune-evading variants of concern (VOCs)

Antimicrobial Peptoids: Design, Synthesis and Biological Applications

The emergence of antimicrobial resistance is a severe threat to global health and new classes of antibiotics are desperately needed. Peptoids, or oligo-N-substituted glycines, are a group of peptidomimetics with increased structural stability and resistance to protease degradation compared to peptide analogues. In Chapter 1, peptoids are introduced and their antimicrobial properties reported to date are summarised. The synthesis and characterisation of one of the largest library of antimicrobial peptoids in existence is outlined in Chapter 2, comprising linear sequences and cyclic compounds. The development of synthetic methodology that allows the on-resin synthesis of novel peptoids containing both lysine- and arginine-type monomers is also described. In Chapter 3, the antiparasitic activity of the peptoid library is assessed against a variety of clinically relevant protozoan targets; including Leishmania mexicana, the causative agent of the neglected tropical disease cutaneous leishmaniasis. Active peptoids were identified against the insect and mammalian life stages of this parasite, including several with low micromolar potency against L. mexicana infected macrophages, an in vivo model of the disease. Additionally, peptoids that have selective activity at sub-micromolar concentrations against Plasmodium falciparum have been identified. Chapter 4 discusses the potent antibacterial and antifungal properties of the peptoid library against planktonic bacteria and also against mixed species, cross kingdom biofilms using a new quantitative polymerase chain reaction approach. Evaluation of peptoid toxicity to mammalian cells is also considered and conjugation of active sequences to the lantibiotic nisin is evaluated as a method to increase peptoid selectivity. To rationalise the activity of the peptoid library, Chapter 5 investigates the relationship between peptoid hydrophobicity, secondary structure and biological activity using circular dichroism spectroscopy and partitioning experiments. Finally, the antimicrobial mode of action is also examined using confocal fluorescence microscopy