Benzylic Dehydroxylation of Echinocandin antifungal drugs restores efficacy against resistance conferred by mutated Glucan Synthase

Each year, infections caused by fungal pathogens claim the lives of about 1.6 million people and affect the health of over a billion people worldwide. Among the most recently developed antifungal drugs are the echinocandins, which noncompetitively inhibit β-glucan synthase, a membrane-bound protein complex that catalyzes the formation of the main polysaccharide component of the fungal cell wall. Resistance to echinocandins is conferred by mutations in FKS genes, which encode the catalytic subunit of the β-glucan synthase complex. Here, we report that selective removal of the benzylic alcohol of the nonproteinogenic amino acid 3S,4S-dihydroxy-l-homotyrosine of the echinocandins anidulafungin and rezafungin, restored their efficacy against a large panel of echinocandin-resistant Candida strains. The dehydroxylated compounds did not significantly affect the viability of human-derived cell culture lines. An analysis of the efficacy of the dehydroxylated echinocandins against resistant Candida strains, which contain mutations in the FKS1 and/or FKS2 genes of the parental strains, identified amino acids of the Fks proteins that are likely to reside in proximity to the l-homotyrosine residue of the bound drug. This study describes the first example of a chemical modification strategy to restore the efficacy of echinocandin drugs, which have a critical place in the arsenal of antifungal drugs, against resistant fungal pathogens.The authors thank J. Berman and D. Perlin for providing Candida strains. This work was supported by the Israel Science Foundation Grant 179/19 (Micha Fridman). TG acknowledges support from the Spanish Ministry of Science and Innovation for grant PGC2018-099921-B-I00 and from the “la Caixa” Foundation under the agreements LCF/PR/GN18/50310010 and LCF/PR/HR21/00737. They also thank E. Ainbinder. O. Singer, and Y. Fried from the Stem Cell Unit of Life Science Core Facilities, the Weizmann Institute of Science. They especially thank H. Barr, head of HTS and Medicinal Chemistry Units at the Maurice and Vivienne Wohl Institute for Drug Discovery, The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science.Peer ReviewedPostprint (published version

Recombinant renewable polyclonal antibodies

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products

Inorganic chemistry:A reducing role for boron

Carbon monoxide molecules are typically coupled together using metal catalysts. The discovery that boron, a non-metal, mediates such a reaction is startling, and raises the prospect of potentially useful carbon–carbon bond-forming processes

Exploiting an Allosteric Binding Site of PRMT3 Yields Potent and Selective Inhibitors

Protein arginine methyltransferases (PRMTs) play an important role in diverse biological processes. Among the nine known human PRMTs, PRMT3 has been implicated in ribosomal biosynthesis via asymmetric dimethylation of the 40S ribosomal protein S2 and in cancer via interaction with the DAL-1 tumor suppressor protein. However, few selective inhibitors of PRMTs have been discovered. We recently disclosed the first selective PRMT3 inhibitor, which occupies a novel allosteric binding site and is noncompetitive with both the peptide substrate and cofactor. Here we report comprehensive structure-activity relationship studies of this series, which resulted in the discovery of multiple PRMT3 inhibitors with submicromolar potencies. An X-ray crystal structure of compound 14u in complex with PRMT3 confirmed that this inhibitor occupied the same allosteric binding site as our initial lead compound. These studies provide the first experimental evidence that potent and selective inhibitors can be created by exploiting the allosteric binding site of PRMT3

RuBisCO-Inspired CO2 Activation and Transformation by an Iridium(I) Complex

The synthesis of a new iridium(I) complex containing an enamido phosphine anion (dbuP(-)) and its unique reactivity with CO2 is reported. The complex binds two equivalents of CO2 and initiates a highly selective reaction cascade. The reaction leads to the reversible cleavage of CO2 and the enamido ligand as well. Computational analysis points to the existence of a relatively stable Ir-CO2 complex as a reaction intermediate prior to CO2 cleavage, which was confirmed experimentally. The observed transformation resembles several aspects of enzymatic CO2 fixation by RuBisCO

Coverage of whole proteome by structural genomics observed through protein homology modeling database

We have been developing FAMSBASE, a protein homology-modeling database of whole ORFs predicted from genome sequences. The latest update of FAMSBASE (http://daisy.nagahama-i-bio.ac.jp/Famsbase/), which is based on the protein three-dimensional (3D) structures released by November 2003, contains modeled 3D structures for 368,724 open reading frames (ORFs) derived from genomes of 276 species, namely 17 archaebacterial, 130 eubacterial, 18 eukaryotic and 111 phage genomes. Those 276 genomes are predicted to have 734,193 ORFs in total and the current FAMSBASE contains protein 3D structure of approximately 50% of the ORF products. However, cases that a modeled 3D structure covers the whole part of an ORF product are rare. When portion of an ORF with 3D structure is compared in three kingdoms of life, in archaebacteria and eubacteria, approximately 60% of the ORFs have modeled 3D structures covering almost the entire amino acid sequences, however, the percentage falls to about 30% in eukaryotes. When annual differences in the number of ORFs with modeled 3D structure are calculated, the fraction of modeled 3D structures of soluble protein for archaebacteria is increased by 5%, and that for eubacteria by 7% in the last 3 years. Assuming that this rate would be maintained and that determination of 3D structures for predicted disordered regions is unattainable, whole soluble protein model structures of prokaryotes without the putative disordered regions will be in hand within 15 years. For eukaryotic proteins, they will be in hand within 25 years. The 3D structures we will have at those times are not the 3D structure of the entire proteins encoded in single ORFs, but the 3D structures of separate structural domains. Measuring or predicting spatial arrangements of structural domains in an ORF will then be a coming issue of structural genomics

Metal-free electrocatalytic hydrogen oxidation using frustrated Lewis pairs and carbon-based Lewis acids

Whilst hydrogen is a potentially clean fuel for energy storage and utilisation technologies, its conversion to electricity comes at a high energetic cost. This demands the use of rare and expensive precious metal electrocatalysts. Electrochemical-frustrated Lewis pairs offer a metal-free, CO tolerant pathway to the electrocatalysis of hydrogen oxidation. They function by combining the hydrogen-activating ability of frustrated Lewis pairs (FLPs) with electrochemical oxidation of the resultant hydride. Here we present an electrochemical–FLP approach that utilises two different Lewis acids – a carbon-based N-methylacridinium cation that possesses excellent electrochemical attributes, and a borane that exhibits fast hydrogen cleavage kinetics and functions as a “hydride shuttle”. This synergistic interaction provides a system that is electrocatalytic with respect to the carbon-based Lewis acid, decreases the required potential for hydrogen oxidation by 1 V, and can be recycled multiple times

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein

A Potent, Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 3 (PRMT3)

PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is essential for maturation of ribosomes, may have a role in lipogenesis, and is implicated in several diseases. A potent, selective, and cell- active PRMT3 inhibitor would be a valuable tool for further investigating PRMT3 biology. Here we report the discovery of the first PRMT3 chemical probe, SGC707, by structure-based optimization of the allosteric PRMT3 inhibitors we reported previously, and thorough characterization of this probe in biochemical, biophysical, and cellular assays. SGC707 is a potent PRMT3 inhibitor (IC50 = 31 ± 2 nm, KD = 53 ± 2 nm) with outstanding selectivity (selective against 31 other methyltransferases and more than 250 non-epigenetic targets). The mechanism of action studies and crystal structure of the PRMT3-SGC707 complex confirm the allosteric inhibition mode. Importantly, SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies. This well- characterized chemical probe is an excellent tool to further study the role of PRMT3 in health and disease