Biology subjects

Malach, E.

Publications and source records attributed to Malach, E..

8 recordsLinked to original sources

bioRxiv2026

Pharmaceutical TAS2R14 Agonists Display Diverse Potency, Efficacy, and Binding-Site Sensitivity

Bitter taste receptors (TAS2Rs) are G-protein coupled receptors that detect chemically diverse compounds, including many clinically used drugs. TAS2R14 is expressed in many extraoral tissues and is activated by hundreds of ligands, including pharmaceutical drugs. Recent cryo-EM structures revealed a previously unrecognized intracellular binding pocket in TAS2R14, raising new questions regarding ligand binding modes. Here, we investigated the activation of TAS2R14 by Tamoxifen, Carbimazole, and Lidocaine using cell-based assays measuring proximal G-protein recruitment (BRET2) and downstream signaling (IP-One). Tamoxifen and Carbimazole activated TAS2R14 with EC50 values in the low micromolar range, whereas Lidocaine required substantially higher concentrations. Targeted receptor mutations were used to evaluate the contribution of extracellular and intracellular binding regions to agonist activity. Carbimazole and Lidocaine showed greater dependence on the intracellular and extracellular positions, respectively, while Tamoxifen displayed assay-dependent, but overall modest sensitivity to the tested mutations. Thus, although existing drugs can activate TAS2R14 through distinct binding modes, TAS2R14-directed repurposing will depend on whether effective local receptor concentrations can be achieved through appropriate delivery strategies.

pharmacology and toxicology↗

bioRxiv2025

Intracellular and Dual-Site Inhibition of a bitter taste GPCR

Bitter taste receptors (TAS2Rs) are G protein-coupled receptors expressed in both gustatory and extraoral tissues and activated by a broad range of compounds. TAS2R14 is among the most promiscuous members of this family, responding to many structurally diverse ligands. Cryo-electron microscopy structures of TAS2R14 have revealed agonists binding in an intracellular pocket, raising the question of the main sites of interaction for known TAS2R14 antagonists. To address this, we examined the effects of mutations at residues located in the extracellular and intracellular regions on receptor inhibition by three antagonist compounds. Mutations in the extracellular region reduced the inhibitory effect of LF22, whereas all three compounds showed reduced inhibition in the intracellular mutants. Computational co-folding of these ligands with TAS2R14 supported these observations, indicating that LF22 interacts with both top and bottom binding sites, whereas LF1 and probenecid engage predominantly the intracellular site adjacent to the G protein interface. Interestingly, LF1 is much more potent for TAS2R16 than its known inhibitor probenecid. These findings reveal distinct inhibitory mechanisms among TAS2R antagonists and provide new insights towards designing inhibitors of bitter taste.

biochemistry↗

bioRxiv2023

Reactivating the complement system using peptidic bacterial labeling tag

The immune system plays a critical role in protecting the host against pathogens, including bacteria, viruses, and parasites. However, pathogens have evolved mechanisms to evade the immune system, for example by altering their surface proteins or by producing enzymes that can interfere with the immune response. These evasion strategies enable pathogens to escape detection and destruction by the immune system, which allows them to establish serious infections. Thus, there is a critical need for new strategies for developing antimicrobial agents. Here, we describe a novel strategy for targeting pathogens, by labeling them with a general peptide functioning as a bacterial binder, conjugated to a protein tag recognizable by the complement system, thereby activating the immune system against the target pathogen. To that end, we screened several pathogenic bacteria to find complement-resistant bacterial strain. A selected peptide binder was crosslinked with the C3b complement protein using glutaraldehyde. We show by an ELISA assay that the resulting complex binds the C5 complement protein with high affinity. We posited that by binding C5, this complex will be capable of initiating the alternative complement downstream proteolytic cascade, thereby inducing the formation of the membrane attack complex. Using this methodology, we were able to eradicate 90% of complement-resistant E. coli bacterial cells. By showing enhancement of complement sensitivity in complement-resistant pathogens, this work demonstrates the basis for new therapeutic approach capable of targeting pathogenic bacteria and activating the immune system against them.

microbiology↗

bioRxiv2023

T1R3 subunit of the sweet taste receptor is activated by D2O in transmembrane domain-dependent manner

Deuterium oxide (D2O) is a water molecule in which both hydrogens are replaced by the heavier and rare isotope deuterium. We have previously shown that D2O has distinct sweet taste, which is mediated by the T1R2/T1R3 sweet taste receptor. Here we explore the effect of heavy water on T1R2 and T1R3 subunits. We show that D2O activates T1R3 transfected HEK293T cells similarly to T1R2/T1R3 transfected cells. The response to glucose dissolved in D2O is higher than to glucose dissolved in water. Mutations of Phenylalanine at position 7305.40 in the transmembrane domain of T1R3 to Alanine, Leucine or Tyrosine, impair or diminish activation by D2O, suggesting a critical role for T1R3 TMD domain in relaying the heavy water signal.

molecular biology↗

bioRxiv2022

Sensitivity of human sweet taste receptor subunits T1R2 and T1R3 to activation by glucose enantiomers

We have previously shown that L-glucose, the non-caloric enantiomer of D-glucose, activates the human sweet taste receptor T1R2/T1R3 transiently expressed in HEK293T cells. Here we show that D- and L-glucose can also activate T1R2 and T1R3 expressed without the counterpart monomer. Serine mutation to alanine in residue 147 in the binding site of T1R3 VFT domain, completely abolishes T1R3S147A activation by either L or D-glucose, while T1R2/T1R3S147A responds in the same way as T1R2 expressed without its counterpart. We further show that the original T1R2 reference sequence (NM_152232.1) is less sensitive by almost an order of magnitude than reference sequence at the time this study was performed (NM_152232.4). We find that out of the four differing positions, it is the R317G in the VFT domain of T1R2, that is responsible for this effect in-vitro. It is important both for practical assay sensitivity, and because glycine is found in this position in [~]20% of the world population. While the effects of the mutations and of the partial transfections were similar for D and L enantiomers, their dose response curves remained distinct, with L-glucose reaching an early plateau.

biochemistry↗

bioRxiv2022

Quorum sensing peptidic inhibitor rescue host immune system eradication: a novel QS infectivity mechanism

Subverting the host immune system is a major task for any given pathogen to assure its survival and proliferation. For the opportunistic human pathogen Bacillus cereus (Bc), immune evasion enables the establishment of potent infections. In various species of the Bc group, the pleiotropic regulator PlcR and its cognate cell-cell signaling peptide PapR7 regulates virulence genes expression in response to fluctuations in population density, i.e., a quorum-sensing (QS) system. However, how QS exerts its effects during infections, and whether PlcR confers the immune evading ability remain unclear. Herein, we report how interception of the QS communication in Bc obliterates the ability to control the host immune system. Here we designed a peptide-based QS inhibitor that suppresses PlcR-dependent virulence factor expression and attenuates Bc infectivity in mouse models. We demonstrate that the QS peptidic inhibitor blocks host immune system-mediated eradication by reducing the expression of PlcR-regulated major toxins. Our findings provide the first evidence that Bc infectivity is regulated by QS circuit mediated destruction of the host immunity, thus reveal a new strategy to limit Bc virulence and enhance host defense. This peptidic quorum-quenching agent constitutes readily accessible chemical tool for studying how other pathogen QS systems modulate host immunity and forms a basis for development of anti-infective therapeutics.

microbiology↗

bioRxiv2020

Sweet chirality: the taste of L and D glucose stereoisomers

Chirality plays a key role in biomolecular recognition. Typically, a change in chirality will dramatically affect ligand-receptor binding. However, both O_SCPLOWDC_SCPLOW-glucose and its enantiomer O_SCPLOWLC_SCPLOW-glucose elicit sweet taste in humans. We show that O_SCPLOWLC_SCPLOW- and O_SCPLOWDC_SCPLOW-glucose are perceived as similarly sweet by humans, and that in cell-based functional assays, both enantiomers activate the human sweet taste receptor TAS1R2/TAS1R3. We hypothesize that both O_SCPLOWLC_SCPLOW- and O_SCPLOWDC_SCPLOW-glucose occupy the orthosteric binding site in the VFT domain of TAS1R2. Using induced-fit docking to a homology model of this domain, we identify two subpockets in this binding site. The model suggests that glucose molecules can bind in either of these subpockets, which overlap with the predicted positions of monosaccharide units of sucrose. One subpocket is close to the hinge between the two lobes, and overlaps with aspartame and neotame site, the second subpocket overlaps with that described for sweetness enhancers. These findings suggest a framework for rational design of sweeteners combinations.

biochemistry↗

bioRxiv2020

Sweet taste of heavy water

Hydrogen to deuterium isotopic substitution has only a minor effect on physical and chemical properties of water and, as such, is not supposed to influence its neutral taste. Here we conclusively demonstrate that humans are, nevertheless, able to distinguish D2O from H2O by taste. Indeed, highly purified heavy water has a distinctly sweeter taste than same-purity normal water and adds to perceived sweetness of sweeteners. In contrast, mice do not prefer D2O over H2O, indicating that they are not likely to perceive heavy water as sweet. HEK 293T cells transfected with the TAS1R2/TAS1R3 heterodimer and chimeric G-proteins are activated by D2O but not by H2O. Lactisole, which is a known sweetness inhibitor acting via the TAS1R3 monomer of the TAS1R2/TAS1R3, suppresses the sweetness of D2O in human sensory tests, as well as the calcium release elicited by D2O in sweet taste receptor-expressing cells. The present multifaceted experimental study, complemented by homology modelling and molecular dynamics simulations, resolves a long-standing controversy about the taste of heavy water, shows that its sweet taste is mediated by the human TAS1R2/TAS1R3 taste receptor, and opens way to future studies of the detailed mechanism of action. One sentence summaryHeavy water elicits sweet taste for humans via the TAS1R2/TAS1R3 taste receptor.

biochemistry↗