Guanidine Moiety Research Articles

Guanidinium-Stapled Helical Peptides for Targeting Protein-Protein Interactions.

Peptide stapling has emerged as a versatile approach in drug discovery to reinforce secondary structure elements especially α-helices and improve properties of linear bioactive peptides. Inspired by the prevalence of arginine in protein-protein and protein-DNA interfaces, we investigated guanidinium-stapling as a means to constrain helical peptides. Guanidinium stapling was readily achieved on solid support, utilizing two orthogonally protected lysine or unatural α-amino acid residues with an amino function. This method allows for easy modulation of the nature and size of the staple as well as helix propensity. Evaluating a set of guanidinium-stapled peptides for their interaction with different protein targets identified several binders with increased target affinity. X-ray structure determination of four complexes revealed that all stapled peptides adopt a helical conformation upon protein binding. Notably, the disubstituted guanidinium generally exhibits a distinct cis/trans conformation and, in one instance, retains a conserved hydrogen bond with the protein surface. By identifying, for the first time, the guanidinium moiety as an effective helical peptide stapling group, this research significantly expands the repertoire of α-helix stapling techniques for the creation of useful protein mimics.

Aminolysis of five-membered cyclic carbonate catalyzed by guanidines: Solvent effects on the formation of guanidine dimers

This paper presents an experimental and computational investigation on the aminolysis reactions of five-membered cyclic carbonate (5CC) catalyzed by mono- or bi-cyclic guanidines in various solvents. For both liquid and solid 5CC substrate employed, higher polar solvent favors the monomeric active guanidine responsible for the catalytic aminolysis reactions. The formation of the dimeric state of the guanidine catalyst, which is seemingly inert to catalytic activity, is prevented by the bulkier substituents connecting to the guanidine moiety. Extensive DFT calculations further confirm that the relative stabilities of dimeric guanidine is depended on both the polarities of the solvents and the bulkiness of the substituents connected to the guanidine moiety, which is consistent with the experimental kinetic results.

Development of Donor-acceptor-type Molecules and Their Application as Analytical Reagents

π-Extended donor-acceptor (D-A)-type molecules, which bear both electron-donor and electron-acceptor substituents on the backbone, exhibit unique optical properties, such as bathochromic shifts in absorption and emission, large Stokes shifts, solvatochromic behavior, and fluorescence quenching in polar solvents. These unique properties are attributed to intramolecular charge transfer (ICT) or twisted intramolecular charge transfer (TICT) in the ground and excited states. This review article introduces three types of D-A-type molecules that are used as detection reagents for (1) methanol, (2) amino acids during solid-phase peptide synthesis (SPPS), and (3) amines present in the biological environment. For methanol detection, D-A-type fluorophores with basic guanidine moieties were developed to differentiate between methanol (MeOH) and ethanol (EtOH) based on the small difference in their pKa values (ΔpKa=0.4). Selective protonation of the guanidine moiety in methanol disrupts the D-A structure, allowing emission in the resultant polar environment. Similarly, an acid-base reaction between the hydrogen chloride (HCl) salts of the D-A-type molecules and amines is applied to detect amines during SPPS. In this method, a colorless solution of an HCl salt of the D-A-type molecule is deprotonated by amines, forming a yellow solution. This is the first reported quantitative and non-destructive colorimetric method for detecting amines. Finally, a turn-on-type amine-labeling reagent was developed for the nucleophilic aromatic substitution (SNAr) reaction. This new reagent enables protein staining of living cells with a large Stokes shift and without solvent-polarity-dependent fluorescence quenching.

Modular Synthesis of Azines Bearing a Guanidine Core from N-Heterocyclic Carbene (NHC)-Derived Selenoureas and Diazo Reagents.

N-Heterocyclic carbene (NHC)-derived selenoureas comprise a fundamentally important class of NHC derivatives, with key applications in coordination chemistry and the determination of NHC electronic properties. Considering the broad reactivity of chalcogen-containing compounds, it is surprising to note that the use of NHC-derived selenoureas as organic synthons remains essentially unexplored. The present contribution introduces a novel, straightforward transformation leading to azines bearing a guanidine moiety, through the reaction of a wide range of NHC-derived selenoureas with commercially available diazo compounds, in the presence of triphenylphosphine. This transformation offers a new approach to such products, having biological, materials chemistry, and organic synthesis applications. The guanidine-bearing azines are obtained in excellent yields, with all manipulations taking place in air. A reaction mechanism is proposed, based on both experimental mechanistic findings and density functional theory (DFT) calculations. A one-pot, multicomponent transesterification reaction between selenoureas, α-diazoesters, alcohols, and triphenylphosphine was also developed, providing highly functionalized azines.

Nanomedicine in Neuroprotection, Neuroregeneration, and Blood-Brain Barrier Modulation: A Narrative Review.

Nanomedicine is a newer, promising approach to promote neuroprotection, neuroregeneration, and modulation of the blood-brain barrier. This review includes the integration of various nanomaterials in neurological disorders. In addition, gelatin-based hydrogels, which have huge potential due to biocompatibility, maintenance of porosity, and enhanced neural process outgrowth, are reviewed. Chemical modification of these hydrogels, especially with guanidine moieties, has shown improved neuron viability and underscores tailored biomaterial design in neural applications. This review further discusses strategies to modulate the blood-brain barrier-a factor critically associated with the effective delivery of drugs to the central nervous system. These advances bring supportive solutions to the solving of neurological conditions and innovative therapies for their treatment. Nanomedicine, as applied to neuroscience, presents a significant leap forward in new therapeutic strategies that might help raise the treatment and management of neurological disorders to much better levels. Our aim was to summarize the current state-of-knowledge in this field.

Self-Assembled Borneol-Guanidine-Based Amphiphilic Polymers as an Efficient Antibiofilm Agent.

Biofilm-associated infections remain a tremendous obstacle to the treatment of microbial infections globally. However, the poor penetrability to a dense extracellular polymeric substance matrix of traditional antibacterial agents limits their antibiofilm activity. Here, we show that nanoaggregates formed by self-assembly of amphiphilic borneol-guanidine-based cationic polymers (BGNx-n) possess strong antibacterial activity and can eliminate mature Staphylococcus aureus (S. aureus) biofilms. The introduction of the guanidine moiety improves the hydrophilicity and membrane penetrability of BGNx-n. The self-assembled nanoaggregates with highly localized positive charges are expected to enhance their interaction with negatively charged bacteria and biofilms. Furthermore, nanoaggregates dissociate on the surface of biofilms into smaller BGNx-n polymers, which enhances their ability to penetrate biofilms. BGNx-n nanoaggregates that exhibit superior antibacterial activity have the minimum inhibitory concentration (MIC) of 62.5 μg·mL-1 against S. aureus and eradicate mature biofilms at 4 × MIC with negligible hemolysis. Taken together, this size-variable self-assembly system offers a promising strategy for the development of effective antibiofilm agents.

Abstract A005: A live cell PRMT5 NanoBRET™ target engagement assay querying competitive and uncompetitive modes of inhibition

Abstract PRMT5 is an essential arginine methyltransferase that regulates a wide spectrum of cellular processes through the methylation of histone and non-histone substrates. In PRMT5 catalysis, SAM serves as the cofactor and methyl group donor, generating a methylated guanidinium moiety on the target substrate. In normal cells, the SAM pools are maintained through the methionine salvage pathway. In 15% of cancers, a key enzyme in this pathway (MTAP) is deleted, leading to an accumulation of the intermediate MTA, which inhibits PRMT5 activity. This genetic loss of function has therefore been pursued as a collateral vulnerability in MTAP deleted cancers. To exploit this synthetic lethality, a number of novel inhibitors (such as MRTX1719) have been developed that bind cooperatively at the PRMT5/MTA complex, offering a compelling pathway to precision medicine. Here we describe a novel, live cell NanoBRET™ Target Engagement assay that enables mechanistic studies on a variety of PRMT5 inhibitors. Using a cell-permeable NanoBRET probe directed to the substrate pocket of PRMT5, both substrate- and cofactor-competitive engagement can be quantified in cells. Moreover, this method can be used to quantify MTA-uncompetitive target engagement in cells, providing a platform to measure the potency of PRMT5-MTA-drug ternary complex formation. This method serves as a first-in-class method to quantify uncompetitive target engagement in live cells, which can be applied to other model systems. Citation Format: Kelly A. Teske, Ani Michaud, Elisabeth Rothweiler, Cesear Corona, Kaitlin Dunn Hoffman, Jennifer Wilkinson, Michael Beck, James Vasta, Kilian Huber, Matthew Robers. A live cell PRMT5 NanoBRET™ target engagement assay querying competitive and uncompetitive modes of inhibition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A005.

Cocatalytic Activity of the Furfuryl and Oxanorbornane-Substituted Guanidines in the Aldol Reaction Catalyzed by (S)-Proline

This work investigated the cocatalytic activity of recently prepared guanidinium salts containing an oxanorbornane subunit in an (S)-proline-catalyzed aldol reaction. The activity was interpreted by the diastereoselectivity of the reaction (anti/syn ratio) and for the most interesting polycyclic guanidinium salt, the enantioselectivity of the reaction was determined. The results indicated a negative impact on the oxanorbornane unit if present as the flexible substituent. For most of the tested aldehydes, the best cocatalysts provided enantioselectivities above 90% and above 95% at room temperature and 0 °C, respectively, culminating in >99.5% for 4–chloro– and 2–nitrobenzaldehyde as the substrate. The barriers for forming four possible enantiomers were calculated and the results for two anti–enantiomers are qualitatively consistent with the experiment. Obtained results suggest that the representatives of furfurylguanidinium and rigid polycyclic oxanorbornane-substituted guanidinium salts are good lead structures for developing new cocatalysts by tuning the chemical space around the guanidine moiety.

A salt bridge of the C-terminal carboxyl group regulates PHPT1 substrate affinity and catalytic activity.

PHPT1 is a histidine phosphatase that modulates signaling in eukaryotes through its catalytic activity. Here, we present an analysis of the structure and dynamics of PHPT1 through a combination of solution NMR, molecular dynamics, and biochemical experiments. We identify a salt bridge formed between the R78 guanidinium moiety and the C-terminal carboxyl group on Y125 that is critical for ligand binding. Disruption of the salt bridge by appending a glycine residue at the C-terminus (G126) leads to a decrease in catalytic activity and binding affinity for the pseudo substrate, para-nitrophenylphosphate (pNPP), as well as the active site inhibitor, phenylphosphonic acid (PPA). We show through NMR chemical shift, 15N relaxation measurements, and analysis of molecular dynamics trajectories, that removal of this salt bridge results in an active site that is altered both structurally and dynamically thereby significantly impacting enzymatic function and confirming the importance of this electrostatic interaction.

Robenidine derivatives as potential antischistosomal drug candidates

Schistosomiasis caused by Schistosoma spp. is a disease that causes a considerable health burden to millions of people worldwide. The limited availability of effective drugs on the market and the increased risk of resistance development due to extensive usage, highlight the urgent need for new antischistosomal drugs. Recent studies have shown that robenidine derivatives, containing an aminoguanidine core, exhibit promising activities against Plasmodium falciparum, motivating further investigation into their efficacy against Schistosoma mansoni, due to their similar habitat and the resulting related cellular mechanisms like the heme detoxification pathway. The conducted phenotypic screening of robenidine and 80 derivatives against newly transformed schistosomula and adult Schistosoma mansoni yielded 11 candidates with low EC50 values for newly transformed schistosomula (1.12–4.63 μM) and adults (2.78–9.47 μM). The structure-activity relationship revealed that electron-withdrawing groups at the phenyl moiety, as well as the presence of methyl groups adjacent to the guanidine moiety, enhanced the activity of derivatives against both stages of Schistosoma mansoni. The two compounds 2,2′-Bis[(3-cyano-4-fluorophenyl)methylene] carbonimidic Dihydrazide Hydrochloride (1) and 2,2′-Bis[(4-difluoromethoxyphenyl) ethylidene] carbonimidic Dihydrazide Hydrochloride (19), were selected for an in vivo study in Schistosoma mansoni-infected mice based on their potency, cytotoxicity, pharmacokinetic-, and physicochemical properties, but failed to reduce the worm burden significantly (worm burden reduction <20%). Thus, robenidine derivatives require further refinements to obtain higher antischistosomal specificity and in vivo activity.

Abstract 2044: A live cell PRMT5 NanoBRET Target Engagement Assay querying competitive and uncompetitive modes of inhibition

Abstract PRMT5 is an essential arginine methyltransferase that regulates a wide spectrum of cellular processes through the methylation of histone and non-histone substrates. In PRMT5 catalysis, SAM serves as the cofactor and methyl group donor, generating a methylated guanidinium moiety on the target substrate. In normal cells, the SAM pools are maintained through the methionine salvage pathway. In 15% of cancers, a key enzyme in this pathway (MTAP) is deleted, leading to an accumulation of the intermediate MTA, which inhibits PRMT5 activity. This genetic loss of function has therefore been pursued as a collateral vulnerability in MTAP deleted cancers. To exploit this synthetic lethality, a number of novel inhibitors have been developed that bind cooperatively at the PRMT5/MTA complex, offering a compelling pathway to precision medicine. Here we describe a novel, live cell NanoBRET Target Engagement assay that enables mechanistic studies on a variety of PRMT5 inhibitors. Using a cell-permeable NanoBRET probe directed to the substrate pocket of PRMT5, both substrate- and cofactor-competitive engagement can be quantified in cells. Moreover, this method can be used to quantify MTA-uncompetitive target engagement in cells, providing a platform to measure the potency of PRMT5-MTA-drug ternary complex formation. This method serves as a first-in-class method to quantify uncompetitive target engagement in live cells, which can be applied to other model systems. Citation Format: Ani Michaud, Elisabeth Rothweiler, Cesear Corona, Michael Beck, Jennifer Wilkinson, James Vasta, Kilian Huber, Matthew Robers. A live cell PRMT5 NanoBRET Target Engagement Assay querying competitive and uncompetitive modes of inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2044.

Self-Assembled Nanocomposite Hydrogels as Carriers for Demineralized Bone Matrix Particles and Enhanced Bone Repair.

Demineralized bone matrix (DBM) has been widely used as an allogeneic alternative to autologous bone graft for bone repair. However, more extensive use of DBM is limited due to its particulate nature after demineralization and rapid particle dispersion following irrigation, resulting in unpredictable osteoinductivity. Here, a new design of injectable hydrogel carriers for DBM that combine self-healing ability and osteogenic properties based on the self-assembly of guanidinylated hyaluronic acid and silica-rich nanoclays is reported. The nanoclays serve as reversible linkages to form a dynamic hydrogel network with the guanidine moieties on the polymer chains. Gelation kinetics and mechanical properties can be controlled by altering nanoclay content in the hydrogel. The resulting hydrogel exerts self-healing ability due to its dynamic crosslinks and well retains its overall performance with high DBM loading. The hydrogel exhibits great cytocompatibility and osteogenic effects mediated by the nanoclays. In vivo delivery of DBM using the nanocomposite hydrogel further demonstrates robust bone regeneration in a mouse calvarial defect model in comparison to DBM delivered with aqueous HA. This work suggests a promising hydrogel platform for many applications including therapeutic delivery and tissue engineering.

A new lanosyl guanidine from Vertebrata lanosa with anti-inflammatory activity

N-Lanosyl guanidine (1), a new bromophenol containing a guanidine moiety was isolated from the red alga Vertebrata lanosa (L.) T.A. Christensen, which is frequently used for cosmetic purposes. Structure elucidation was performed by means of mass spectrometry as well as 1D and 2D NMR spectroscopy. Due to its structural features, 1 might share a common biosynthetic route with known bromophenolic ureido derivatives. Regarding potential bioactivities, 1 has shown clear anti-inflammatory properties, reducing cytokine release in lipopolysaccharide-stimulated phorbol 12-myristate 13-acetate-differentiated THP-1 macrophages. No signs of toxicity were observed, in either the cell line nor in the nematode Caenorhabditis elegans. However, 1 was inactive against the gram-negative bacterium Pseudomonas aeruginosa.

Synthesis, biological activity, and binding modes of novel guanidino-containing neonicotinoid derivatives

In the present study, the guanidine moiety from natural products was introduced into neonicotinoid insecticides, and a series of nicotine derivatives with guanidine functional groups were synthesized.

Electron transfer kinetics of a series of copper complexes with tripodal tetradentate guanidine quinolinyl ligands.

Copper complexes of tripodal ligands have been used as model systems for electron transfer proteins for decades, displaying a broad range of electron self-exchange rates. We herein report a group of six tripodal tetradentate triarylamine ligands which display a varying number of guanidine and 2-methylquinolinyl moieties. Their corresponding Cu(I) complexes have been (re)synthesized and studied with regard to their electron transfer properties. While their molecular structures in the solid state are four-coordinate and display an uncommon umbrella distortion, DFT studies of the Cu(II) systems reveal that they gain an additional ligand in the form of a solvent molecule and exhibit a range of possible conformers that likely co-exist in thermal equilibrium. The redox-couples' electron self-exchange rates were analyzed using Marcus theory and vary over four orders of magnitude which cyclic voltammetry studies suggest to be due to a gated addition-oxidation electron transfer mechanism. This mechanism deviates from previously studied systems, likely due to the structural anomalies of the Cu(I) systems. This demonstrates that the chosen path of tripodal model systems can be influenced by molecular design.

Colour tuneability of heteroleptic iridium complexes through second-sphere coordination.

Novel H-bond-rich iridium(iii) complexes with different systems of cyclometalating ligands bearing the general formula [Ir(C^N)2(N^N)] were synthesised, characterised and their photophysical properties determined. The incorporated guanidine moieties allow us to introduce a hydrogen-bonding array for the formation of self-assembled iridium-bound hydrogen-bonded systems. Through a series of experimental and computational studies we demonstrated the host-guest chemistry of these systems. These hydrogen-bonding interactions were shown to have significant impacts on chromaticity, quantum yields and lifetimes with emphasis on energy transfer studies based on this second-sphere coordination approach.

Preclinical studies of RA475, a guanidine-substituted spirocyclic candidate RPN13/ADRM1 inhibitor for treatment of ovarian cancer.

There is an urgent unmet need for more targeted and effective treatments for advanced epithelial ovarian cancer (EOC). The emergence of drug resistance is a particular challenge, but small molecule covalent inhibitors have promise for difficult targets and appear less prone to resistance. Michael acceptors are covalent inhibitors that form bonds with cysteines or other nucleophilic residues in the target protein. However, many are categorized as pan-assay interference compounds (PAINS) and considered unsuitable as drugs due to their tendency to react non-specifically. Targeting RPN13/ADRM1-mediated substrate recognition and deubiquitination by the proteasome 19S Regulatory Particle (RP) is a promising treatment strategy. Early candidate RPN13 inhibitors (iRPN13) produced a toxic accumulation of very high molecular weight polyubiquitinated substrates, resulting in therapeutic activity in mice bearing liquid or solid tumor models, including ovarian cancer; however, they were not drug-like (PAINS) because of their central piperidone core. Up284 instead has a central spiro-carbon ring. We hypothesized that adding a guanidine moiety to the central ring nitrogen of Up284 would produce a compound, RA475, with improved drug-like properties and therapeutic activity in murine models of ovarian cancer. RA475 produced a rapid accumulation of high molecular polyubiquitinated proteins in cancer cell lines associated with apoptosis, similar to Up284 although it was 3-fold less cytotoxic. RA475 competed binding of biotinylated Up284 to RPN13. RA475 shows improved solubility and distinct pharmacodynamic properties compared to Up284. Specifically, tetraubiquitin firefly luciferase expressed in leg muscle was stabilized in mice more effectively upon IP treatment with RA475 than with Up284. However, pharmacologic analysis showed that RA475 was more rapidly cleared from the circulation, and less orally available than Up284. RA475 shows reduced ability to cross the blood-brain barrier and in vitro inhibition of HERG. Treatment of mice with RA475 profoundly inhibited the intraperitoneal growth of the ID8-luciferase ovarian tumor model. Likewise, RA475 treatment of immunocompetent mice inhibited the growth of spontaneous genetically-engineered peritoneal tumor, as did weekly cisplatin dosing. The combination of RA475 and cisplatin significantly extended survival compared to individual treatments, consistent with synergistic cytotoxicity in vitro. In sum, RA475 is a promising candidate covalent RPN13i with potential utility for treatment of patients with advanced EOC in combination with cisplatin.

Synthesis and antimicrobial properties of guanidine-functionalized labdane type diterpenoids

The occurrence of increased antibiotic resistance has reduced the availability of drugs effective in the control of infectious diseases, especially those caused by various combinations of bacteria and/or fungi that are often associated with poorer patient outcomes. In the hunt for novel antibiotics of interest to treat polymicrobial diseases, molecules bearing guanidine moieties have recently come to the fore in designing and optimizing antimicrobial agents. Due to their remarkable antibacterial and antifungal activities, labdane diterpenes are also attracting increasing interest in antimicrobial drug discovery. In this study, six different guanidines prenylated with labdanic fragments were synthesized and evaluated for their antimicrobial properties. Assays were carried out against both non-resistant and antibiotic-resistant bacteria strains, while their possible antifungal activities have been tested on the yeast Candida albicans. Two of the synthesized compounds, namely labdan-8,13(R)-epoxy-15-oyl guanidine and labdan-8,13(S)-epoxy-15-oyl guanidine, were finally selected as the best candidates for further developments in drug discovery, due to their antimicrobial effects on both Gram-negative and Gram-positive bacterial strains, their fungicide action, and their moderate toxicity in vivo on zebrafish embryos. The study also provides insights into the structure-activity relationships of the guanidine-functionalized labdane-type diterpenoids.

Synthesis of Novel Arginine Building Blocks with Increased Lipophilicity Compatible with Solid-Phase Peptide Synthesis

Arginine, due to the guanidine moiety, increases peptides’ hydrophilicity and enables interactions with charged molecules, but at the same time, its presence in a peptide chain might reduce its permeability through biological membranes. This might be resolved by temporary coverage of the peptide charge by lipophilic, enzyme-sensitive alkoxycarbonyl groups. Unfortunately, such a modification of a guanidine moiety has not been reported to date and turned out to be challenging. Here, we present a new, optimized strategy to obtain arginine building blocks with increased lipophilicity that were successfully utilized in the solid-phase peptide synthesis of novel arginine vasopressin prodrugs.

Targeted Delivery of Celastrol via Chondroitin Sulfate Derived Hybrid Micelles for Alleviating Symptoms in Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is caused by an accumulation of excess fat in the liver leading to oxidative stress and liver cell injury, as well as overproduction of inflammatory cytokines. CD44 has been identified as a potential therapeutic target in the development of NAFLD to nonalcoholic steatohepatitis. Here, chondroitin sulfate (CS) is selected to construct a CD44-targeted delivery system for the treatment of NAFLD. Specifically, two CS-derived amphiphilic materials including CS conjugated with either 4-aminophenylboronic acid pinacol ester (CS-PBE) or phenformin (CS-PFM) were synthesized, respectively. The presence of PBE moieties on CS-PBE rendered the vehicle with enhanced loading capacity and scavenging potential against reactive oxygen species, while the presence of guanidine moieties on CS-PFM enhanced the internalization of vehicles in the differentiated hepatocytes. Next, celastrol (CLT) was encapsulated in the hybrid micelle to afford CS-Hybrid/CLT, which demonstrates sufficient stability, enhanced cellular uptake efficiencies in differentiated HepG2 cells, and therapeutic potential to alleviate lipid accumulation in differentiated HepG2 cells. In a high-fat-diet-induced NAFLD rat model, CS-Hybrid/CLT micelles demonstrated the capacity to dramatically decrease hepatic lipid accumulation and free fatty acid levels with greatly improved pathologic liver histology and downregulated hepatic inflammation levels. These results suggest that CS-based amphiphilic micelles may offer a promising strategy to effectively deliver therapeutic cargos to the liver for the treatment of NAFLD.