Organ- and Cell-Selective Delivery of mRNA In Vivo Using Guanidinylated Serinol Charge-Altering Releasable Transporters.

Selective RNA delivery is required for the broad implementation of RNA clinical applications, including prophylactic and therapeutic vaccinations, immunotherapies for cancer, and genome editing. Current polyanion delivery relies heavily on cationic amines, while cationic guanidinium systems have received limited attention due in part to their strong polyanion association, which impedes intracellular polyanion release. Here, we disclose a general solution to this problem in which cationic guanidinium groups are used to form stable RNA complexes upon formulation but at physiological pH undergo a novel charge-neutralization process, resulting in RNA release. This new delivery system consists of guanidinylated serinol moieties incorporated into a charge-altering releasable transporter (GSer-CARTs). Significantly, systematic variations in structure and formulation resulted in GSer-CARTs that exhibit highly selective mRNA delivery to the lung (∼97%) and spleen (∼98%) without targeting ligands. Illustrative of their breadth and translational potential, GSer-CARTs deliver circRNA, providing the basis for a cancer vaccination strategy, which in a murine model resulted in antigen-specific immune responses and effective suppression of established tumors.

Novel guanidine derivatives targeting leukemia as selective Src/Abl dual inhibitors: Design, synthesis and anti-proliferative activity.

A new series of benzene-sulfonamide derivatives 3a-i was designed and synthesized via the reaction of N-(pyrimidin-2-yl)cyanamides 1a-i with sulfamethazine sodium salt 2 as dual Src/Abl inhibitors. Spectral data IR, 1H-, 13C- NMR and elemental analyses were used to confirm the structures of all the newly synthesized compounds 3a-i and 4a-i. Crucially, we screened all the synthesized compounds 3a-i against NCI 60 cancer cell lines. Among all, compound 3b was the most potent, with IC50 of 0.018 µM for normoxia, and 0.001 µM for hypoxia, compared to staurosporine against HL-60 leukemia cell line. To verify the selectivity of this derivative, it was assessed against a panel of tyrosine kinase EGFR, VEGFR-2, B-raf, ERK, CK1, p38-MAPK, Src and Abl enzymes. Results revealed that compound 3b can effectively and selectively inhibit Src/Abl with IC500.25 µM and Abl inhibitory activity with IC500.08 µM, respectively, and was found to be more potent on these enzymes than other kinases that showed the following results: EGFR IC500.31 µM, VEGFR-2 IC500.68 µM, B-raf IC500.33 µM, ERK IC501.41 µM, CK1 IC500.29 µM and p38-MAPK IC500.38 µM. Moreover, cell cycle analysis and apoptosis performed to compound 3b against HL-60 suggesting its antiproliferative activity through Src/Abl inhibition. Finally, molecular docking studies and physicochemical properties prediction for compounds 3b, 3c, and 3 h were carried out to investigate their biological activities and clarify their bioavailability.

Scaffold-Oriented Asymmetric Catalysis: Conformational Modulation of Transition State Multivalency during a Catalyst-Controlled Assembly of a Pharmaceutically Relevant Atropisomer.

A new class of superbasic, bifunctional peptidyl guanidine catalysts is presented, which enables the organocatalytic, atroposelective synthesis of axially chiral quinazolinediones. Computational modeling unveiled the conformational modulation of the catalyst by a novel phenyl urea N-cap, that preorganizes the structure into the active, folded state. A previously unanticipated noncovalent interaction involving a difluoroacetamide acting as a hybrid mono- or bidentate hydrogen bond donor emerged as a decisive control element inducing atroposelectivity. These discoveries spurred from a scaffold-oriented project inspired from a fascinating investigational BTK inhibitor featuring two stable chiral axes and relies on a mechanistic framework that was foreign to the extant lexicon of asymmetric catalysis.

Human apo-metallothionein 1a is not a random coil: Evidence from guanidinium chloride, high temperature, and acidic pH unfolding studies.

The structures of apo-metallothioneins (apo-MTs) have been relatively elusive due to their fluxional, disordered state which has been difficult to characterize. However, intrinsically disordered protein (IDP) structures are rather diverse, which raises questions about where the structure of apo-MTs fit into the protein structural spectrum. In this paper, the unfolding transitions of apo-MT1a are discussed with respect to the effect of the chemical denaturant GdmCl, temperature conditions, and pH environment. Cysteine modification in combination with electrospray ionization mass spectrometry was used to probe the unfolding transition of apo-MT1a in terms of cysteine exposure. Circular dichroism spectroscopy was also used to monitor the change in secondary structure as a function of GdmCl concentration. For both of these techniques, cooperative unfolding was observed, suggesting that apo-MT1a is not a random coil. More GdmCl was required to unfold the protein backbone than to expose the cysteines, indicating that cysteine exposure is likely an early step in the unfolding of apo-MT1a. MD simulations complement the experimental results, suggesting that apo-MT1a adopts a more compact structure than expected for a random coil. Overall, these results provide further insight into the intrinsically disordered structure of apo-MT.

Fluorescent Guanidinium-Azacarbazole for Oxoanion Binding in Water.

Oxoanions such as carboxylates, phosphates, and sulfates play important roles in both chemistry and biology and are abundant on the cell surface. We report on the synthesis and properties of a rationally designed guanidinium-containing oxoanion binder, 1-guanidino-8-amino-2,7-diazacarbazole (GADAC). GADAC binds to a carboxylate, phosphate, and sulfate in pure water with affinities of 3.6 × 104, 1.1 × 103, and 4.2 × 103 M-1, respectively. Like 2-azacarbazole, which is a natural product that enables scorpions to fluoresce, GADAC is fluorescent in water (λabs = 356 nm, λem = 403 nm, ε = 13,400 M-1 cm-1). The quantum yield of GADAC is pH-sensitive, increasing from Φ = 0.12 at pH 7.4 to Φ = 0.53 at pH 4.0 as a result of the protonation of the aminopyridine moiety. The uptake of GADAC into live human melanoma cells is detectable in the DAPI channel at low micromolar concentrations. Its properties make GADAC a promising candidate for applications in oxoanion binding and fluorescence labeling in biological (e.g., the delivery of cargo into cells) and other contexts.

Guanidine-modified albumin-MMAE conjugates with enhanced endocytosis ability.

Aiming to address the insufficient endocytosis ability of traditional albumin drug conjugates, this paper reports elegant guanidine modification to improve efficacy for the first time. A series of modified albumin drug conjugates were designed and synthesized with different structures, including guanidine (GA), biguanides (BGA) and phenyl (BA), and different quantities of modifications. Then, the endocytosis ability and in vitro/vivo potency of albumin drug conjugates were systematically studied. Finally, a preferred conjugate A4 was screened, which contained 15 BGA modifications. Conjugate A4 maintains spatial stability similar to that of the unmodified conjugate AVM and could significantly enhance endocytosis ability (p*** = 0.0009) compared with the unmodified conjugate AVM. Additionally, the in vitro potency of conjugate A4 (EC50 = 71.78 nmol in SKOV3 cells) was greatly enhanced (approximately 4 times) compared with that of the unmodified conjugate AVM (EC50 = 286.00 nmol in SKOV3 cells). The in vivo efficacy of conjugate A4 completely eliminated 50% of tumors at 33 mg/kg, which was significantly better than the efficacy of conjugate AVM at the same dose (P** = 0.0026). In addition, theranostic albumin drug conjugate A8 was designed to intuitively realize drug release and maintain antitumor activity similar to conjugate A4. In summary, the guanidine modification strategy could provide new ideas for the development of new generational albumin drug conjugates.

Synthetic and natural guanidine derivatives as antitumor and antimicrobial agents: A review.

Guanidines are fascinating small nitrogen-rich organic compounds, which have been frequently associated with a wide range of biological activities. This is mainly due to their interesting chemical features. For these reasons, for the past decades, researchers have been synthesizing and evaluating guanidine derivatives. In fact, there are currently on the market several guanidine-bearing drugs. Given the broad panoply of pharmacological activities displayed by guanidine compounds, in this review, we chose to focus on antitumor, antibacterial, antiviral, antifungal, and antiprotozoal activities presented by several natural and synthetic guanidine derivatives, which are undergoing preclinical and clinical studies from January 2010 to January 2023. Moreover, we also present guanidine-containing drugs currently in the market for the treatment of cancer and several infectious diseases. In the preclinical and clinical setting, most of the synthesized and natural guanidine derivatives are being evaluated as antitumor and antibacterial agents. Even though DNA is the most known target of this type of compounds, their cytotoxicity also involves several other different mechanisms, such as interference with bacterial cell membranes, reactive oxygen species (ROS) formation, mitochondrial-mediated apoptosis, mediated-Rac1 inhibition, among others. As for the compounds already used as pharmacological drugs, their main application is in the treatment of different types of cancer, such as breast, lung, prostate, and leukemia. Guanidine-containing drugs are also being used for the treatment of bacterial, antiprotozoal, antiviral infections and, recently, have been proposed for the treatment of COVID-19. To conclude, the guanidine group is a privileged scaffold in drug design. Its remarkable cytotoxic activities, especially in the field of oncology, still make it suitable for a deeper investigation to afford more efficient and target-specific drugs.

Synthesis and evaluation of a deltic guanidinium analogue of a cyclic RGD peptide.

A guanidine group is abundantly found in natural products and drugs. Guanidine has the highest basicity among many common functional groups in nature. Because of its high basicity, it generally exists as a protonated guanidinium and functions as a cationic hydrogen bond donor. Finding an appropriate bioisostere of guanidinium is challenging because of its high basicity and unique trigonal planar shape. In this study, we explored the possibility of "deltic guanidinium" as a bioisostere of guanidinium using a cyclic arginine-glycine-aspartic acid (RGD) peptide as a parent compound. We synthesized c(deltic RGDyK), in which a guanidinium group of an arginine residue in c(RGDyK) is replaced with deltic guanidinium. A target binding assay, biodistribution study, and metabolic stability assay were conducted with c(deltic RGDyK) and its radioiodinated variant. The deltic guanidinium analog peptides exhibited similar biological properties to the parent peptides and improved in vivo stability, indicating that deltic guanidinium could work as a unique bioisostere of guanidinium.

New Guanidine Alkaloids Batzelladines O and P from the Marine Sponge Monanchora pulchra Induce Apoptosis and Autophagy in Prostate Cancer Cells.

Two new guanidine alkaloids, batzelladines O (1) and P (2), were isolated from the deep-water marine sponge Monanchora pulchra. The structures of these metabolites were determined by NMR spectroscopy, mass spectrometry, and ECD. The isolated compounds exhibited cytotoxic activity in human prostate cancer cells PC3, PC3-DR, and 22Rv1 at low micromolar concentrations and inhibited colony formation and survival of the cancer cells. Batzelladines O (1) and P (2) induced apoptosis, which was detected by Western blotting as caspase-3 and PARP cleavage. Additionally, induction of pro-survival autophagy indicated as upregulation of LC3B-II and suppression of mTOR was observed in the treated cells. In line with this, the combination with autophagy inhibitor 3-methyladenine synergistically increased the cytotoxic activity of batzelladines O (1) and P (2). Both compounds were equally active in docetaxel-sensitive and docetaxel-resistant prostate cancer cells, despite exhibiting a slight p-glycoprotein substrate-like activity. In combination with docetaxel, an additive effect was observed. In conclusion, the isolated new guanidine alkaloids are promising drug candidates for the treatment of taxane-resistant prostate cancer.

Insights into the binding of arginine to adenosine phosphate from mimetic complexes.

The amino acid arginine plays a key role in the interaction of proteins with adenosine phosphates, as its protonated guanidinium side group is capable of building multipodal H-bonding interactions with the oxygen atoms of the phosphate, phosphoester and ribose moieties and with the nitrogen atoms of adenine. Protein interactions often take place in competition with other ionic species, typically metal cations, which are prone to build concerted coordination arrangements with the same centers of negative charge as guanidinium. We report on a vibrational spectroscopy and computational investigation of a positively charged ternary complex formed by adenosine monophosphate (AMP) with methyl guanidinium and Na+. Following a bottom-up approach, an analogous complex with ribose phosphate is characterized as well, which serves to assess the individual role of the phosphate, sugar and adenine moieties in the binding process and to compare, within a single complex, the interactions associated with diffuse versus localized charge distributions of guanidinium and the alkali cation, respectively. The results indicate that Na+ is preferentially hosted in a semi-rigid pocket formed by the phosphoester-adenosine backbone of AMP and displaces guanidinium to a peripheral binding to the phosphate anionic end group. This suggests that the control of the salt concentration may constitute an effective route to modulate protein-AMP complexation.

Residual Structure in the Denatured State of the Fast-Folding UBA(1) Domain from the Human DNA Excision Repair Protein HHR23A.

The structure of the first ubiquitin-associated domain from HHR23A, UBA(1), was determined by X-ray crystallography at a 1.60 Å resolution, and its stability, folding kinetics, and residual structure under denaturing conditions have been investigated. The concentration dependence of thermal denaturation and size-exclusion chromatography indicate that UBA(1) is monomeric. Guanidine hydrochloride (GdnHCl) denaturation experiments reveal that the unfolding free energy, ΔGu°'(H2O), of UBA(1) is 2.4 kcal mol-1. Stopped-flow folding kinetics indicates sub-millisecond folding with only proline isomerization phases detectable at 25 °C. The full folding kinetics are observable at 4 °C, yielding a folding rate constant, kf, in the absence of a denaturant of 13,000 s-1 and a Tanford ß-value of 0.80, consistent with a compact transition state. Evaluation of the secondary structure via circular dichroism shows that the residual helical structure in the denatured state is replaced by polyproline II structure as the GdnHCl concentration increases. Analysis of NMR secondary chemical shifts for backbone 15NH, 13CO, and 13Cα atoms between 4 and 7 M GdnHCl shows three islands of residual helical secondary structure that align in sequence with the three native-state helices. Extrapolation of the NMR data to 0 M GdnHCl demonstrates that helical structure would populate to 17-33% in the denatured state under folding conditions. Comparison with NMR data for a peptide corresponding to helix 1 indicates that this helix is stabilized by transient tertiary interactions in the denatured state of UBA(1). The high helical content in the denatured state, which is enhanced by transient tertiary interactions, suggests a diffusion-collision folding mechanism.

Direct Cellular Delivery of Exogenous Genetic Material and Protein via Colloidal Nano-Assemblies with Biopolymer.

Direct cytosolic delivery of large biomolecules that bypass the endocytic pathways is a promising strategy for therapeutic applications. Recent works have shown that small-molecule, nanoparticle, and polymer-based carriers can be designed for direct cytosolic delivery. It has been shown that the specific surface chemistry of the carrier, nanoscale assembly between the carrier and cargo molecule, good colloidal stability, and low surface charge of the nano-assembly are critical for non-endocytic uptake processes. Here we report a guanidinium-terminated polyaspartic acid micelle for direct cytosolic delivery of protein and DNA. The polymer delivers the protein/DNA directly to the cytosol by forming a nano-assembly, and it is observed that <200 nm size of colloidal assembly with near-zero surface charge is critical for efficient cytosolic delivery. This work shows the importance of size and colloidal property of the nano-assembly for carrier-based cytosolic delivery of large biomolecules.

A Bivalent Supramolecular GCP Ligand Enables Blocking of the Taspase1/Importin α Interaction.

Taspase1 is a unique protease not only pivotal for embryonic development but also implicated in leukemia as well as solid tumors. As such, it is a promising target in cancer therapy, although only a limited number of Taspase1 inhibitors lacking general applicability are currently available. Here we present a bivalent guanidiniocarbonyl-pyrrole (GCP)-containing supramolecular ligand that is capable of disrupting the essential interaction between Taspase1 and its cognate import receptor Importin α in a concentration-dependent manner in vitro with an IC50 of 35 µM. Here, size of the bivalent vs the monovalent construct as well as its derivation with an aromatic cbz-group arose as critical determinants for efficient interference of 2GC. This was also evident when we investigated the effects in different tumor cell lines, resulting in comparable EC50 values (∼40-70 µM). Of note, in higher concentrations, 2GC also interfered with Taspase1's proteolytic activity. We thus believe to set the stage for a novel class of Taspase1 inhibitors targeting a pivotal protein-protein interaction prerequisite for its cancer-associated proteolytic function.

Poly(allylguanidine)-Coated Surfaces Regulate TGF-ß in Glioblastoma Cells to Induce Apoptosis via NF-κB Pathway Activation.

Polycationic biomaterials are currently widely applied in neuronal cell cultures to promote cell adhesion and viability. However, polycations generally have cytotoxic properties that limit their application in the field of biomaterials. In this study, we examined the use of a novel polycation poly(allylguanidine) (PAG), which contains a guanidine group in the side chain and a structure similar to poly(allylamine hydrochloride) (PAH), an example of another commonly used polycation. Our findings showed that exposure to PAG induced apoptosis in glioblastoma (GBM) cells, while exposure to PAH induced necrosis. Compared to control groups, the PAG coating significantly limited the proliferation of GBM8901 in vitro and in vivo. Furthermore, GBM8901 cells exposed to the PAG coating exhibited increased levels of phospho-p65 and phosphor-IκB, implying that GBM8901 cells underwent apoptotic cell death via the NF-κB pathway by the regulation of TGF-ß. This result was further confirmed to be consistent with the experimental results from western blot protein analysis and apoptosis/necrosis assays. These findings indicate that the polycation PAG has the potential to not only suppress the proliferation of GBM8901 cancer cells but also improve the neural viability and promote the differentiation of neural stem/precursor cells into mature neurons. In conclusion, biomaterials such as PAG act as extremely potent options for applications in the treatment of pathological conditions such as brain cancer.

Methylglyoxal Forms Diverse Mercaptomethylimidazole Crosslinks with Thiol and Guanidine Pairs in Endogenous Metabolites and Proteins.

Methylglyoxal (MGO) is a reactive byproduct formed by several metabolic precursors, the most notable being triosephosphates in glycolysis. While many MGO-mediated adducts have been described, the reactivity and specific biomolecular targets of MGO remain incompletely mapped. Based on our recent discovery that MGO can form stable mercaptomethylimidazole crosslinks between cysteine and arginine (MICA) in proteins, we hypothesized that MGO may participate in myriad reactions with biologically relevant guanidines and thiols in proteins, metabolites, and perhaps other biomolecules. Herein, we performed steady-state and kinetic analyses of MGO reactivity with several model thiols, guanidines, and biguanide drugs to establish the plausible and prevalent adducts formed by MGO in proteins, peptides, and abundant cellular metabolites. We identified several novel, stable MICA metabolites that form in vitro and in cells, as well as a novel intermolecular post-translational MICA modification of surface cysteines in proteins. These data confirm that kinetic trapping of free MGO by thiols occurs rapidly and can decrease formation of more stable imidazolone (MG-H1) arginine adducts. However, reversible hemithioacetal adducts can go on to form stable MICA modifications in an inter- and intramolecular fashion with abundant or proximal guanidines, respectively. Finally, we discovered that intracellular MICA-glutathione metabolites are recognized and exported by the efflux pump MRP1, providing a parallel and perhaps complementary pathway for MGO detoxification working alongside the glyoxalase pathway. These data provide new insights into the plausible reactions involving MGO in cells and tissues, as well as several new molecular species in proteins and metabolites for further study.

Insight into the protein salting-in mechanism of arginine, magnesium chloride and ethylene glycol: Solvent interaction with aromatic solutes.

Key factors in the salting-in effects on proteins of additives are their interactions with aromatic groups. We studied the interaction of four aromatic solutes, benzyl alcohol (BA), phenol, 4-hydroxybenzyl alcohol (4-HBA) and methyl gallate (MG), with different salting-in additives, arginine hydrochloride (ArgHCl), magnesium chloride (MgCl2), ethylene glycol (EG), and guanidine hydrochloride (GdnHCl) using solubility measurements. We used sodium chloride (NaCl) as a control. MgCl2 decreased the solubility of the four aromatic solutes with weak solute dependence. In contrast, ArgHCl, GdnHCl, and EG increased the solubility of four aromatic solutes with a similar solute dependence. Their salting-in effects were weaker on BA and 4-HBA and stronger on phenol and MG. These results indicate that attached groups alter the aromatic properties, affecting the interactions between the benzene ring and these three additives. More importantly, the observed results demonstrate that the salting-in mechanism is different between MgCl2, EG and ArgHCl, which should play a role in their effects on protein solubility.

Expression of HCV genotype-4 core antigen in prokaryotic E. coli system for diagnosis of HCV infection in Egypt.

BACKGROUND: Egypt has a high prevalence of hepatitis C virus (HCV) infection with 92.5% of genotype-4. AIM: This study aimed to clone and express the core gene of HCV genotype-4 for using it to develop a highly sensitive, specific, and cost-effective diagnostic assay for detecting HCV infection. METHODS: Using synthetic HCV genotype-4 core gene, pET15b as E. coli expression vector, and 1 mM lactose as inducer, the HCV core protein (MW 17 kDa) was expressed in the form of inclusion bodies (IBs) that was purified and solubilized using 8 M guanidinium HCl. The recombinant core protein was in vitro refolded by a rapid dilution method for further purification using weak cation exchange liquid chromatography. The immunogenicity of the purified protein was tested by ELISA using 129 serum samples. RESULTS: The recombinant core protein was successfully expressed and purified. The results also showed that the in-house anti-HCV core assay is accurate, specific (~96.6%), and highly sensitive (~100%) in accordance with the commercial ELISA kit. CONCLUSION: The sensitivity, specificity, and reproducibility of the developed assay were high and promising to be used as a screening assay for detecting HCV infection.

In Situ Monitored Vortex Fluidic-Mediated Protein Refolding/Unfolding Using an Aggregation-Induced Emission Bioprobe.

Protein folding is important for protein homeostasis/proteostasis in the human body. We have established the ability to manipulate protein unfolding/refolding for ß-lactoglobulin using the induced mechanical energy in the thin film microfluidic vortex fluidic device (VFD) with monitoring as such using an aggregation-induced emission luminogen (AIEgen), TPE-MI. When denaturant (guanidine hydrochloride) is present with ß-lactoglobulin, the VFD accelerates the denaturation reaction in a controlled way. Conversely, rapid renaturation of the unfolded protein occurs in the VFD in the absence of the denaturant. The novel TPE-MI reacts with exposed cysteine thiol when the protein unfolds, as established with an increase in fluorescence intensity. TPE-MI provides an easy and accurate way to monitor the protein folding, with comparable results established using conventional circular dichroism. The controlled VFD-mediated protein folding coupled with in situ bioprobe AIEgen monitoring is a viable methodology for studying the denaturing of proteins.

PEGylation of Guanidinium and Indole Bearing Poly(methacrylamide)s - Biocompatible Terpolymers for pDNA Delivery.

This study describes the first example for shielding of a high performing terpolymer that consists of N-(2-hydroxypropyl)methacrylamide (HPMA), N-(3-guanidinopropyl)methacrylamide (GPMA), and N-(2-indolethyl)methacrylamide monomers (IEMA) by block copolymerization of a polyethylene glycol derivative - poly(nona(ethylene glycol)methyl ether methacrylate) (P(MEO9 MA)) via reversible addition-fragmentation chain transfer (RAFT) polymerization. The molecular weight of P(MEO9 MA) is varied from 3 to 40 kg mol-1 while the comonomer content of HPMA, GPMA, and IEMA is kept comparable. The influence of P(MEO9 MA) block with various molecular weights is investigated over cytotoxicity, plasmid DNA (pDNA) binding, and transfection efficiency of the resulting polyplexes. Overall, the increase in molecular weight of P(MEO9 MA) block demonstrates excellent biocompatibility with higher cell viability in L-929 cells and an efficient binding to pDNA at N/P ratio of 2. The significant transfection efficiency in CHO-K1 cells at N/P ratio 20 is obtained for block copolymers with molecular weight of P(MEO9 MA) up to 10 kg mol-1 . Moreover, a fluorescently labeled analogue of P(MEO9 MA), bearing perylene monoimide methacrylamide (PMIM), is introduced as a comonomer in RAFT polymerization. Polyplexes consisting of labeled block copolymer with 20 kg mol-1 of P(MEO9 MA) and pDNA are incubated in Hela cells and investigated through structured illumination microscopy (SIM).

Synthesis and antiproliferative screening of novel doubly modified colchicines containing urea, thiourea and guanidine moieties.

A new series of 10-demethoxy-10-methylaminocolchicines bearing urea, thiourea or aguanidine moieties at position C7 has been designed, synthesized and evaluated for in vitro anticancer activity against different cancer cell lines (A549, MCF-7, LoVo, LoVo/DX). The majority of the new derivatives were active in the nanomolar range and were characterized by lower IC50 values than cisplatin or doxorubicin. Two ureas (4 and 8) and thioureas (19 and 25) were found to be good antiproliferative agents (low IC50 values and high SI) and could prove to be promising candidates for further research in the field of anticancer drugs based on the colchicine skeleton.