Allosteric Research Articles

CDK2 Allosteric Modulators: The New Route in the Design of New Drugs as Anticancer and Non-hormonal Contraceptives.

CDK2 plays a pivotal role in controlling the cell cycle progression in eukaryotes and for this reason, it has been the subject of several studies for suitable inhibitors in the last decades. But more than 30 years of basic research have not generated an inhibitor as marketed drugs. Some inhibitors are to date in early phase clinical development. Moreover, most efforts to develop CDK2 inhibitors have been oriented towards orthosteric inhibitors, which block the kinase activity by binding to the ATP binding site, competing directly with ATP. These compounds have off-target kinase activity, because of the structural homology of the active sites of several other kinases. Targeting the CDK2 allosteric binding pocket could produce successful CDK2 inhibitors. Few examples of high-affinity allosteric CDK2 inhibitors are known. Despite promising research results, none has been approved for marketing. In recent years, various methodologies have been reported capable of identifying new and never-discovered portions of the target protein, which present adequate druggability characteristics. In this paper, we have highlighted and discussed the more recent findings on allosteric inhibitors intending to encouraging further exploration mainly focused on in silico drug discovery.

Asciminib resistance of a new BCR::ABL1 p.I293_K294insSSLRD mutant detected in a Ph + ALL patient.

Chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia patients largely benefit from an expanding tyrosine kinase inhibitors (TKIs) toolbox that has improved the outcome of both diseases. However, TKI success is continuously challenged by mutation-driven acquired resistance and therefore, close monitoring of clonal genetic diversity is necessary to ensure proper clinical management and adequate response to treatment. Here, we report the case of a ponatinib-resistant Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL) patient harboring a BCR::ABL1 p.I293_K294insSLLRD mutation. Using in vitro proliferation assays on newly generated Ba/F3 cell lines, we confirmed that the mutation confers moderate resistance to ponatinib, and to imatinib and nilotinib. In contrast, BCR::ABL1SLLRD Ba/F3 cells remain highly sensitive to dasatinib. Unexpectedly, the insertion also provides resistance to asciminib with no inhibitory effect up to 1000nM. Based on predicted structural models, we speculate that the p.I293_K294insSLLRD disrupts the interaction between the SH3 domain and the kinase domain, shifting the equilibrium toward the active conformation. This shift confers resistance to TKIs that preferentially bind to the inactive conformation, as well as to the allosteric asciminib inhibitor. However, the mutation retains sensitivity to dasatinib, which targets the active form of the kinase.

Structural basis for the inhibition of PRC2 by active transcription histone posttranslational modifications.

Polycomb repressive complex 2 (PRC2) trimethylates histone H3 on K27 (H3K27me3) leading to gene silencing that is essential for embryonic development and maintenance of cell identity. PRC2 is regulated by protein cofactors and their crosstalk with histone modifications. Trimethylated histone H3 on K4 (H3K4me3) and K36 (H3K36me3) localize to sites of active transcription and inhibit PRC2 activity through unknown mechanisms. Using cryo-electron microscopy, we reveal that histone H3 tails containing H3K36me3 engage poorly with PRC2 and preclude its effective interaction with chromatin, while H3K4me3 binds to the allosteric site in the EED subunit, acting as an antagonist that competes with activators required for spreading of the H3K27me3 repressive mark. Thus, the location of the H3K4me3 and H3K36me3 modifications along the H3 tail allows them to target two requirements for efficient trimethylation of H3K27 by PRC2. We further show that the JARID2 cofactor modulates PRC2 activity in the presence of these histone modifications.

Novel Orthosteric/Allosteric Ligands of Cannabinoid Receptors: An Unexpected Pharmacological Profile.

The design of dualsteric/bitopic receptor ligands as compounds capable of simultaneously interacting with both the orthosteric and an allosteric binding site has gained importance to achieve enhanced receptor specificity and minimize off-target effects. In this work, we reported the synthesis and biological evaluation of a new series of compounds, namely, the RF series, obtained by chemically combining the CB1R ago-positive allosteric modulators (PAM) GAT211 with the cannabinoid receptors (CBRs) orthosteric agonist FM6b. Therefore, RF compounds were designed as dualsteric/bitopic ligands for hCB1R with the aim of obtaining stronger hCB1R agonists or ago-PAMs, with improved receptor subtype selectivity and reduction of central side effects. Unexpectedly, in vitro assays on hCB1R indicated RF compounds were inverse agonists/antagonists, exhibiting different profiles compared to those of parent compounds FM6b and GAT211 and, furthermore, two compounds behaved as hCB2R PAMs. The unpredictable change in the function of these new ligands suggests that the function of cannabinoids is not simply predicted.

Examining Arginase-1 Trimerization Uncovers a Promising Allosteric Site for Inhibition.

Arginase-1 (ARG-1) is a promising target for cancer immunotherapy, but the small size and the highly polar nature of its catalytic site present significant challenges for inhibitor development. An alternative strategy to induce enzyme inhibition by targeting protein oligomerization has been developed recently, offering several advantages such as increased selectivity, promotion of protein degradation, and potential substoichiometric inhibition. In this study, we demonstrated that only trimeric ARG-1 is active, which was confirmed by producing monomeric arginase-1. Through in silico-driven site-directed mutagenesis, we identified an allosteric site involving five key amino acids responsible for ARG-1 trimerization. We further demonstrated the covalent modification of a key arginine residue within this pocket using phenylglyoxal disrupted ARG-1 oligomerization. Although phenylglyoxal has limited potency, it effectively supports the concept of ARG-1 inhibition via homomeric disruption, validating this allosteric targeting approach.

Targeting cryptic allosteric sites of G protein-coupled receptors as a novel strategy for biased drug discovery.

G protein-coupled receptors (GPCRs) represent the largest family of membrane receptors and are highly effective targets for therapeutic drugs. GPCRs couple different downstream effectors, including G proteins (such as Gi/o, Gs, G12, and Gq) and β-arrestins (such as β-arrestin 1 and β-arrestin 2) to mediate diverse cellular and physiological responses. Biased signaling allows for the specific activation of certain pathways from the full range of receptors' signaling capabilities. Targeting more variable allosteric sites, which are spatially different from the highly conserved orthosteric sites, represents a novel approach in biased GPCR drug discovery, leading to innovative strategies for targeting GPCRs. Notably, the emergence of cryptic allosteric sites on GPCRs has expanded the repertoire of available drug targets and improved receptor subtype selectivity. Here, we conduct a summary of recent progress in the structural determination of cryptic allosteric sites on GPCRs and elucidate the biased signaling mechanisms induced by allosteric modulators. Additionally, we discuss means to identify cryptic allosteric sites and design biased allosteric modulators based on cryptic allosteric sites through structure-based drug design, which is an advanced pharmacotherapeutic approach for treating GPCR-associated diseases.

Non-Steroidal Anti-Inflammatory Drugs Are Inhibitors of the Intestinal Proton-Coupled Amino Acid Transporter (PAT1): Ibuprofen and Diclofenac Are Non-Translocated Inhibitors.

Background/Objectives: The proton-coupled amino acid transporter (PAT1) is an intestinal absorptive solute carrier responsible for the oral bioavailability of some GABA-mimetic drug substances such as vigabatrin and gaboxadol. In the present work, we investigate if non-steroidal anti-inflammatory drug substances (NSAIDs) interact with substrate transport via human (h)PAT1. Methods: The transport of substrates via hPAT1 was investigated in Caco-2 cells using radiolabeled substrate uptake and in X. laevis oocytes injected with hPAT1 cRNA, measuring induced currents using the two-electrode voltage clamp technique. The molecular interaction between NSAIDs and hPAT1 was investigated using an AlphaFold2 model and molecular docking. Results: NSAIDs such as ibuprofen, diclofenac, and flurbiprofen inhibited proline uptake via hPAT1, with IC50 values of 954 (logIC50 2.98 ± 0.1) µM, 272 (logIC50 2.43 ± 0.1) µM, and 280 (logIC50 2.45 ± 0.1) µM, respectively. Ibuprofen acted as a non-competitive inhibitor of hPAT1-mediated proline transport. In hPAT1-expressing oocytes, ibuprofen and diclofenac did not induce inward currents, and inhibited inward currents caused by proline. Molecular modeling pointed to a binding mode involving an allosteric site. Conclusions: NSAIDs interact with hPAT1 as non-translocated non-competitive inhibitors, and molecular modeling points to a binding mode involving an allosteric site distinct from the substrate binding site. The present findings could be used as a starting point for developing specific hPAT1 inhibitors.

Allosteric inhibition of PTP1B by bromocatechol-chalcone derivatives.

Development of allosteric inhibitors may be a viable strategy to discover hypoglycemic drugs targeting PTP1B. Allosteric inhibitors occupying the BB site that is a hydrophobic pocket restrict the WPD loop in an open conformation, preventing the physiological dephosphorylation reaction. Toward the BB site, sixty bromocatechol-chalcone derivatives were designed and synthesized as allosteric inhibitors of PTP1B against diabetes mellitus. The most potent compound LXQ-87 (C8) inhibited PTP1B noncompetitively with an IC50 value of 1.061±0.202μM. Oral administration of LXQ-87 reduces the fasting blood glucose level and improves glucose tolerance and dyslipidemia in BKS db/db mice suffering from T2DM. LXQ-87 alleviates insulin resistance and promotes cellular glucose uptake by directly binding to intracellular PTP1B.

Design and synthesis of (E)-3-benzylideneindolin-2-one derivatives as potential allosteric inhibitors of Aurora A kinase.

The mitotic kinase Aurora A, a pivotal regulator of the cell cycle, is overexpressed in various cancers and has emerged as one of the most promising targets for anticancer drug discovery. However, the lack of specificity and potential toxicity have impeded clinical trials involving orthosteric inhibitors. In this study, allosteric sites of Aurora A were predicted using the AlloReverse web server. Based on the non-ATP competitive inhibitor Tripolin A and molecular docking information targeting the desired allosteric site 3 of Aurora A, a series of (E)-3-benzylideneindolin-2-one derivatives were designed and synthesized. Compared to Tripolin A, our compounds AK09, AK34 and AK35 have stronger inhibitory effects and can be further investigated as potential allosteric inhibitors. Moreover, the compound AK34 with the strongest inhibitory activity (IC50 = 1.68 μM) has a high affinity for Aurora A (K D = 216 nM). According to the analysis of the structure-activity relationship of the compounds and the results of their molecular docking models, these compounds tend to act on the allosteric site 3 of Aurora A.

Molecular dynamics simulations to decipher the hotspots at the allosteric site of human 5-lipoxygenase.

Human 5-lipoxygenase (LOX) is a non-heme, Fe-containing LOX which catalyses the conversion of arachidonic acid (AA) to leukotriene A4 (LTA4). LTA4 is subsequently converted to cysteinyl-LTs and LTB4 that cause bronchoconstriction and act as chemotactic and chemokinetic agent on human leukocytes, respectively. Leukotrienes play significant roles in inflammation in asthma, cardiovascular diseases, allergic rhinitis, atopic dermatitis, inflammatory bowel disease, rheumatoid arthritis, psoriasis and many more. Thus, in order to suppress LT formation for the management of such diseases, the intrinsic details of the structure of 5-LOX are crucial for the design/development of 5-LOX inhibitors. Here, we deciphered the role of various amino acids at the allosteric site of 5-LOX through molecular dynamics simulations. 3-O-Acetyl-11-keto-beta-boswellic acid (AKBA), a well-recognized allosteric inhibitor of 5-LOX, was used as reference compound. The consequences of amino acid mutations (R101, E108, H130, E134) on AKBA binding have been studied in silico. The changes were characterized at the interaction level. Our observations provide structural insights into crucial residues which are important for stabilizing the ligand at the allosteric site. Principal component analysis (PCA) was applied to the molecular dynamics simulation data to identify the structural fluctuations in the 5-LOX structure. The derived mechanistic details of allosteric 5-LOX inhibition may facilitate the development of novel therapeutics targeting 5-LOX.

Dynamic regulation of ion transport through a bis(1,3-propanediol)-based channel via allosteric azobenzene photoswitching.

The transportation of ions across cell membranes is vital in biological functions and is frequently controlled by external triggers like light, ligands, and voltage. Synthetic ion transport systems, particularly those featuring gating mechanisms, have attracted considerable interest. In this research, we engineered self-assembled barrel rosette ion channels using a photoresponsive azobenzene integrated at an allosteric site. Morphological studies verified more effective self-assembly of the trans form in contrast to the cis form. The restricted self-assembly of the cis form can be ascribed to the nonplanar structure of cis azobenzene moieties, which inhibits favorable π-π stacking interactions. The ion transport studies demonstrated the formation of ion channels by the trans form with anion antiport as the primary transport mechanism. In contrast, the cis form exhibited lower efficiency. Based on these observations, dynamically gated ion transport was achieved by employing two sets of electromagnetic radiation at 365 nm and 450 nm, respectively. Molecular dynamics simulation studies demonstrated that the channel formed by assembling trans monomers exhibited greater stability when compared to the channel formed by cis monomers. Additionally, the trans channel was found to recognize and transport chloride effectively.

Targeting N-Myc in neuroblastoma with selective Aurora kinase A degraders.

The N-Myc transcription factor, encoded by MYCN, is a mechanistically validated, yet challenging, target for neuroblastoma (NB) therapy development. In normal neuronal progenitors, N-Myc undergoes rapid degradation, while, in MYCN-amplified NB cells, Aurora kinase A (Aurora-A) binds to and stabilizes N-Myc, resulting in elevated protein levels. Here, we demonstrate that targeted protein degradation of Aurora-A decreases N-Myc levels. A potent Aurora-A degrader, HLB-0532259 (compound 4), was developed from an Aurora-A-binding ligand that engages the Aurora-A/N-Myc complex. HLB-0532259 promotes the degradation of Aurora-A, which elicits concomitant N-Myc degradation, with nanomolar potency and excellent selectivity. HLB-0532259 surpasses the cellular efficacy of established allosteric Aurora-A inhibitors, exhibits favorable pharmacokinetic properties, and elicits tumor reduction in a murine xenograft NB model. This study broadly delineates a strategy for targeting "undruggable" proteins that are reliant on accessory proteins for cellular stabilization.

ACVR1 mediates renal tubular EMT in kidney fibrosis via AKT activation.

Tubulointerstitial fibrosis in the kidneys is a chronic and progressive process. Although studies suggested that tubular epithelial-mesenchymal transition (EMT) plays a key role in the development of kidney fibrosis, whether ACVR1, a member of the TGFβ superfamily, is involved in the EMT needs to be illustrated. Using bioinformatics analysis of bulk-seq data (GSE23338 and GSE168876), we found that TGF-β1 perhaps activated the PI3K/AKT signaling pathway and induced the mRNA expression of ACVR1, fibronectin, and Collagen I in HK-2 cells (human renal tubular epithelial cell line). Furthermore, qPCR and western blotting results confirmed the high expressions of ACVR1 and EMT markers in TGFβ-induced HK-2 cells. Similar results were also found in the UUO mouse model. Besides, different time-point immunofluorescent staining indicated a positive correlation between the expression of the ACVR1 and EMT marker vimentin in TGF-β1-induced HK-2 cells. Consequently, knockdown ACVR1 effectively inhibited the expression of TGF-β1-induced EMT markers and AKT phosphorylation in HK-2 cells. Moreover, treatment of HK-2 cells with MK2206 (an allosteric inhibitor of AKT) decreased the activation of AKT and the expression of α-SMA while treatment of cells with SC79 (a AKT activator) enhanced the expression of α-SMA. These findings suggest that ACVR1 regulated the EMT of renal tubular epithelial cells through activation of the AKT signaling pathway and that ACVR1 could be considered novel therapeutic targets for renal fibrosis.

Virtual screening and binding mechanism of allosteric inhibitors based on SHP2E76A

Virtual screening and binding mechanism of allosteric inhibitors based on SHP2E76A

Glutamine metabolism is essential for coronavirus replication in host cells and in mice.

Understanding the fundamental biochemical and metabolic requirements for the replication of coronaviruses within infected cells is of notable interest for the development of broad-based therapeutic strategies, given the likelihood of the emergence of new pandemic-potential virus species, as well as future variants of SARS-CoV-2. Here we demonstrate members of the glutaminase family of enzymes (GLS and GLS2), which catalyze the hydrolysis of glutamine to glutamate (i.e., the first step in glutamine metabolism), play key roles in coronavirus replication in host cells. Using a range of human seasonal and zoonotic coronaviruses, we show three examples where GLS expression increases during coronavirus infection of host cells, and another where GLS2 is upregulated. The viruses hijack the metabolic machinery responsible for glutamine metabolism to generate the building blocks for biosynthetic processes and satisfy the bioenergetic requirements demanded by the "glutamine addiction" of virus-infected cells. We demonstrate that genetic silencing of glutaminase enzymes reduces coronavirus infection and that newer members of two classes of allosteric inhibitors targeting these enzymes, designated as SU1, a pan-GLS/GLS2 inhibitor, and UP4, a specific GLS inhibitor, block viral replication in epithelial cells. Moreover, treatment of SARS-CoV-2 infected K18-human ACE2 transgenic mice with SU1 resulted in their complete survival compared to untreated control animals, which succumbed within 10days post-infection. Overall, these findings highlight the importance of glutamine metabolism for coronavirus replication in human cells and mice and show that glutaminase inhibitors can block coronavirus infection and thereby may represent a novel class of broad-based anti-viral drug candidates.

Deciphering Potent Protein Tyrosine Phosphatase‐1B Inhibitors Through In silico Molecular Docking, MMGBSA, and Molecular Dynamics

AbstractAlzheimer's disease (AD) stands as the most prevalent progressive neurodegenerative disorder, marked by cognitive impairment, memory loss, and cognitive deficits. In the present study, the targeted protein tyrosine phosphatase 1B (PTP1B), a crucial enzyme involved in various signaling pathways, is associated with AD pathogenesis. Our study employs the virtual screening of the compounds from an extensive database. Based on the docking score, the molecular dynamics simulation has been performed for top three compounds, donepezil and ertiprotafib for the comparative study of the molecules. As a result, the molecules CNP0377119, CNP0390654, CNP0377195, donepezil, and ertiprotafib were identified with the favorable docking scores. Further analyses, including PRIME MMGB‐SA and ADMET properties, focused on standard amino acids like Asp193 and Glu276. In the 100 ns molecular simulation trajectory, the three compounds exhibited stability through effective binding properties with ligands. These interactions included favorable binding conformations and hydrogen bond formations with Asn193, Glu276, Lys197, Glu200, and Tyr46. The π‐π stacking interactions were also observed with amino acids Phe196 and Phe280. The allosteric inhibitor of PTP1B directly attaches to the WPD loop when the cysteine‐phosphate intermediate is in its open conformation hindering the closure of the WPD loop, thereby inhibiting the PTP1B activity.

Trametinib and M17, a novel small molecule inhibitor of AKT, display a synergistic antitumor effect in triple negative breast cancer cells through the AKT/mTOR and MEK/ERK pathways.

Triple negative breast cancer (TNBC) is associated with a poor prognosis and limited response to traditional chemotherapy, necessitating the exploration of novel treatment approaches. Recent researches have highlighted the interconnected roles of the PI3K/AKT pathway and MAPK pathway in TNBC cells, contributing to the efficacy of treatments. Therefore, the concurrent inhibition of both pathways presents a potential new therapeutic strategy for TNBC patients. This study aimed to evaluate the antitumor efficacy of M17, an AKT allosteric inhibitor and a new synthesized shikonin derivative, both alone and in combination with the MEK inhibitor trametinib. We applied various cellular assays and a subcutaneous 4T1 tumor bearing BALB/c mice model were utilized to assess the in vitro and in vivo antitumor effects. Computational docking and Bio-Layer Interferometry (BLI) were employed to investigate the binding of M17 with AKT. Additionally, flow cytometry, transwell assays, western blotting, and tumor xenograft assays were conducted to explore the potential synergistic mechanisms of the combined therapy. The results demonstrated that M17 exhibited moderate antitumor activity against TNBC cells, but significantly enhanced the apoptotic effects and inhibited proliferation and migration when combined with trametinib. Furthermore, the combination of M17 and trametinib showed even more pronounced antitumor activity in vivo. Mechanistically, the dual therapy synergistically suppressed TNBC by targeting the AKT/mTOR and MEK/ERK signaling pathways and inhibiting epithelial-mesenchymal transition. In conclusion, the findings suggested that the combination of M17 and trametinib holds promise as a synergistic treatment option for TNBC patients.

Lycorine esters exert anti-HCoV-OC43 effect through reversibly acylating cysteine residue in the nsp 12 NiRAN domain.

By introducing ester warheads into the hydroxyl groups in lycorine (1), three types of lycorine mono-ester or di-ester analogues were synthesized and evaluated for their antiviral activities against HCoV-OC43. Most of them showed higher selective indexes (SI) than 1, up to nearly 14 times. Using compound 6b as a probe, we firstly demonstrated that lycorine esters directly targeted nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain in the non-structural protein 12 (nsp 12) by reversibly acylating Cys12 to induce the shrink of NiRAN pocket and block the viral replication, different from the known RdRp inhibitors. Meanwhile, the reversible acylation mode of lycorine esters guaranteed the higher SI values and long-acting effects of its kind. Thus, in addition to acting as prodrugs, ester compounds with a highly acylating warhead can be used as covalent probes to explore the detailed mode of action and improve the safety window. Compound 6b has been identified as a new reversible covalent RdRp allosteric inhibitor for further investigation.

The activity regulation of lipase from Aspergillus fumigatus by ligand through allosteric exploration.

Lipase activity from Aspergillus fumigatus (AFL) were modulated by an effector (HMD) that was discovered for an allosteric site on the bioactive macromolecule. Experimental evaluation and computational modeling of allosteric effects revealed alterations in the structure of AFL. It was found that AFL's activity in HMD solution increased by approximately 46 % due to mainly enhanced lid mobility. HMD-AFL interaction was driven by enthalpy and entropy. However, when AFL was coupled to HMD-modified microspheres (PS-HMD-p), its hydrolysis activity decreased by ~14.3 % due to reduced lid mobility. After immobilization, AFL's ester-synthesis activity also decreased, due to changes in the conformational dynamics and the geometric characteristics of active site. Investigating the structural dynamics of allosteric regulation of the lipase not only reveals its structural changes underlying the functional variation but also enhances the understanding of the allosteric property that is underappreciated in exoenzymes.

Allosteric site engagement and cooperativity mechanism by PHI1 for BRAFV600E kinase inhibition

Allosteric site engagement and cooperativity mechanism by PHI1 for BRAFV600E kinase inhibition