Inhibition Of Binding Research Articles

Amivantamab efficacy in wild-type EGFR NSCLC tumors correlates with levels of ligand expression

Amivantamab is an FDA-approved bispecific antibody targeting EGF and Met receptors, with clinical activity against EGFR mutant non-small cell lung cancer (NSCLC). Amivantamab efficacy has been demonstrated to be linked to three mechanisms of action (MOA): immune cell-mediated killing, receptor internalization and degradation, and inhibition of ligand binding to both EGFR and Met receptors. Among the EGFR ligands, we demonstrated that amphiregulin (AREG) is highly expressed in wild-type (WT) EGFR (EGFRWT) NSCLC primary tumors, with significantly higher circulating protein levels in NSCLC patients than in healthy volunteers. Treatment of AREG-stimulated EGFRWT cells/tumors with amivantamab or with an AREG-targeting antibody inhibited ligand-induced signaling and cell/tumor proliferation/growth. Across 11 EGFRWT NSCLC patient-derived xenograft models, amivantamab efficacy correlated with AREG RNA levels. Interestingly, in these models, amivantamab anti-tumor activity was independent of Fc engagement with immune cells, suggesting that, in this context, the ligand-blocking function is sufficient for amivantamab maximal efficacy. Finally, we demonstrated that in lung adenocarcinoma patients, high expression of AREG and EGFR mutations were mutually exclusive. In conclusion, these data 1) highlight EGFR ligand AREG as a driver of tumor growth in some EGFRWT NSCLC models, 2) illustrate the preclinical efficacy of amivantamab in ligand-driven EGFRWT NSCLC, and 3) identify AREG as a potential predictive biomarker for amivantamab activity in EGFRWT NSCLC.

Toward Unveiling Putative Binding Sites of Interleukin-33: Insights from Mixed-Solvent Molecular Dynamics Simulations of the Interleukin-1 Family.

The interleukin (IL)-1 family is a major proinflammatory cytokine family, ranging from the well-studied IL-1s to the most recently discovered IL-33. As a new focus, IL-33 has attracted extensive research for its crucial immunoregulatory roles, leading to the development of notable monoclonal antibodies as clinical candidates. Efforts to develop small molecules disrupting IL-33/ST2 interaction remain highly desired but encounter challenges due to the shallow and featureless interfaces. The information from relative cytokines has shown that traditional binding site identification methods still struggle in mapping cryptic sites, necessitating dynamic approaches to uncover druggable pockets on IL-33. Here, we employed mixed-solvent molecular dynamics (MixMD) simulations with diverse-property probes to map the hotspots of IL-33 and identify potential binding sites. The protocol was first validated using the known binding sites of two IL-1 family members and then applied to the structure of IL-33. Our simulations revealed several binding sites and proposed side-chain rearrangements essential for the binding of a known inhibitor, aligning well with experimental NMR findings. Further microsecond-time scale simulations of this IL-33-protein complex unveiled distinct binding modes with varying occurrences. These results could facilitate future efforts in developing ligands to target challenging flexible pockets of IL-33 and IL-1 family cytokines in general.

Structure and Inhibition of Insect UDP-N-acetylglucosamine Pyrophosphorylase: A Key Enzyme in the Hexosamine Biosynthesis Pathway.

UDP-N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the last step in the hexosamine biosynthesis pathway to directly produce UDP-N-acetylglucosamine (UDP-GlcNAc). Because UAPs play important physiological and pathological roles in organisms, they are considered potential targets for drug and pesticide development. However, the lack of efficient and selective inhibitors is a bottleneck that must be overcome. This study reports the first crystal structure of the insect UAP from Spodoptera frugiperda (SfUAP) in complex with UDP-GlcNAc. SfUAP has two insect-specific structural characteristics in the active pocket, namely, a free Cys (Cys334) and a Mg2+ binding site, which differentiate it from human UAP (HsAGX1) and fungal UAP (AfUAP) in terms of substrate and inhibitor binding. N-(4-Nitrophenyl)maleimide (pNPMI) and myricetin are discovered as potent covalent and noncovalent inhibitors of SfUAP, respectively. Moreover, myricetin can significantly reduce the level of cellular O-GlcNAcylation by inhibiting both UAP and O-GlcNAc transferase. These findings provide novel insights into the development of UAP-based drugs and pesticides.

Inhibitory effect of human indoleamine 2,3-dioxygenase 1 (hIDO1) by kazinols of 1,3-diphenylpropane derivatives

This study focused on identifying and characterizing 1,3-diphenylpropane derivatives from flavonoids that inhibit human indoleamine 2,3-dioxygenase 1 (hIDO1) enzymes, which play a role in immune regulation and are associated with various diseases. A series of isolated metabolites (1–7) demonstrated modest to high inhibition of hIDO1, with binding degree values ranging from 26.31 to 72.17%. In particular, during a target-based screening of natural products using hIDO1, kazinol J (6, a 1,3-diphenylpropane derivative) was found to potently inhibit hIDO1, with a binding degree of 72.17% at 1 ppm. Kazinol J (6) showed concentration-dependent and mixed inhibition kinetics and achieved slow and time-dependent inhibition of hIDO1. Additionally, docking simulations were performed to evaluate the inhibitory potential and binding interactions of the compounds with hIDO1. These findings suggest that these 1,3-diphenylpropane derivatives can serve as therapeutic agents for conditions involving hIDO1 dysregulation, such as cancer, autoimmune disorders, and infectious diseases.

Effects of a Paleolithic diet compared to a diabetes diet on leptin binding inhibition in secondary analysis of a randomised cross-over study

BackgroundBeneficial effects from practising a Paleolithic diet as compared to a diabetes diet on weight, waist circumference, satiety, leptin, HbA1c and glucose control in randomised controlled trial participants with type 2 diabetes could be due to lower leptin resistance. Support for this hypothesis comes from an in vitro experiment that showed that digested wheat gluten, which is excluded from a Paleolithic diet, inhibits leptin from binding to its receptor, thus indicating a possible dietary cause of leptin resistance. However, the clinical relevance of the latter finding is unclear since removal of enzyme activity from the gluten digest by heat treatment also abolished leptin binding inhibition. Assessment of leptin binding inhibition in vivo is possible by comparison of total leptin levels with those of ‘biologically active’ leptin bound to its receptor (bioLep).ObjectivesTo assess the effects of a Paleolithic diet compared to a diabetes diet on leptin binding inhibition and to replicate our in vitro study.MethodsBioLep and total leptin levels were measured in secondary analysis of fasting plasma samples from our open label random order three plus three-month long cross-over trial performed in 2005–2007, that compared a Paleolithic diet with a diabetes diet in participants with type 2 diabetes without insulin treatment (per protocol). BioLep was also measured in vitro for known recombinant leptin concentrations incubated with a series of concentrations of 10 kDa spin-filtered digested wheat gluten, with or without prior heat treatment, at 100ºC for 30 min and centrifugation.ResultsThere was no difference between diets when comparing differences between bioLep and total leptin levels and their ratio in the 13 participants, three women and 10 men, aged 52–74 years with a mean BMI of 30 kg/m2 and a mean diabetes duration of eight years. We found no carry-over or period effect for bioLep and total leptin. In vitro, wheat gluten digest inhibited leptin binding in a dose-dependent manner but not after heat treatment.ConclusionsWe found no leptin binding inhibition after the Paleolithic or diabetes diet, possibly due to its abolishment from cooking-related heat treatment of wheat gluten.Trial registrationRegistered on 14/02/2007 at ClinicalTrials.gov Identifier: NCT00435240.

Ligand-induced conformational changes in protein molecules detected by sum-frequency generation

We present the first demonstration of ligand-induced conformational changes in a biological molecule, a protein, by sum-frequency generation (SFG). Constructs of KRasG12D protein were prepared by selectively deuterating residues of a single amino acid type using isotope-labeled amino acids and cell-free protein synthesis. By attaching labeled protein to a supported bilayer membrane via a His-tag to Ni-NTA-bearing lipids, we ensured that single layers of ordered molecules were formed while preserving the protein’s native structure. Exceptionally large SFG amide I signals were produced in both labeled and unlabeled proteins, demonstrating a high degree of orientational order upon attachment to the bilayer. Deuterated protein also produced SFG signals in the CDx spectral region, which were not present in the unlabeled protein. The CDx signals were measured before and after binding a peptide inhibitor, KRpep-2d, revealing shifts in SFG intensity due to conformational changes at the labeled sites. In particular, peaks associated with CDx stretching vibrations for alanine, valine, and glycine changed substantially in amplitude upon inhibitor binding. By inspection of the crystal structure, these three residues are uniquely colocated on the protein surface in and near the nucleotide binding site, which is in allosteric communication with the site of peptide inhibitor binding, suggesting an approach to identify a ligand’s binding site. The technique offers a highly sensitive, nonperturbative method of mapping ligand-induced conformational changes and allosteric networks in biological molecules for studies of the relationship between structure and function and mechanisms of action in drug discovery.

B cell targeting in IgA nephropathy.

The "multi-hit theory/4-hit theory" pathogenesis hypothesis is widely accepted and IgA nephropathy (IgAN) is understood to be a disease originating from Hit 1, galactose deficient IgA1 (GdIgA1). The chronic repetitive activation of the complement pathway (alternative and lectin pathways) and the subsequent inflammation results in progressive glomerular damage that spills over into increased intraglomerular pressure and other hemodynamic changes, increased urinary protein, glomerulosclerosis, and tubulointerstitial fibrosis. The basic pathophysiology of this disease is the progression of a mixture of such acute and chronic pathologies. Currently, a number of new drugs has emerged as promising agents, such as complement regulators, endothelin receptor antagonists, and SGLT2 inhibitors, which are associated with each pathological step after glomerular deposition of GdIgA1/immune complexes. On the other hand, the molecular mechanisms of GdIgA1 production are gradually being elucidated, and the development of several novel therapeutic agents targeting the responsible B cells and their international clinical trials are progressing. These agents that inhibit or control the production of the Hit1, GdIgA1, are highly expected as essential therapies for this disease. The large body of clinical and basic research findings to date strongly suggest that nephritogenic GdIgA1 is a polymeric IgA1 of mucosal origin. In addition, the B cells involved in its nephritogenic GdIgA1 production are mainly differentiated mature B cells such as plasma cells, which may migrate to the bone marrow as well as the mucosa. The innate immune system in the mucosa, especially Toll-like receptors (TLRs), is thought to be involved in their production. Among TLRs, TLT9 and TLR7, which recognize bacterial and viral unmethylated DNA and RNA, have been reported to be involved. The mucosal activation of these TLRs is associated with the production of APRIL (A Proliferation Inducing Ligand) and BAFF (B cell activating factor), which are TNF superfamily cytokines involved in B cell maturation, survival, and IgA class switching, and may also be involved in the production of nephritogenic GdIgA1. It is still inconclusive whether APRIL or BAFF is more closely involved in the production of nephritogenic GdIgA1. Phenotypes in transgenic animal models suggest BAFF involvement, however, a genome wide association study (GWAS) analysis of human IgAN has identified APRIL, not BAFF, as a candidate gene. Based on the above background, several international clinical trials are underway for drugs such as TLR regulators (hydroxychloroquine), anti-APRIL drugs, anti-BAFF drugs, APRIL/BAFF receptor (TACI) binding inhibitors, and cytoreductive drugs (proteasome inhibitors, anti-CD38 antibodies) to inhibit nephritogenic GdIgA1 production in responsible B cells. This session will provide an overview of the responsible B cells, their GdIgA1 production mechanism, and ongoing drugs.

Structural visualization of inhibitor binding in prolyl oligopeptidase.

The association and dissociation of proteins and ligands are crucial in biophysics for potential drug development [Baron and McCammon, Annu. Rev. Phys. Chem. 64, 151-175 (2013)]. However, identifying and characterizing the reaction pathways for these rare events has been a long-standing challenge. Molecular dynamics (MD) simulations are limited in exploring biophysical processes on experimental timescales, so ligand transport processes through complex transient tunnels formed by proteins during dynamics are often simulated using enhanced sampling MD [Rydzewski and Nowak, Phys. Life Rev. 22-23, 58-74 (2017)]. Erroneously identified ligand binding pathways can affect thermodynamic and kinetic characteristics calculated from MD trajectories. A system that has the potential to be a therapeutic target for neurodegenerative diseases is prolyl oligopeptidase (PREP). This is due to its involvement in promoting protein aggregation and disrupting cellular function through affecting protein-protein interactions (PPI). The recent discovery of a new type of PREP inhibitor that targets PPI raises important questions about the diversity of ligand binding pathways in PREP and their impact on protein dynamics [Pätsi et al., J. Med. Chem. 67, 5421-5436 (2024); Kilpeläinen et al., J. Med. Chem. 66, 7475-7496 (2023); and Walczewska-Szewc et al., Phys. Chem. Chem. Phys. 24, 4366-4373 (2022)]. In this article, using results from enhanced sampling MD, we visually present how the binding process in PREP depends on subtle changes in inhibitors, which could be crucial in treating neurodegenerative disorders.

Molecular basis of bacterial lectin recognition of eukaryotic glycans: The case of Mycoplasma pneumoniae and Mycoplasma genitalium cytoadhesins

Mycoplasma pneumoniae and Mycoplasma genitalium are two emerging bacterial pathogens that colonize the human respiratory and urogenital epithelia, respectively. Both pathogens express cell surface cytoadhesins that play a crucial role in the interaction with the host, mediating the attachment to sialylated glycan receptors and triggering infection. The design of competitive binding inhibitors of Mycoplasma cytoadhesins has potential to disrupt these interactions and lessen bacterial pathogenesis. To this end, we report here molecular insights into the adhesion mechanisms of M. pneumoniae and M. genitalium, which are largely mediated by sialylated glycans on the host cell surface. In detail, a combination of Nuclear Magnetic Resonance (NMR) spectroscopy, fluorescence analysis and computational studies allowed us to explore the recognition by the cytoadhesins P40/P90 in M. pneumoniae and P110 in M. genitalium of sialylated N- and O-glycans. We reveal that, unlike other bacterial adhesins, which are characterized by a wide binding pocket, Mycoplasma cytoadhesins principally accommodate the sialic acid residue, in a similar manner to mammalian Siglecs. These findings represent crucial insight into the future development of novel compounds to counteract Mycoplasma infections by inhibiting bacterial adherence to host tissues.

The Inhibiting Effect of GB-2, (+)-Catechin, Theaflavin, and Theaflavin 3-Gallate on Interaction between ACE2 and SARS-CoV-2 EG.5.1 and HV.1 Variants.

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, continues to pose significant global health challenges. The results demonstrated that GB-2 at 200 μg/mL effectively increased the population of 293T-ACE2 cells with low RBD binding for both SARS-CoV-2 Omicron EG.5.1 and HV.1 variants by dual-color flow cytometry, indicating its ability to inhibit virus attachment. Further investigation revealed that (+)-catechin at 25 and 50 μg/mL did not significantly alter the ACE2-RBD interaction for the EG.5.1 variant. In contrast, theaflavin showed inhibitory effects at both 25 and 50 μg/mL for EG.5.1, while only the higher concentration was effective for HV.1. Notably, theaflavin 3-gallate exhibited a potent inhibition of ACE2-RBD binding for both variants at both concentrations tested. Molecular docking studies provided insight into the binding mechanisms of theaflavin and theaflavin 3-gallate with the RBD of EG.5.1 and HV.1 variants. Both compounds showed favorable docking scores, with theaflavin 3-gallate demonstrating slightly lower scores (-8 kcal/mol) compared to theaflavin (-7 kcal/mol) for both variants. These results suggest stable interactions between the compounds and key residues in the RBD, potentially explaining their inhibitory effects on virus attachment. In conclusion, GB-2, theaflavin, and theaflavin 3-gallate demonstrate significant potential as inhibitors of the ACE2-RBD interaction in Omicron variants, highlighting their therapeutic promise against COVID-19. However, these findings are primarily based on computational and in vitro studies, necessitating further in vivo research and clinical trials to confirm their efficacy and safety in humans.

Structural changes of wheat starch and activity inhibition of α-glucosidase by persimmon (Diospyros kaki Thunb.) leaves extract retarding starch digestibility

Elevated blood sugar levels caused by starch digestion was a target for controlling diabetes mellitus. The in vitro and in vivo digestibility of wheat starch was evaluated to find that adding 15 % persimmon leaf extract (PLE) to starch reduced its digestibility by 69.50 % and the peak postprandial blood glucose by 23.63 %. Subsequently, we observed under scanning electron microscopy and atomic force microscopy that the presence of PLE led to the destruction of starch structure and the aggregation of α-glucosidase so as to decrease starch digestion and hinder the binding of starch to α-glucosidase. Through multi-spectral analysis, PLE hindered the clathrate of iodine and starch, and also increased the crystallinity of starch by 48.58 %. For α-glucosidase inhibitory activity (IC50 = 72.49 μg/mL), PLE preferentially occupied the active center of α-glucosidase, changed its fluorescence characteristics and secondary structure through hydrogen bonding and hydrophobic interaction. Moreover, among the 23 potential α-glucosidase inhibitors screened from PLE, combined with molecular simulation, Procyanidin B2 had the strongest inhibitory effect (IC50 = 33.22 μg/mL) and binding energy (−7.09 kcal/mol), which was most effectively inhibitory on digestion. These results indicated the potential of PLE in hypoglycemia targeting both starch and α-glucosidase.

The transcription regulator ID3 maintains tumor-specific memory CD8+ T cells in draining lymph nodes during tumorigenesis

During tumorigenesis, the recently identified tumor-specific memory Tcells in draining lymph nodes (TdLN-TTSM cells) play a pivotal role in tumor repression that gives rise to progenitor exhausted T (TPEX) cells and further replenishes tumor-specific CD8+ Tcells residing in the tumor microenvironment (TME). However, how TTSM cells are maintained in TdLN is largely unknown. Here, we show that the transcription regulator ID3 (inhibitor of DNA binding 3) is highly expressed by TTSM cells compared with other CD8+ Tcell subsets. The deficiency of ID3 significantly interrupts the maintenance of TTSM and TPEX cells, resulting in decreased tumor-infiltrating CD8+ Tcells and impaired tumor control. Consistent with this, overexpression of ID3 in CD8+ Tcells increases the TTSM cell population and enhances the anti-tumor immune response.

Direct inhibition of tumor hypoxia response with synthetic transcriptional repressors.

Many oncogenic transcription factors (TFs) are considered to be undruggable because of their reliance on large protein-protein and protein-DNA interfaces. TFs such as hypoxia-inducible factors (HIFs) and X-box-binding protein 1 (XBP1) are induced by hypoxia and other stressors in solid tumors and bind to unfolded protein response element (UPRE) and hypoxia-induced response element (HRE) motifs to control oncogenic gene programs. Here, we report a strategy to create synthetic transcriptional repressors (STRs) that mimic the basic leucine zipper domain of XBP1 and recognize UPRE and HRE motifs. A lead molecule, STR22, binds UPRE and HRE DNA sequences with high fidelity and competes with both TFs in cells. Under hypoxia, STR22 globally suppresses HIF1α binding to HRE-containing promoters and enhancers, inhibits hypoxia-induced gene expression and blocks protumorigenic phenotypes in triple-negative breast cancer (TNBC) cells. In vivo, intratumoral and systemic STR22 treatment inhibited hypoxia-dependent gene expression, primary tumor growth and metastasis of TNBC tumors. These data validate a novel strategy to target the tumor hypoxia response through coordinated inhibition of TF-DNA binding.

2-amino-6-ethoxy-4-arylpyridine-3,5-dicarbonitrile Scaffolds as potential acetylcholinesterase and butyrylcholinesterase inhibitors

Inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes are of prime importance for treating Alzheimerʼs disease (AD). Apart from many organic scaffolds, pyridine-based compounds have previously been reported as potential α-glucosidase inhibitors. The current study reports a series of pyridine-based synthetic molecules for their acetylcholinesterase AChE and butyrylcholinesterase BChE inhibitory potential assessed via in vitro, kinetics, and in silico studies. For this purpose, 2-amino-6-ethoxy-4-arylpyridine-3,5-dicarbonitrile analogs 1–23 were synthesized by using a two-step reaction scheme. In the first step, different aryl aldehydes were treated with malononitrile to afford the 2-benzylidenemalononitrile in the presence of catalyst ammonium acetate. In the next step, 2-benzylidenemalononitrile intermediates were reacted again with malononitrile, catalyzed by potassium hydroxide, to synthesize a range of functionalized pyridine scaffolds in good yields. Compounds were subjected to in vitro screening against AChE and BChE enzymes. Several derivatives, including 2, 3, 11, 14–16, 19, 20, and 22, showed many folds of increased inhibitory potential and binding affinity in the ranges of Ki = 1.62 ± 0.13 nM to 15.84 ± 0.10 nM for AChE and Ki = 1.35 ± 0.31 nM to 13.52 ± 0.61 nM for BChE, as compared to the standard tacrine Ki = 53.31 ± 11.32 nM for AChE, and Ki = 58.16 ± 7.24 nM for BChE. Further, molecular docking studies deduced key interactions between the ligands (compounds) and the active pocket of enzymes. The current research study has identified several potential AChE and BChE inhibitors that may serve as lead candidates after targeted advanced research for exploring anti-Alzheimer agents.

Exploring the correlation between cardiovascular adverse events and antidepressant use: A retrospective pharmacovigilance analysis based on the FDA Adverse Event Reporting System database

BackgroundThe high comorbidity and mutually reinforcing relation between depression and cardiovascular disease have raised concerns about the cardiovascular risk of antidepressants. To gain a better understanding of this correlation, we performed a comprehensive evaluation regarding the types and degrees of cardiovascular adverse events (AEs) associated with 37 commonly prescribed antidepressants. MethodsAE reports from January 2004 to December 2023 were retrieved from the FDA Adverse Event Reporting System (FAERS) database. Disproportionality analysis was performed to identify antidepressant-related cardiovascular signals using the reporting odds ratio, proportional reporting ratio, and information component. Influencing factors of cardiovascular death, including age, sex, antidepressant choice, and concomitant medication, were explored. The underlying mechanisms of antidepressant-associated cardiovascular risk related to neurotransmitter transporters/receptors were further explored. ResultsThe use of antidepressants was associated with eight categories of Standardized MedDRA Queries of cardiovascular events. Different antidepressants exerted varying types and degrees of cardiovascular risks along with contributions to death in reports with cardiovascular AEs. Among them, monoamine oxidase inhibitors had the highest risk of developing six cardiovascular event categories: torsades de pointes (TdP)/QT prolongation, hypertension, cardiac arrhythmias, cardiomyopathy, pulmonary hypertension, and ischaemic heart disease. Age, male and the use of 24 types of antidepressants and concomitant medications were positively correlated with death in cardiovascular AEs. The highest risk associated with antidepressants was found in amoxapine (OR = 5.00 [2.13, 11.75], P < 0.001), followed by moclobemide (OR = 3.66 [1.85, 7.24], P < 0.001). Correlation analysis indicated the occurrence of antidepressant-related TdP/QT prolongation, hypertension and cardiomyopathy was associated with the binding and uptake inhibition of dopamine and norepinephrine transporters as well as their selectivity over serotonin transporters. ConclusionThe retrospective analysis revealed that cardiovascular AEs were connected with antidepressant use, and the binding/uptake inhibitory potency and selectivity of neurotransmitters of antidepressants played an important role, providing a preliminary basis for further in-depth study of antidepressant-related cardiovascular toxicity. However, as an exploratory study, prospective studies are needed to validate our findings in the future.

Mutational analysis reveals the importance of residues of the access tunnel inhibitor site to human P-glycoprotein (ABCB1)-mediated transport.

Human P-glycoprotein (P-gp) utilizes energy from ATP hydrolysis for the efflux of chemically dissimilar amphipathic small molecules and plays an important role in the development of resistance to chemotherapeutic agents in most cancers. Efforts to overcome drug resistance have focused on inhibiting P-gp-mediated drug efflux. Understanding the features distinguishing P-gp inhibitors from substrates is critical. Cryo-electron microscopy has revealed distinct binding patterns, emphasizing the role of the L-site or access tunnel in inhibition. We substituted 5-9 residues of the L-site with alanine to investigate whether the binding of a second inhibitor molecule to the L-site is required for inhibiting drug efflux. We reveal, for the first time, that mutations in the L-site affect the drug efflux activity of P-gp, despite their distance from the substrate-binding pocket (SBP). Surprisingly, after the mutations were introduced, inhibitors such as tariquidar and zosuquidar still inhibited drug efflux by mutant P-gps. Communication between the transmembrane helices (TMHs) and nucleotide-binding domains (NBDs) was evaluated using the ATPase assay, revealing distinct modulation patterns by inhibitors for the mutants, with zosuquidar exhibiting substrate-like stimulation of ATPase. Furthermore, L-site mutations abolished ATP-dependent thermal stabilization. In silico molecular docking studies corroborated the altered inhibitor binding due to mutations in the L-site residues, shedding light on their critical role in substrate transport and inhibitor interactions with P-gp. These findings suggest that inhibitors bind either to the SBP alone, and/or to alternate site(s) when the L-site is disabled by mutagenesis.

The Role of Water Networks in Phosphodiesterase Inhibitor Dissociation and Kinetic Selectivity.

In search of new opportunities to develop Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) inhibitors that have selectivity over the off-target human PDE4 (hPDE4), different stages of a fragment-growing campaign were studied using a variety of biochemical, structural, thermodynamic, and kinetic binding assays. Remarkable differences in binding kinetics were identified and this kinetic selectivity was explored with computational methods, including molecular dynamics and interaction fingerprint analyses. These studies indicate that a key hydrogen bond between GlnQ.50 and the inhibitors is exposed to a water channel in TbrPDEB1, leading to fast unbinding. This water channel is not present in hPDE4, leading to inhibitors with a longer residence time. The computer-aided drug design protocols were applied to a recently disclosed TbrPDEB1 inhibitor with a different scaffold and our results confirm that shielding this key hydrogen bond through disruption of the water channel represents a viable design strategy to develop more selective inhibitors of TbrPDEB1. Our work shows how computational protocols can be used to understand the contribution of solvent dynamics to inhibitor binding, and our results can be applied in the design of selective inhibitors for homologous PDEs found in related parasites.

Structural and Biochemical Insights into the Mechanism of Action of the Clinical USP1 Inhibitor, KSQ-4279.

DNA damage triggers cell signaling cascades that mediate repair. This signaling is frequently dysregulated in cancers. The proteins that mediate this signaling are potential targets for therapeutic intervention. Ubiquitin-specific protease 1 (USP1) is one such target, with small-molecule inhibitors already in clinical trials. Here, we use biochemical assays and cryo-electron microscopy (cryo-EM) to study the clinical USP1 inhibitor, KSQ-4279 (RO7623066), and compare this to the well-established tool compound, ML323. We find that KSQ-4279 binds to the same cryptic site of USP1 as ML323 but disrupts the protein structure in subtly different ways. Inhibitor binding drives a substantial increase in thermal stability of USP1, which may be mediated through the inhibitors filling a hydrophobic tunnel-like pocket in USP1. Our results contribute to the understanding of the mechanism of action of USP1 inhibitors at the molecular level.

Quinone and inhibitor binding in Escherichia coli complex I

Quinone and inhibitor binding in Escherichia coli complex I

Identification of the Inhibitor Binding Site of the Human Mitochondrial Pyruvate Carrier

Identification of the Inhibitor Binding Site of the Human Mitochondrial Pyruvate Carrier