Evaluation of phytoconstituents of Tinospora cordifolia against K417N and N501Y mutant spike glycoprotein and main protease of SARS-CoV-2- an in silico study.

Coronavirus disease 2019 (COVID-19) caused appalling conditions over the globe, which is currently faced by the entire human population. One of the primary reasons behind the uncontrollable situation is the lack of specific therapeutics. In such conditions, drug repurposing of available drugs (viz. Chloroquine, Lopinavir, etc.) has been proposed, but various clinical and preclinical investigations indicated the toxicity and adverse side effects of these drugs. This study explores the inhibition potency of phytochemicals from Tinospora cordifolia (Giloy) against SARS CoV-2 drugable targets (spike glycoprotein and Mpro proteins) using molecular docking and MD simulation studies. ADMET, virtual screening, MD simulation, postsimulation analysis (RMSD, RMSF, Rg, SASA, PCA, FES) and MM-PBSA calculations were carried out to predict the inhibition efficacy of the phytochemicals against SARS CoV-2 targets. Tinospora compounds showed better binding affinity than the corresponding reference. Their binding affinity ranges from -9.63 to -5.68 kcal/mole with spike protein and -10.27 to -7.25 kcal/mole with main protease. Further 100 ns exhaustive simulation studies and MM-PBSA calculations supported favorable and stable binding of them. This work identifies Nine Tinospora compounds as potential inhibitors. Among those, 7-desacetoxy-6,7-dehydrogedunin was found to inhibit both spike (7NEG) and Mpro (7MGS and 6LU7) proteins, and Columbin was found to inhibit selected spike targets (7NEG and 7NX7). In all the analyses, these compounds performed well and confirms the stable binding. Hence the identified compounds, advocated as potential inhibitors can be taken for further in vitro and in vivo experimental validation to determine their anti-SARS-CoV-2 potential.Communicated by Ramaswamy H. Sarma.

In silico design and computational evaluation of novel 2-arylaminopyrimidine-based compounds as potential multi-targeted protein kinase inhibitors: application for the native and mutant (T315I) Bcr-Abl tyrosine kinase.

An integrated computational approach to drug discovery was used to identify novel potential inhibitors of the native and mutant (T315I) Bcr-Abl tyrosine kinase, the enzyme playing a key role in the pathogenesis of chronic myeloid leukemia (CML). This approach included i) design of chimeric molecules based on the 2-arylaminopyrimidine fragment, the main pharmacophore of the Abl kinase inhibitors imatinib and nilotinib used in the clinic for the CML treatment, ii) molecular docking of these compounds with the ATP-binding site of the native and mutant Abl kinase, iii) refinement of the ligand-binding poses by the quantum chemical method PM7, iv) molecular dynamics simulations of the ligand/Abl complexes, and v) prediction of the ligand/Abl binding affinity in terms of scoring functions of molecular docking, machine learning, quantum chemistry, and molecular dynamics. As a result, five top-ranking compounds able to effectively block the enzyme catalytic site were identified. According to the data obtained, these compounds exhibit close modes of binding to the Abl kinase active site that are mainly provided by hydrogen bonds and multiple van der Waals contacts. The identified compounds show high binding affinity to the native and mutant Abl kinase comparable with the one calculated for the FDA-approved kinase-targeted inhibitors imatinib, nilotinib, and ponatinib used in the calculations as a positive control. The results obtained testify to the predicted drug candidates against CML may serve as good scaffolds for the design of novel anticancer agents able to target the ATP-binding pocket of the native and mutant Abl kinase.Communicated by Ramaswamy H. Sarma.

The effects of mutation on the drug binding affinity of Neuraminidase: case study of Capsaicin using steered molecular dynamics simulation.

The influenza virus is an important respiratory pathogen that causes many incidences of diseases and even death each year. One of the primary factors of this virus is the Neuraminidase surface protein, which causes the virus to leave the host cell and spread to new target cells. The main antiviral medication for influenza is designed as a protein inhibitor ligand that prevents further spread of the disease, and eventually relieves the emerged symptoms. The effectiveness of such inhibitory drugs is highly associated with their binding affinity. In this paper, the binding affinity of an herbal ligand of Capsaicin bound to Neuraminidase of the influenza virus is investigated using steered molecular dynamics (SMD) simulation. Since mutations of the virus directly impact the binding affinity of the inhibitory drugs, different mutations were generated by using Mutagenesis module. The rapid spread of infection during the avian influenza A/H5N1 epidemic has raised concerns about far more dangerous consequences if the virus becomes resistant to current drugs. Currently, oseltamivir (Tamiflu), zanamivir (Relenza), pramivir (Rapivab), and laninamivir (Inavir) are increasingly used to treat the flu. However, with the rapid evolution of the virus, some drug-resistant strains are emerging. Therefore, it is very important to seek alternative therapies and identify the roots of drug resistance. Obtained results demonstrated a reduced binding affinity for the applied mutations. This reduction in binding affinity will cause the virus mutation to become resistant to the drug, which will spread the disease and make it more difficult to treat. From a molecular prospect, this decrease in binding affinity is due to the loss of a number of effective bonds between the ligand and the receptor, which occurs with mutations of the wild-type (WT) species. The results of the present study can be used in the rational design of novel drugs that are compatible with specific mutations.

Mutant firefly luciferase enzymes resistant to the inhibition by sodium chloride.

OBJECTIVES: Firefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study was aimed at obtaining mutant luciferase enzymes resistant to the sodium chloride inhibition. RESULTS: We first obtained two mutant luciferase enzymes whose inhibition were alleviated and determined the mutations to be Val288Ile and Glu488Val. Under medical dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride. CONCLUSIONS: The mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The luciferase CR may be extensively useful in any bioassay which includes firefly luciferase and is employed in the presence of sodium chloride.

Selective and noncovalent targeting of RAS mutants for inhibition and degradation.

Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. Here, we report a monobody, termed 12VC1, that recognizes the active state of both KRAS(G12V) and KRAS(G12C) up to 400-times more tightly than wild-type KRAS. The crystal structures reveal that 12VC1 recognizes the mutations through a shallow pocket, and 12VC1 competes against RAS-effector interaction. When expressed intracellularly, 12VC1 potently inhibits ERK activation and the proliferation of RAS-driven cancer cell lines in vitro and in mouse xenograft models. 12VC1 fused to VHL selectively degrades the KRAS mutants and provides more extended suppression of mutant RAS activity than inhibition by 12VC1 alone. These results demonstrate the feasibility of selective targeting and degradation of KRAS mutants in the active state with noncovalent reagents and provide a starting point for designing noncovalent therapeutics against oncogenic RAS mutants.

Novel Mechanistic Observations and NES-Binding Groove Features Revealed by the CRM1 Inhibitors Plumbagin and Oridonin.

The protein chromosome region maintenance 1 (CRM1) is an important nuclear export factor and drug target in diseases such as cancer and viral infections. Several plant-derived CRM1 inhibitors including plumbagin and oridonin possess potent antitumor activities. However, their modes of CRM1 inhibition remain unclear. Here, a multimutant CRM1 was engineered to enable crystallization of these two small molecules in its NES groove. Plumbagin and oridonin share the same three conjugation sites in CRM1. In solution, these two inhibitors targeted more CRM1 sites and inhibited its activity through promoting its aggregation, in addition to directly targeting the NES groove. While the plumbagin-bound NES groove resembles the NES-bound groove state, the oridonin complex reveals for the first time a more open NES groove. The observed greater NES groove dynamics may improve cargo loading through a "capture-and-tighten" mechanism. This work thus provides new insights on the mechanism of CRM1 inhibition by two natural products and a structural basis for further development of these or other CRM1 inhibitors.

Identification and characterization of a novel mutant isocitrate dehydrogenase 1 inhibitor for glioma treatment.

Isocitrate dehydrogenase 1 (IDH1) mutant R132H, promoting the oncometabolite D-2-hydroxyglutarate (D2HG), is a driver mutation and an emerging therapeutic target in glioma. This study identified a novel mutant IDH1 inhibitor, WM17, by virtual screening and enzymatic confirmation. It could bind to and increase mutant IDH1 protein's thermostability in both endogenous heterozygous cells and exogenous overexpressed cells. Consequently, WM17 reversed the accumulation of D2HG and histone hypermethylation in IDH1 mutated cells. Finally, we concluded that WM17 significantly inhibited cell migration in IDH1 mutated glioma cells, although it has no apparent effect on cell proliferation. Further studies are guaranteed toward the development of WM17 as a therapeutic agent for IDH1 mutated glioma.

Bispecific antibodies targeting mutant RAS neoantigens.

Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS-derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.

Inhibition of BRAF Sensitizes Thyroid Carcinoma to Immunotherapy by Enhancing tsMHCII-mediated Immune Recognition.

CONTEXT: Multiple mechanisms play roles in restricting the ability of T-cells to recognize and eliminate tumor cells. OBJECTIVE: To identify immune escape mechanisms involved in papillary thyroid carcinoma (PTC) to optimize immunotherapy. SETTING AND DESIGN: iTRAQ analysis was conducted to identify proteins differentially expressed in PTC samples with or without BRAFV600E mutation. Molecular mechanisms regulating tumor cell evasion were investigated by in vitro modulations of BRAF/MAPK and related pathways. The pathological significance of identified tumor-specific major histocompatibility complex class II (tsMHCII) molecules in mediating tumor cell immune escape and targeted immune therapy was further evaluated in a transgenic mouse model of spontaneous thyroid cancer. RESULTS: Proteomic analysis showed that tsMHCII level was significantly lower in BRAFV600E-associated PTCs and negatively correlated with BRAF mutation status. Constitutive activation of BRAF decreased tsMHCII surface expression on tumor cells, which inhibited activation of CD4+ T-cells and led to immune escape. Pathway analysis indicated that the transforming growth factor (TGF)-ß1/SMAD3-mediated repression of tsMHCII, which could be reversed by BRAF inhibition (BRAFi). Targeting this pathway with a combined therapy of BRAF inhibitor PLX4032 and anti-PD-1 antibody efficiently blocked tumor growth by increasing CD4+ T-cell infiltration in a transgenic PTC mouse model. CONCLUSIONS: Our results suggest that BRAFV600E mutation in PTC impairs the expression of tsMHCII through the TGF-ß1/SMAD3 pathway to enhance immune escape. Combined treatment with PLX4032 and anti-PD-1 antibody promotes recognition and elimination of PTC by the immune system in a pre-clinical mouse model, and therefore offers an effective therapeutic strategy for patients with advanced PTC.

Structural And Computational Perspectives of Selectively Targeting Mutant Proteins.

Diseases are often caused by mutant proteins. Many drugs have limited effectiveness and/or toxic side effects because of a failure to selectively target the disease-causing mutant variant, rather than the functional wild type protein. Otherwise, the drugs may even target different proteins with similar structural features. Designing drugs that successfully target mutant proteins selectively represents a major challenge. Decades of cancer research have led to an abundance of potential therapeutic targets, often touted to be "master regulators". For many of these proteins, there are no FDA-approved drugs available; for others, off-target effects result in dose-limiting toxicity. Cancer-related proteins are an excellent medium to carry the story of mutant-specific targeting, as the disease is both initiated and sustained by mutant proteins; furthermore, current chemotherapies generally fail at adequate selective distinction. This review discusses some of the challenges associated with selective targeting from a structural biology perspective, as well as some of the developments in algorithm approach and computational workflow that can be applied to address those issues. One of the most widely researched proteins in cancer biology is p53, a tumor suppressor. Here, p53 is discussed as a specific example of a challenging target, with contemporary drugs and methodologies used as examples of burgeoning successes. The oncogene KRAS, which has been described as "undruggable", is another extensively investigated protein in cancer biology. This review also examines KRAS to exemplify progress made towards selective targeting of diseasecausing mutant proteins. Finally, possible future directions relevant to the topic are discussed.

BCL7C suppresses ovarian cancer growth by inactivating mutant p53.

B-cell CLL/lymphoma 7 protein family member C (BCL7C) located at chromosome 16p11.2 shares partial sequence homology with the other two family members, BCL7A and BCL7B. Its role in cancer remains completely unknown. Here, we report our finding of its tumor-suppressive role in ovarian cancer. Supporting this is that BCL7C is downregulated in human ovarian carcinomas, and its underexpression is associated with unfavorable prognosis of ovarian cancer as well as some other types of human cancers. Also, ectopic BCL7C restrains cell proliferation and invasion of ovarian cancer cells. Consistently, depletion of BCL7C reduces apoptosis and promotes cell proliferation and invasion of these cancer cells. Mechanistically, BCL7C suppresses mutant p53-mediated gene transcription by binding to mutant p53, while knockdown of BCL7C enhances the expression of mutant p53 target genes in ovarian cancer cells. Primary ovarian carcinomas that sustain low levels of BCL7C often show the elevated expression of mutant p53 target genes. In line with these results, BCL7C abrogates mutant p53-induced cell proliferation and invasion, but had no impact on proliferation and invasion of cancer cells with depleted p53 or harboring wild-type p53. Altogether, our results demonstrate that BCL7C can act as a tumor suppressor to prevent ovarian tumorigenesis and progression by counteracting mutant p53 activity.

Role of lysine residue of islet amyloid polypeptide in fibril formation, membrane binding, and inhibitor binding.

The aggregation of islet amyloid polypeptide (IAPP) is implicated in the pathogenesis of type 2 diabetes (T2D). In T2D, this peptide aggregates to form amyloid fibrils; the mechanism responsible for islet amyloid formation is unclear. However, it is known that the aggregation propensity of IAPP is highly related to its primary sequence. Several residues have been suggested to be critical in modulating IAPP amyloid formation, but role of the sole lysine residue at position 1 (Lys-1) in IAPP has not been discussed. In our previous study, we found that glycated IAPP can form amyloid faster than normal IAPP and induce normal IAPP to expedite the aggregation process. To gain more insight into the contribution of Lys-1 in the kinetics of fibril formation, we synthesized another two IAPP variants, K1E-IAPP and K1Nle-IAPP, in which the Lys residue was mutated to glutamate and norleucine, respectively. Interestingly, we observed that the negative or neutral charged side chain at this position was preferred for amyloid formation. The findings suggested this residue may take part in the inter- or intra-molecular interaction during IAPP aggregation, even though it was proposed not to be in part of fibril core structure. Our data also revealed that the inhibitory mechanism of some inhibitors for IAPP aggregation require reaction with Lys-1. Modifications of Lys-1, such as protein glycation, may affect the effectiveness of the inhibitory action of some potential drugs in the treatment of amyloidosis.

Pharmacological inhibition of ataxia-telangiectasia mutated exacerbates acute kidney injury by activating p53 signaling in mice.

The DNA damage response after kidney injury induces cell cycle arrest in renal tubular epithelial cells, resulting in the secretion of pro-fibrotic cytokines, thereby promoting interstitial fibrosis in a paracrine manner. Phosphorylation of ataxia-telangiectasia mutated (ATM) is the initial step in the DNA damage response and subsequent cell cycle arrest; however, the effects of ATM inhibition on the injured kidney have not been explored. Pharmacological ATM inhibition by KU55933 in cisplatin-treated mice did not ameliorate, but instead exacerbated cisplatin-induced DNA damage and tubular injury, thereby increasing mortality. Analysis of isolated tubular epithelia by FACS from bigenic SLC34a1-CreERt2; R26tdTomato proximal tubular-specific reporter mice revealed that KU55933 upregulated p53 and subsequent pro-apoptotic signaling in tubular epithelia of cisplatin-treated mice, leading to marked mitochondrial injury and apoptosis. In addition, KU55933 attenuated several DNA repair processes after cisplatin treatment, including single-strand DNA repair and Fanconi anemia pathways, suggesting that DNA repair after dual treatment of cisplatin and KU55933 was not sufficient to prevent the cisplatin-induced tubular injury. Our study suggested that ATM inhibition does not increase DNA repair after cisplatin-induced DNA damage and exacerbates tubular injury through the upregulation of p53-dependent pro-apoptotic signaling. Acute kidney injury must be carefully monitored when ATM inhibitors become available in clinical practice in the future.

A small molecule chaperone rescues the stability and activity of a cancer-associated variant of NAD(P)H:quinone oxidoreductase 1 in vitro.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a human FAD-dependent enzyme that plays a crucial role in the antioxidant defense system. A naturally occurring single-nucleotide polymorphism (NQO1*2) in the NQO1 gene leads to an amino acid substitution (P187S), which severely compromises the activity and stability of the enzyme. The NQO1*2 genotype has been linked to a higher risk for several types of cancer and poor survival rate after anthracycline-based chemotherapy. In this study, we show that a small molecular chaperone (N-(2-bromophenyl)pyrrolidine-1-sulfonamide) repopulates the native wild-type conformation. As a consequence of the stabilizing effect, the enzymatic activity of the P187S variant protein is strongly improved in the presence of the molecular chaperone in vitro.

Newly Developed CK1-Specific Inhibitors Show Specifically Stronger Effects on CK1 Mutants and Colon Cancer Cell Lines.

Protein kinases of the CK1 family can be involved in numerous physiological and pathophysiological processes. Dysregulated expression and/or activity as well as mutation of CK1 isoforms have previously been linked to tumorigenesis. Among all neoplastic diseases, colon and rectal cancer (CRC) represent the fourth leading cause of cancer related deaths. Since mutations in CK1δ previously found in CRC patients exhibited increased oncogenic features, inhibition of CK1δ is supposed to have promising therapeutic potential for tumors, which present overexpression or mutations of this CK1 isoform. Therefore, it is important to develop new small molecule inhibitors exhibiting higher affinity toward CK1δ mutants. In the present study, we first characterized the kinetic properties of CK1δ mutants, which were detected in different tumor entities. Subsequently, we characterized the ability of several newly developed IWP-based inhibitors to inhibit wild type and CK1δ mutants and we furthermore analyzed their effects on growth inhibition of various cultured colon cancer cell lines. Our results indicate, that these compounds represent a promising base for the development of novel CRC therapy concepts.

Metal-induced change in catalytic loop positioning in Helicobacter pylori arginase alters catalytic function.

Arginase is a bimetallic enzyme that utilizes mainly Mn2+ or Co2+ for catalytic function. In human homolog, the substitution of Mn2+ with Co2+ significantly reduces the Km value without affecting the kcat. However, in the Helicobacter pylori counterpart (important for pathogenesis), the kcat increases nearly 4-fold with Co2+ ions both in the recombinant holoenzyme and arginase isolated from H. pylori grown with Co2+ or Mn2+. This suggests that the active site of arginase in the two homologs is modulated differently by these two metal ions. To investigate the underlying mechanism for metal-induced difference in catalytic activity in the H. pylori enzyme, we used biochemical, biophysical and microsecond molecular dynamics simulations studies. The study shows that the difference in binding affinity of Co2+ and Mn2+ ions with the protein is linked to a different positioning of a loop (-122HTAYDSDSKHIHG134-) that contains a conserved catalytic His133. Consequently, the proximity of His133 and conserved Glu281 is varied. We found that the Glu281-His133 interaction is crucial for catalytic function and was previously unexplored in other homologs. We suggest that the proximity difference between these two residues in the Co2+- and Mn2+-proteins alters the proportion of protonated His133 via variation in its pKa. This affects the efficiency of proton transfer - an essential step of l-arginine hydrolysis reaction catalyzed by arginase and thus activity. Unlike in human arginase, the flexibility of the above segment observed in H. pylori homolog suggests that this region in the H. pylori enzyme may be explored to design its specific inhibitors.

Ruxolitinib binding to human serum albumin: bioinformatics, biochemical and functional characterization in JAK2V617F+ cell models.

Ruxolitinib is a type I JAK inhibitor approved by FDA for targeted therapy of Philadelphia-negative myeloproliferative neoplasms (MPNs), all characterized by mutations activating the JAK2/STAT signaling pathway. Treatment with ruxolitinib improves constitutional symptoms and splenomegaly. However, patients can become resistant to treatment and chronic therapy has only a mild effect on molecular/pathologic remissions. Drugs interaction with plasma proteins, i.e. human serum albumin (HSA), is an important factor affecting the intensity and duration of their pharmacological actions. Here, the ruxolitinib recognition by the fatty acid binding sites (FAs) 1, 6, 7, and 9 of HSA has been investigated from the bioinformatics, biochemical and/or biological viewpoints. Docking simulations indicate that ruxolitinib binds to multiple sites of HSA. Ruxolitinib binds to the FA1 and FA7 sites of HSA with high affinity (Kr = 3.1 µM and 4.6 µM, respectively, at pH 7.3 and 37.0 °C). Moreover, HSA selectively blocks, in a dose dependent manner, the cytotoxic activity of ruxolitinib in JAK2V617F+ cellular models for MPN, in vitro. Furthermore this event is accompanied by changes in the cell cycle, p27Kip1 and cyclin D3 levels, and JAK/STAT signaling. Given the high plasma concentration of HSA, ruxolitinib trapping may be relevant in vivo.

Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds.

Accumulation of mutant proteins is a major cause of many diseases (collectively called proteopathies), and lowering the level of these proteins can be useful for treatment of these diseases. We hypothesized that compounds that interact with both the autophagosome protein microtubule-associated protein 1A/1B light chain 3 (LC3)1 and the disease-causing protein may target the latter for autophagic clearance. Mutant huntingtin protein (mHTT) contains an expanded polyglutamine (polyQ) tract and causes Huntington's disease, an incurable neurodegenerative disorder2. Here, using small-molecule-microarray-based screening, we identified four compounds that interact with both LC3 and mHTT, but not with the wild-type HTT protein. Some of these compounds targeted mHTT to autophagosomes, reduced mHTT levels in an allele-selective manner, and rescued disease-relevant phenotypes in cells and in vivo in fly and mouse models of Huntington's disease. We further show that these compounds interact with the expanded polyQ stretch and could lower the level of mutant ataxin-3 (ATXN3), another disease-causing protein with an expanded polyQ tract3. This study presents candidate compounds for lowering mHTT and potentially other disease-causing proteins with polyQ expansions, demonstrating the concept of lowering levels of disease-causing proteins using autophagosome-tethering compounds.

A Mechanistic Study of a Potent and Selective Epidermal Growth Factor Receptor Inhibitor against the L858R/T790M Resistance Mutation.

Covalent targeting is a promising strategy for increasing the potency and selectivity of potential drug candidates. This therapeutic approach was recently reported for the epidermal growth factor receptor (EGFR), wherein a covalent binder, 20g [N-(3-{7-[2-methoxy-4-(4-methylpiperazin-1-yl)phenylamino]-3,4-dihydro-3-isopropyl-2,4-dioxopyrimido[4,5-d]pyrimidin-1(2H)-yl}phenyl)acrylamide], demonstrated significant selectivity and inhibitory activity toward the EGFR L858R/T790M double mutant (EGFRDM) relative to the EGFR wild-type form (EGFRWT). The enhanced therapeutic potency of 20g against EGFRDM is 263 times greater than that against EGFRWT, which necessitates a rational explanation for the underlying selective and inhibitory mechanisms. In this work, we investigate the differential binding modes of 20g with EGFRWT and EGFRDM using molecular dynamics simulations coupled with free energy calculations and further identify key residues involved in the selective targeting, binding, and inhibitory mechanisms mediated by 20g. We find that systematic orientational and conformational changes in the α-loop, p-loop, active loop, and αC-helix are responsible for the disparate binding mechanisms and inhibitory prowess of 20g with respect to EGFRWT and EGFRDM. The calculated binding free energies show good correlation with the experimental biological activity. The total binding free energy difference between EGFRWT-20g and EGFRDM-20g is -11.47 kcal/mol, implying that 20g binds more strongly to EGFRDM. This enhanced binding affinity of 20g for EGFRDM is a result of a large increase in the van der Waals and electrostatic interactions with three critical residues (Met790, Gln791, and Met793) that are chiefly responsible for the high-affinity interactions mediated by 20g with EGFRDM relative to EGFRWT.

Mechanistic and Structural Insights into Cysteine-Mediated Inhibition of Pyruvate Kinase Muscle Isoform 2.

Cancer cells regulate key enzymes in the glycolytic pathway to control the glycolytic flux, which is necessary for their growth and proliferation. One of the enzymes is pyruvate kinase muscle isoform 2 (PKM2), which is allosterically regulated by various small molecules. Using detailed biochemical and kinetic studies, we demonstrate that cysteine inhibits wild-type (wt) PKM2 by shifting from an active tetramer to a mixture of a tetramer and a less active dimer/monomer equilibrium and that the inhibition is dependent on cysteine concentration. The cysteine-mediated PKM2 inhibition is reversed by fructose 1,6-bisphosphate, an allosteric activator of PKM2. Furthermore, kinetic studies using two dimeric PKM2 variants, S437Y PKM2 and G415R PKM2, show that the reversal is caused by the tetramerization of wtPKM2. The crystal structure of the wtPKM2-Cys complex was determined at 2.25 Å, which showed that cysteine is held to the amino acid binding site via its main chain groups, similar to that observed for phenylalanine, alanine, serine, and tryptophan. Notably, ligand binding studies using fluorescence and isothermal titration calorimetry show that the presence of phosphoenolpyruvate alters the binding affinities of amino acids for wtPKM2 and vice versa, thereby unravelling the existence of a functionally bidirectional coupling between the amino acid binding site and the active site of wtPKM2.