Targeting SHP2 Cryptic Allosteric Sites for Effective Cancer Therapy.

SHP2, a pivotal component downstream of both receptor and non-receptor tyrosine kinases, has been underscored in the progression of various human cancers and neurodevelopmental disorders. Allosteric inhibitors have been proposed to regulate its autoinhibition. However, oncogenic mutations, such as E76K, convert SHP2 into its open state, wherein the catalytic cleft becomes fully exposed to its ligands. This study elucidates the dynamic properties of SHP2 structures across different states, with a focus on the effects of oncogenic mutation on two known binding sites of allosteric inhibitors. Through extensive modeling and simulations, we further identified an alternative allosteric binding pocket in solution structures. Additional analysis provides insights into the dynamics and stability of the potential site. In addition, multi-tier screening was deployed to identify potential binders targeting the potential site. Our efforts to identify a new allosteric site contribute to community-wide initiatives developing therapies using multiple allosteric inhibitors to target distinct pockets on SHP2, in the hope of potentially inhibiting or slowing tumor growth associated with SHP2.

Discovery of First-in-Class Small Molecule Inhibitors of Lymphocyte Activation Gene 3 (LAG-3).

Lymphocyte activation gene 3 (LAG-3) is a negative immune checkpoint that plays a key role in downregulating the immune response to cancer. Inhibition of LAG-3 interactions allows T cells to regain cytotoxic activity and reduce the immunosuppressive function of regulating T cells. We utilized a combination approach of focused screening and "SAR by catalog" to identify small molecules that function as dual inhibitors of the interactions of LAG-3 with major histocompatibility complex (MHC) class II and fibrinogen-like protein 1 (FGL1). Our top hit compound inhibited both LAG-3/MHCII and LAG-3/FGL1 interactions in biochemical binding assays with IC50 values of 4.21 ± 0.84 and 6.52 ± 0.47 µM, respectively. Moreover, we have demonstrated the ability of our top hit compound to block LAG-3 interactions in cell-based assays. This work will pave the way for future drug discovery efforts aiming at the development of LAG-3-based small molecules for cancer immunotherapy.

Identification of novel peptide inhibitors for oncogenic KRAS G12D as therapeutic options using mutagenesis-based remodeling and MD simulations.

The Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) serves as a molecular switch, cycling between guanosine triphosphate (GTP)-bound and inactive guanosine diphosphate (GDP)-bound states. KRAS modulates numerous signal transduction pathways including the conventional RAF-MEK-ERK pathway. Mutations in the RAS genes have been linked to the formation of malignant tumors. Human malignancies typically show mutations in the Ras gene including HRAS, KRAS, and NRAS. Among all the mutations in exon 12 and exon 13 of the KRAS gene, the G12D mutation is more prevalent in pancreatic and lung cancer and accounts for around 41% of all G12 mutations, making them potential anticancer therapeutic targets. The present study is aimed at repurposing the peptide inhibitor KD2 of the KRAS G12D mutant. We employed an in-silico mutagenesis approach to design novel peptide inhibitors from the experimentally reported peptide inhibitor, and it was found that substitutions (N8W, N8I, and N8Y) might enhance the peptide's binding affinity toward the KRAS. Molecular dynamics simulations and binding energy calculations confirmed that the newly designed peptide inhibitors are stable and that their binding affinities are stronger as compared to the wild-type peptide. The detailed analysis revealed that newly designed peptides have the potential to inhibit KRAS/Raf interaction and the oncogenic signal of the KRAS G12D mutant. Our findings strongly suggest that these peptides should be tested and clinically validated to combat the oncogenic activity of KRAS.Communicated by Ramaswamy H. Sarma.

Synthesis of thiazole-based-thiourea analogs: as anticancer, antiglycation and antioxidant agents, structure activity relationship analysis and docking study.

This work reports the convenient approach for the synthesis of thiazole based thiourea derivatives (1-21) from 2-bromo-1-(4-fluorophenyl)thiazole-1-one and phenyl isothiocyanates. The scope and diversity were achieved from readily available phenyl isothiocyanates. This protocol involves an oxidative C-S bond formation. Moreover, hybrid thiazole based thiourea scaffolds (1-21) according to literature known protocol were screened in vitro for anticancer Potential against breast cancer, antiglycation and antioxidant inhibitory profile. All newly developed scaffolds were showed moderate to good inhibitory potentials ranging from 0.10 ± 0.01 µM to 11.40 ± 0.20 µM, 64.20 ± 0.40 µM to 385.10 ± 1.70 µM and 8.90 ± 0.20 µM to 39.20 ± 0.50 µM against anticancer, antiglycation and antioxidant respectively. Among the series, compounds 12 (IC50 = 0.10 ± 0.01 µM), 10 (IC50 = 64.20 ± 0.40 µM) and 12 (IC50 = 8.90 ± 0.20 µM) with flouro substitution at phenyl ring of thiourea were identified to be the most potent among the series having excellent anticancer, antiglycation and antioxidant potential. The structure of all the newly synthetics scaffolds were confirmed by using different types of spectroscopic techniques such as HREI-MS, 1H- and 13C-NMR spectroscopy. To find structure-activity relationship, molecular docking studies were carried out to understand the binding mode of active inhibitors with active site of enzymes and results supported the experimental data.Communicated by Ramaswamy H. Sarma.

Computational screening and analysis of deleterious nsSNPs in human p14ARF (CDKN2A gene) protein using molecular dynamic simulation approach.

Cyclin-dependent kinase inhibitor 2 A (CDKN2A) gene belongs to the cyclin-dependent kinase family that code for two transcripts (p16INK4A and p14ARF), both work as tumor suppressors proteins. The mutation that occurs in the p14ARF protein can lead to different types of cancers. Single nucleotide polymorphisms (SNPs) are an important type of genetic alteration that can lead to different types of diseases. In this study, we applied the computational strategy on human p14ARF protein to identify the potential deleterious nsSNPs and check their impact on the structure, function, and protein stability. We applied more than ten prediction tools to screen the retrieved 288 nsSNPs, consequently extracting four deleterious nsSNPs i.e., rs139725688 (R10G), rs139725688 (R21W), rs374360796 (F23L) and rs747717236 (L124R). Homology modeling, conservation and conformational analysis of mutant models were performed to examine the divergence of these variants from the native p14ARF structure. All-atom molecular dynamics simulation revealed a significant impact of these mutations on protein stability, compactness, globularity, solvent accessibility and secondary structure elements. Protein-protein interactions indicated that p14ARF operates as a hub linking clusters of different proteins and that changes in p14ARF may result in the disassociation of numerous signal cascades. Our current study is the first survey of computational analysis on p14ARF protein that determines the association of these nsSNPs with the altered function of p14ARF protein and leads to the development of various types of cancers. This research proposes the described functional SNPs as possible targets for proteomic investigations, diagnostic procedures, and treatments.Communicated by Ramaswamy H. Sarma.

Identification of novel peptide inhibitors for the KRas-G12C variant to prevent oncogenic signaling.

Kirsten rat sarcoma viral oncogene homolog (KRas) activating mutations are common in solid tumors, accounting for 90%, 45%, and 35% of pancreatic, colorectal, and lung cancers (LC), respectively. Each year, nearly 150k new cases (both men and women) of KRas-mutated malignancies are reported in the United States. NSCLC (non-small cell lung cancer) accounts for 80% of all LC cases. KRas mutations are found in 15% to 25% of NSCLC patients. The main cause of NSCLC is the KRas-G12C mutation. The drugs Sotorasib and Adagrasib were recently developed to treat advanced NSCLC caused by the KRas-G12C mutation. Most patients do not respond to KRas-G12C inhibitors due to cellular, molecular, and genetic resistance. Because of their safety, efficacy, and selectivity, peptide inhibitors have the potential to treat newly developing KRas mutations. Based on the KRas mutations, peptide inhibitors that are highly selective and specific to individual lung cancers can be rationally designed. The current study uses an alanine and residue scanning approach to design peptide inhibitors for KRas-G12C based on the known peptide. Our findings show that substitution of F3K, G11T, L8C, T14C, K13D, G11S, and G11P considerably enhances the binding affinity of the novel peptides, whereas F3K, G11T, L8C, and T14C peptides have higher stability and favorable binding to the altered peptides. Overall, our study paves the road for the development of potential therapeutic peptidomimetics that target the KRas-G12C complex and may inhibit the KRas and SOS complex from interacting.Communicated by Ramaswamy H. Sarma.

New lead compounds identification against KRas mediated cancers through pharmacophore-based virtual screening and in vitro assays.

Cancer remains the leading cause of mortality and morbidity in the world, with 19.3 million new diagnoses and 10.1 million deaths in 2020. Cancer is caused due to mutations in proto-oncogenes and tumor-suppressor genes. Genetic analyses found that Ras (Rat sarcoma) is one of the most deregulated oncogenes in human cancers. The Ras oncogene family members including NRas (Neuroblastoma ras viral oncogene homolog), HRas (Harvey rat sarcoma) and KRas are involved in different types of human cancers. The mutant KRas is considered as the most frequent oncogene implicated in the development of lung, pancreatic and colon cancers. However, there is no efficient clinical drug even though it has been identified as an oncogene for 30 years. Therefore there is an emerging need to develop potent, new anticancer drugs. In this study, computer-aided drug designing approaches as well as experimental methods were employed to find new and potential anti-cancer drugs. The pharmacophore model was developed from an already known FDA approved anti-cancer drug Bortezomib using the software MOE. The validated pharmacophore model was then used to screen the in-house and commercially available databases. The pharmacophore-based virtual screening resulted in 26 and 86 hits from in-house and commercial databases respectively. Finally, 6/13 (in-house database) and 24/64 hits (commercial databases) were selected with different scaffolds having good interactions with the significant active residues of KRasG12D protein that were predicted as potent lead compounds. Finally, the results of pharmacophore-based virtual screening were further validated by molecular dynamics simulation analysis. The 6 hits of the in-house database were further evaluated experimentally. The experimental results showed that these compounds have good anti-cancer activity which validate the protocol of our in silico studies. KRasG12D protein is a very important anti-cancer target and potent inhibitors for this target are still not available, so small lead compound inhibitors were identified to inhibit the activity of this protein by blocking the GTP-binding pocket.Communicated by Ramaswamy H. Sarma.

Xanthine oxidase inhibitory study of eight structurally diverse phenolic compounds.

This project was designed to explore the xanthine oxidase (XO) inhibitory mechanism of eight structurally diverse phenolic compounds [quercetin: C1, quercetin-3-rhamnoside: C2, 4, 5-O-dicaffeoylquinic acid: C3, 3, 5-O-dicaffeoylquinic acid: C4, 3, 4-O-di-caffeoylquinic acid: C5, 4-O-caffeoylquinic acid (C6), 3-O-caffeoylquinic acid: C7, and caffeic acid: C8]. For this purpose, in-vitro and different computational methods were applied to determine the xanthine oxidase (XO) inhibitory potential of eight structurally diverse phenolic compounds. The results revealed that phenolic compounds (C1-C8) possess strong to weak XO inhibitory activity. These results were further confirmed by atomic force microscopy (AFM) and 1H NMR analysis. Furthermore, computational study results revealed that phenolic compounds (C1-C8) bind with the surrounding amino acids of XO at the molybdenum (MO) site. These in-vitro and in-silico results divulge that phenolic compounds have a strong potential to lower uric acid levels via interacting with the XO enzyme and can be used to combat hyperuricemia.

Machine Learning-based Virtual Screening for STAT3 Anticancer Drug Target.

BACKGROUND: Signal transducers and activators of the transcription (STAT) family is composed of seven structurally similar and highly conserved members, including STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. The STAT3 signaling cascade is activated by upstream kinase signals and undergoes phosphorylation, homo-dimerization, nuclear translocation, and DNA binding, resulting in the expression of target genes involved in tumor cell proliferation, metastasis, angiogenesis, and immune editing. STAT3 hyperactivation has been documented in a number of tumors, including head and neck, breast, lung, liver, kidney, prostate, pancreas cancer, multiple myeloma, and acute myeloid leukemia. Drug discovery is a timeconsuming and costly process; it may take ten to fifteen years to bring a single drug to the market. Machine learning algorithms are very fast and effective and commonly used in the field, such as drug discovery. These algorithms are ideal for the virtual screening of large compound libraries to classify molecules as active or inactive. OBJECTIVE: The present work aims to perform machine learning-based virtual screening for the STAT3 drug target. METHODS: Machine learning models, such as k-nearest neighbor, support vector machine, Gaussian naïve Bayes, and random forest for classifying the active and inactive inhibitors against a STAT3 drug target, were developed. Ten-fold cross-validation was used for model validation. Then the test dataset prepared from the zinc database was screened using the random forest model. A total of 20 compounds with 88% accuracy was predicted as active against STAT3. Furthermore, these twenty compounds were docked into the active site of STAT3. The two complexes with good docking scores as well as the reference compound were subjected to MD simulation. A total of 100ns MD simulation was performed. RESULTS: Compared to all other models, the random forest model revealed better results. Compared to the standard reference compound, the top two hits revealed greater stability and compactness. CONCLUSION: In conclusion, our predicted hits have the ability to inhibit STAT3 overexpression to combat STAT3-associated diseases.

Strategies for Targeting KRAS: A Challenging Drug Target.

In the developed world, cancer is the most common cause of death. Among the 36 human genes of the RAS family, KRAS, NRAS, and HRAS play a prominent role in human cancer. KRAS belongs to the Ras superfamily of proteins and is a small GTPase signal transduction protein. Among the RAS isoform, KRAS is the dominant mutant that induces approximately 86% of the RAS mutations. The most frequently mutated KRAS isoform is KRAS4B. About 90% of pancreatic cancer, 30-40% of colon cancer, and 15 to 20% of lung cancers are caused by mutations KRAS4B isoform. Liver cancer, bladder cancer, breast cancer, and myeloid leukaemia are also caused by mutations in KRAS but are rare. The FDA has recently approved sotorasib for the treatement of KRASG12C-mutated advanced non-small cell lung cancer (NSCLC) patients. However, no FDAapproved drugs are available for other KRAS-driven cancer. As the KRAS proteins lack a druggable pocket accessible to the chemical inhibitors, the cancer-causing mutant proteins are almost identical to their essential wild-type counterparts. Therefore, they are considered undruggable. The new insights into the structure and function of RAS have changed this understanding and encouraged the development of many drug candidates. This review provides information about the different strategies for targeting KRAS, a challenging drug target that might be valuable for the scientific community.

A unique amphiphilic triblock copolymer, nontoxic to human blood and potential supramolecular drug delivery system for dexamethasone.

The drug delivery system (DDS) often causes toxicity, triggering undesired cellular injuries. Thus, developing supramolecules used as DDS with tunable self-assembly and nontoxic behavior is highly desired. To address this, we aimed to develop a tunable amphiphilic ABA-type triblock copolymer that is nontoxic to human blood cells but also capable of self-assembling, binding and releasing the clinically used drug dexamethasone. We synthesized an ABA-type amphiphilic triblock copolymer (P2L) by incorporating tetra(aniline) TANI as a hydrophobic and redox active segment along with monomethoxy end-capped polyethylene glycol (mPEG2k; Mw = 2000 g mol-1) as biocompatible, flexible and hydrophilic part. Cell cytotoxicity was measured in whole human blood in vitro and lung cancer cells. Polymer-drug interactions were investigated by UV-Vis spectroscopy and computational analysis. Our synthesized copolymer P2L exhibited tuned self-assembly behavior with and without external stimuli and showed no toxicity in human blood samples. Computational analysis showed that P2L can encapsulate the clinically used drug dexamethasone and that drug uptake or release can also be triggered under oxidation or low pH conditions. In conclusion, copolymer P2L is nontoxic to human blood cells with the potential to carry and release anticancer/anti-inflammatory drug dexamethasone. These findings may open up further investigations into implantable drug delivery systems/devices with precise drug administration and controlled release at specific locations.

Disaggregation mechanism of prion amyloid for tweezer inhibitor.

The aggregation of amyloid has been an important event in the pathology of amyloidogenicity. A number of small molecules have been designed for Amyloidosis treatment. Molecular tweezer CLR01, a potential drug for misfolded ß-amyloids inhibition, was reportedly bind directly to Lysine residues and interrupt oligomerization. However, the disaggregation mechanism of amyloid for this inhibitor is unclear. Here we used long timescale of molecular dynamic simulation to reveal the mechanism of disaggregation for pentamer prion amyloid. Molecular docking and molecular dynamics simulation demonstrate that CLR01 is attached with Lysine222 nitrogen by π-cation interaction of its nine aromatic rings and formation of salt bridge/hydrogen bond of one of the two rotatable peripheral anionic phosphate groups. Upon CLR01 binding, we found a major shifting occurs in initial conformation of the oligomer and stretch out the N-terminal chain A from the rest of the amyloid which seems to be the first stage of disaggregated the fibrils slowly yet efficiently. Moreover, the CLR01 remodelled the pentamer Prion220-272 into a compact structure which might be the resistant conformation for further oligomerization. Our work will contribute to better understand the interaction and deterioration mechanism of molecular tweezer for prions and similar amyloids, and offer significant insights into therapeutic development for Amyloidosis treatment.

Dual roles of ATP-binding site in protein kinases: Orthosteric inhibition and allosteric regulation.

Protein kinases use ATP to phosphorylate other proteins. Phosphorylation (p) universally orchestrates a fine-tuned network modulating a multitude of biological processes. Moreover, the start of networks, ATP-binding site, has been recognized dual roles to impact protein kinases function: (i) orthosteric inhibition, via being blocked to interference ATP occupying and (ii) allosteric regulation, via being altered first to induce further conformational changes. The above two terminologies are widely used in drug design, which has acquired quite a significant progress in the protein kinases field over the past 2 decades. Most small molecular inhibitors directly compete with ATP to implement orthosteric inhibition, still exhibiting irreplaceable and promising therapeutic effects. Additionally, numerous inhibitors can paradoxically lead protein kinases to hyperphosphorylation, even activation, indicative of the allosteric regulation role of the ATP-binding site. Here, we review the quintessential examples that apply for the dual roles in diverse ways. Our work provides an insight into the molecular mechanisms under the dual roles and will be promisingly instructive for future drug development.

In vitro and in silico Xanthine Oxidase Inhibitory Activity of Selected Phytochemicals Widely Present in Various Edible Plants.

AIM AND OBJECTIVE: The present study was designed to evaluate the xanthine oxidase (XO) inhibitory and antioxidant activities of 30 bioactive compounds present in edible food plants for the possible treatment of hyperuricemia. MATERIALS AND METHODS: The XO inhibitory, SO and DPPH radical scavenging activities of selected dietary polyphenols were determined by using colorimetric assays. The molecular docking analysis was performed to evaluate the insight into inhibitory mode of action of bioactive compounds against XO. RESULTS: The results show that apigenin, galangin, kaempferol, quercetin, genistein and resveratrol potently inhibit XO enzyme among all tested compounds. Flavonoids exhibit higher, anthocyanins and hydroxycinnamic acids moderate, maslinic acid, ellagic acid, salicylic acid, [6]-gingerol and flavan-3-ols showed weak XO inhibitory activity. The results of molecular docking study revealed that these bioactive compounds bind with the active site of XO and occupy the active site which further prevents the entrance of substrate and results in the inhibition of XO. CONCLUSION: Inhibition of XO gives a robust biochemical basis for management of hyperuricemia, gout and other associated diseases via controlling uric acid synthesis.

Immunoproteasome inhibitor DPLG3 attenuates experimental colitis by restraining NF-κB activation.

Inflammatory bowel disease is a chronic and pathologic autoimmune condition. And immunoproteasome is becoming an attractive therapeutic target for autoimmune inflammatory diseases. In this study, we evaluated the therapeutic effects of a specific small molecule inhibitor of the chymotryptic-like ß5i subunits of the immunoproteasome, DPLG3, in a preclinical murine colitis model and explored the underlying molecular mechanism for the immune suppression. DPLG3 showed significant effects in attenuating the disease progression in experimental colitis, reducing the body and spleen weight losses, and colon length shortening compared to vehicle-treated controls and to the well studied immunoproteasome inhibitor ONX-0914. Mechanistically, DPLG3 decreased inflammatory cytokines and the influx of effector T cells and macrophages in colon tissues while increasing the number of regulatory T cells. Molecular docking analysis of the protein-ligand interaction profile revealed that the ß5i-DPLG3 complex was more stable and efficient in the binding sites compared to those formed with ONX-0914 and LU-005i. Furthermore, DPLG3 reduced the protein levels of the canonical NF-κB p50 and p65, as well as the nuclear p65. Thus, DPLG3 constitutes a potentially efficacious clinical agent for autoimmune inflammatory diseases.

Decoding allosteric communication pathways in protein lysine acetyltransferase.

In bacteria, protein lysine acetylation circuits can control core processes such as carbon metabolism. In E. coli, cyclic adenosine monophosphate (cAMP) controls the transcription level and activity of protein lysine acetyltransferase (PAT). The M. tuberculosis PatA (Mt-PatA) resides in two different conformations; the activated state and autoinhibited state. However, the mechanism of cAMP allosteric regulation of Mt-PatA remains mysterious. Here, we performed extensive all-atom molecular dynamics (MD) simulations (three independent run for each system) and built a residue-residue dynamic correlation network to show how cAMP mediates allosteric activation. cAMP binds at the regulatory site in the regulatory domain, which is 32 Å away from the catalytic site. An extensive conformational restructuring relieves autoinhibition caused by a molecular Lid (residues 161-203) that shelters the substrate-binding surface. In the activated state, the regulatory domain rotates (~40°) around Ser144, which links both domains. Rotation removes the C-terminus from the cAMP site and relieves the autoinhibited state. Also, the molecular Lid refolds and creates an activator binding site. A conserved residue, His173, was mutated into Lys in the Lid, and during an MD trajectory of the activated state, positioned itself near an acetyl donor molecule in the catalytic domain, suggesting a direct mechanism for acetylation. This study describes the allosteric framework for Mt-PatA and prerequisite intermediate states that permit long-distance signal transmission.

In-silico design of peptide inhibitors of K-Ras target in cancer disease.

Cancer is a leading cause of death, over one million individuals analyzed, and around 500,000 deaths happen due to cancer every year alone in the United States. The Ras is a significant protein in the signaling transduction pathways and has a leading role in cell proliferation. Above 30% of all human tumors arises due to the mutations in genes that encode a Ras protein that operate signaling cascades necessary for malignant transformation, tumor angiogenesis, and metastasis. The Ras gene family comprised of 36 total genes in human. The N-Ras, K-Ras, and H-Ras are accounted for to assume noticeable function in human cancer. The mutation in K-Ras protein is most commonly found in tumors. K-Ras is the most crucial driver in lung and pancreatic cancers. Among the mutations of N-Ras, H-Ras, and K-Ras, the mutant K-Ras is the most prevalent target for the development of Lungs, colon, and pancreatic cancers. The study aimed to develop the peptide inhibitors of the K-Ras G12D. The crystal structure of the mutant K-Ras/R11.1.6 G12D complex was retrieved from the protein databank. The protein R11.1.6 directly blocks interaction with Raf and diminishes signaling through the Raf-MEK-ERK signaling pathway. Here, in this study, we designed novel peptides from the truncated reference peptide (R11.1.6) through residue scan methodology. The top ten designed peptides (based on binding free energies) were subjected to molecular dynamics simulations using AMBER to evaluate stability. Our results indicate that the top ten selected peptides have strong interactions with K-Ras than the reference peptide (R11.1.6) and have the potency to prevent the binding of Raf and K-Ras.Communicated by Ramaswamy H. Sarma.

Laboratory Evolution of GH11 Endoxylanase Through DNA Shuffling: Effects of Distal Residue Substitution on Catalytic Activity and Active Site Architecture.

Endoxylanase with high specific activity, thermostability, and broad pH adaptability is in huge demand. The mutant library of GH11 endoxylanase was constructed via DNA shuffling by using the catalytic domain of Bacillus amyloliquefaciens xylanase A (BaxA) and Thermomonospora fusca TF xylanase A (TfxA) as parents. A total of 2,250 colonies were collected and 756 of them were sequenced. Three novel mutants (DS153: N29S, DS241: S31R and DS428: I51V) were identified and characterized in detail. For these mutants, three residues of BaxA were substituted by the corresponding one of TfxA_CD. The specific activity of DS153, DS241, and DS428 in the optimal condition was 4.54, 4.35, and 3.9 times compared with the recombinant BaxA (reBaxA), respectively. The optimum temperature of the three mutants was 50°C. The optimum pH for DS153, DS241, and DS428 was 6.0, 7.0, and 6.0, respectively. The catalytic efficiency of DS153, DS241, and DS428 enhanced as well, while their sensitivity to recombinant rice xylanase inhibitor (RIXI) was lower than that of reBaxA. Three mutants have identical hydrolytic function as reBaxA, which released xylobiose-xylopentaose from oat spelt, birchwood, and beechwood xylan. Furthermore, molecular dynamics simulations were performed on BaxA and three mutants to explore the precise impact of gain-of-function on xylanase activity. The tertiary structure of BaxA was not altered under the substitution of distal residues (N29S, S31R, and I51V); it induced slightly changes in active site architecture. The distal impact rescued the BaxA from native conformation ("closed state") through weakening interactions between "gate" residues (R112, N35 in DS241 and DS428; W9, P116 in DS153) and active site residues (E78, E172, Y69, and Y80), favoring conformations with an "open state" and providing improved activity. The current findings would provide a better and more in-depth understanding of how distal single residue substitution improved the catalytic activity of xylanase at the atomic level.

Allosteric Modulation of Intrinsically Disordered Proteins.

The allosteric property of globular proteins is applauded as their intrinsic ability to regulate distant sites, and this property further plays a critical role in a wide variety of cellular regulatory mechanisms. Recent advancements and studies have revealed the manifestation of allostery in intrinsically disordered proteins or regions as allosteric sites present within or mediated by IDP/IDRs facilitates the signaling interactions for various biological mechanisms which would otherwise be impossible for globular proteins to regulate. This thematic review has highlighted the biological outcomes that can be achieved by the mechanism of allosteric regulation of intrinsically disordered proteins or regions. The similar mechanism has been implemented on Adenovirus 5 early region 1A and tumor apoptosis protein p53 in correspondence with other partners in binary and ternary complexes, which are the subject of the current review. Both these proteins regulate once they bind to their partners, consequently, forming either a binary or a ternary complex. Allosteric regulation by IDPs is currently a subject undergoing intense study, and the ongoing research work will ensure a better understanding of precision and efficiency of cellular regulation by them. Allosteric regulation mechanism can also be researched by intrinsically disordered protein-specific force field.

Gain-of-Function SHP2 E76Q Mutant Rescuing Autoinhibition Mechanism Associated with Juvenile Myelomonocytic Leukemia.

Juvenile myelomonocytic leukemia (JMML) is an invasive myeloproliferative neoplasm and is a childhood disease with very high clinical lethality. The SHP2 is encoded by the PTPN11 gene, which is a nonreceptor (pY)-phosphatase and mutation causes JMML. The structural hierarchy of SHP2 includes protein tyrosine phosphatase domain (PTP) and Src-homology 2 domain (N-SH2 and C-SH2). Somatic mutation (E76Q) in the interface of SH2-PTP domain is the most commonly identified mutation found in up to 35% of patients with JMML. The mechanism of this mutant associated with JMML is poorly understood. Here, molecular dynamics simulation was performed on wild-type and mutant (E76Q) of SHP2 to explore the precise impact of gain-of-function on PTP's activity. Consequently, such impact rescues the SHP2 protein from autoinhibition state through losing the interface interactions of Q256/F7 and S502/Q76 or weakening interactions of Q256/R4, Q510/G60, and Q506/A72 between N-SH2 and PTP domains. The consequences of these interactions further relieve the D'E loop away from the PTP catalytic site. The following study would provide a mechanistic insight for better understanding of how individual SHP2 mutations alter the PTP's activity at the atomic level.