Computer‐Guided Design,Synthesis, and Biological Evaluation of Quinoxalinebisarylureas as FLT3 Inhibitors

Activating mutations of FMS‐like tyrosine kinase 3 (FLT3) are present in ～30 % of patients with acute myeloid leukemia (AML) and are associated with poor prognosis. Point mutations in the tyrosine kinase domain (TKD) are observed as primary mutations or are acquired as secondary mutations in FLT3 with internal tandem duplications (ITDs) after treatment with tyrosine kinase inhibitors (TKIs). Although dozens of potent inhibitors against FLT3 ITD have been reported, activating TKD point mutations, especially at residues F691 and D835, remain the leading cause for therapy resistance, highlighting the consistent need for new potent inhibitors. Herein we report the identification and characterization of novel quinoxaline‐based FLT3 inhibitors. We used the pharmacophore features of diverse known inhibitors as a starting point for a new optimization algorithm for type II TKIs, starting from an in silico library pharmacophore search and induced‐fit docking in the known FLT3 structure. This led to the design of a set of diverse quinoxalinebisarylureas, which were profiled in an FLT3 kinase activity assay. The most promising compounds were further evaluated in a zebrafish embryo phenotype assay.     

Detection of FLT3 Oncogene Mutations in Acute Myeloid Leukemia Using Conformation Sensitive Gel Electrophoresis

FLT3 (fms-related tyrosine kinase 3) is a receptor tyrosine kinase class III that is expressed on by early hematopoietic progenitor cells and plays an important role in hematopoietic stem cell proliferation, differentiation and survival. FLT3 is also expressed on leukemia blasts in most cases of acute myeloid leukemia (AML). In order to determine the frequency of FLT3 oncogene mutations, we analyzed genomic DNA of adult de novo acute myeloid leukemia (AML). Polymerase chain reaction (PCR) and conformation-sensitive gel electrophoresis (CSGE) were used for FLT3 exons 11, 14, and 15, followed by direct DNA sequencing. Two different types of functionally important FLT 3 mutations have been identified. Those mutations were unique to patients with inv(16), t(15:17) or t(8;21) and comprised fifteen cases with internal tandem duplication (ITD) mutation in the juxtamembrane domain and eleven cases with point mutation (exon 20, Asp835Tyr). The high frequency of the flt3 proto-oncogene mutations in acute myeloid leukemia AML suggests a key role for the receptor function. The association of FLT3 mutations with chromosomal abnormalities invites speculation as to the link between these two changes in the pathogenesis of acute myeloid leukemiaAML. Furthermore, CSGE method has shown to be a rapid and sensitive screening method for detection of nucleotide alteration in FLT3 gene. Finally, this study reports, for the first time in Saudi Arabia, mutations in the human FLT3 gene in acute myeloid leukemia AML patients.     

Targeted Therapeutic Approach Based on Understanding of Aberrant Molecular Pathways Leading to Leukemic Proliferation in Patients with Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogenous hematopoietic neoplasm with various genetic abnormalities in myeloid stem cells leading to differentiation arrest and accumulation of leukemic cells in bone marrow (BM). The multiple genetic alterations identified in leukemic cells at diagnosis are the mainstay of World Health Organization classification for AML and have important prognostic implications. Recently, understanding of heterogeneous and complicated molecular abnormalities of the disease could lead to the development of novel targeted therapeutic agents. In the past years, gemtuzumab ozogamicin, BCL-2 inhibitors (venetovlax), IDH 1/2 inhibitors (ivosidenib and enasidenib) FLT3 inhibitors (midostaurin, gilteritinib, and enasidenib), and hedgehog signaling pathway inhibitors (gladegib) have received US Food and Drug Administration (FDA) approval for the treatment of AML. Especially, AML patients with elderly age and/or significant comorbidities are not currently suitable for intensive chemotherapy. Thus, novel therapeutic planning including the abovementioned target therapies could lead to improve clinical outcomes in the patients. In the review, we will present various important and frequent molecular abnormalities of AML and introduce the targeted agents of AML that received FDA approval based on the previous studies.     

Molecular Modeling Studies of N-phenylpyrimidine-4-amine Derivatives for Inhibiting FMS-like Tyrosine Kinase-3

Overexpression and frequent mutations in FMS-like tyrosine kinase-3 (FLT3) are considered risk factors for severe acute myeloid leukemia (AML). Hyperactive FLT3 induces premature activation of multiple intracellular signaling pathways, resulting in cell proliferation and anti-apoptosis. We conducted the computational modeling studies of 40 pyrimidine-4,6-diamine-based compounds by integrating docking, molecular dynamics, and three-dimensional structure–activity relationship (3D-QSAR). Molecular docking showed that K644, C694, F691, E692, N701, D829, and F830 are critical residues for the binding of ligands at the hydrophobic active site. Molecular dynamics (MD), together with Molecular Mechanics Poison–Boltzmann/Generalized Born Surface Area, i.e., MM-PB(GB)SA, and linear interaction energy (LIE) estimation, provided critical information on the stability and binding affinity of the selected docked compounds. The MD study suggested that the mutation in the gatekeeper residue F691 exhibited a lower binding affinity to the ligand. Although, the mutation in D835 in the activation loop did not exhibit any significant change in the binding energy to the most active compound. We developed the ligand-based comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) models. CoMFA (q² = 0.802, r² = 0.983, and QF32 = 0.698) and CoMSIA (q² = 0.725, r² = 0.965 and QF32 = 0.668) established the structure–activity relationship (SAR) and showed a reasonable external predictive power. The contour maps from the CoMFA and CoMSIA models could explain valuable information about the favorable and unfavorable positions for chemical group substitution, which can increase or decrease the inhibitory activity of the compounds. In addition, we designed 30 novel compounds, and their predicted pIC₅₀ values were assessed with the CoMSIA model, followed by the assessment of their physicochemical properties, bioavailability, and free energy calculation. The overall outcome could provide valuable information for designing and synthesizing more potent FLT3 inhibitors.     

FLT3 Tyrosine Kinase Inhibitors for the Treatment of Fit and Unfit Patients with FLT3-Mutated AML: A Systematic Review

FLT3-mutated acute myeloid leukemia accounts for around 30% of acute myeloid leukemia (AML). The mutation carried a poor prognosis until the rise of tyrosine kinase inhibitors (TKIs). New potent and specific inhibitors have successfully altered the course of the disease, increasing the complete response rate and the survival of patients with FLT3-mutated AML. The aim of this article is to review all the current knowledge on these game-changing drugs as well as the unsolved issues raised by their use for fit and unfit FLT3-mutated AML patients. To this end, we analyzed the results of phase I, II, III clinical trials evaluating FLT3-TKI both in the first-line, relapse monotherapy or in combination referenced in the PubMed, the American Society of Hematology, the European Hematology Association, and the Clinicaltrials.gov databases, as well as basic science reports on TKI resistance from the same databases. The review follows a chronological presentation of the different trials that allowed the development of first- and second-generation TKI and ends with a review of the current lines of evidence on leukemic blasts resistance mechanisms that allow them to escape TKI.     

Rationale for a Combination Therapy with the STAT5 Inhibitor AC-4-130 and the MCL1 Inhibitor S63845 in the Treatment of FLT3-Mutated or TET2-Mutated Acute Myeloid Leukemia

The FMS-like tyrosine kinase 3 (FLT3) gene is mutated in one-third of patients with de novo acute myeloid leukemia (AML). Mutated FLT3 variants are constitutively active kinases signaling via AKT kinase, MAP kinases, and STAT5. FLT3 inhibitors have been approved for the treatment of FLT3-mutated AML. However, treatment response to FLT3 inhibitors may be short-lived, and resistance may emerge. Compounds targeting STAT5 may enhance and prolong effects of FLT3 inhibitors in this subset of patients with FLT3-mutated AML. Here STAT5-inhibitor AC-4-130, FLT3 inhibitor midostaurin (PKC412), BMI-1 inhibitor PTC596, MEK-inhibitor trametinib, MCL1-inhibitor {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845, and BCL-2 inhibitor venetoclax were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells grown in the absence or presence of bone marrow stroma. Synergistic effects on cell viability were detected in both FLT3-mutated and FLT3-wild-type AML cells treated with AC-4-130 in combination with the MCL1 inhibitor {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845. AML patient samples with a strong response to AC-4-130 and {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845 combination treatment were characterized by mutated FLT3 or mutated TET2 genes. Susceptibility of AML cells to AC-4-130, PTC596, trametinib, PKC412, and venetoclax was altered in the presence of HS-5 stroma. Only the MCL1 inhibitor {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845 induced cell death with equal efficacy in the absence or presence of bone marrow stroma. The combination of the STAT5-inhibitor AC-4-130 and the MCL1 inhibitor {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845 may be an effective treatment targeting FLT3-mutated or TET2-mutated AML.     

Emerging Targeted Therapy for Specific Genomic Abnormalities in Acute Myeloid Leukemia

We describe recent updates of existing molecular-targeting agents and emerging novel gene-specific strategies. FLT3 and IDH inhibitors are being tested in combination with conventional chemotherapy for both medically fit patients and patients who are ineligible for intensive therapy. FLT3 inhibitors combined with non-cytotoxic agents, such as BCL-2 inhibitors, have potential therapeutic applicability. The menin-MLL complex pathway is an emerging therapeutic target. The pathway accounts for the leukemogenesis in AML with MLL-rearrangement, NPM1 mutation, and NUP98 fusion genes. Potent menin-MLL inhibitors have demonstrated promising anti-leukemic effects in preclinical studies. The downstream signaling molecule SYK represents an additional target. However, the TP53 mutation continues to remain a challenge. While the p53 stabilizer APR-246 in combination with azacitidine failed to show superiority compared to azacitidine monotherapy in a phase 3 trial, next-generation p53 stabilizers are now under development. Among a number of non-canonical approaches to TP53-mutated AML, the anti-CD47 antibody magrolimab in combination with azacitidine showed promising results in a phase 1b trial. Further, the efficacy was somewhat better in patients with the TP53 mutation. Although clinical evidence has not been accumulated sufficiently, targeting activating KIT mutations and RAS pathway-related molecules can be a future therapeutic strategy.     

Virtual Alanine Scan of the Main Protease Active Site in Severe Acute Respiratory Syndrome Coronavirus 2

Recently, inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) have been proposed as potential therapeutic agents for COVID-19. Studying effects of amino acid mutations in the conformation of drug targets is necessary for anticipating drug resistance. In this study, with the structure of the SARS-CoV-2 Mpro complexed with a non-covalent inhibitor, we performed molecular dynamics (MD) simulations to determine the conformation of the complex when single amino acid residue in the active site is mutated. As a model of amino acid mutation, we constructed mutant proteins with one residue in the active site mutated to alanine. This method is called virtual alanine scan. The results of the MD simulations showed that the conformation and configuration of the ligand was changed for mutants H163A and E166A, although the structure of the whole protein and of the catalytic dyad did not change significantly, suggesting that mutations in His163 and Glu166 may be linked to drug resistance.     

Assessment of pseudo-energy potentials by the best-five test: a new use of the three-dimensional profiles of proteins

We propose a new assessment, called the best-five test, for the pseudo- energy potential empirically derived from the protein structural database. The object of the test is the three-dimensional (3D) profiles of proteins, which are directly connected to the pseudo-energy potentials. In the 3D profile, the fitness of each amino acid type is ranked at each residue site of a protein. A site whose native residue type is ranked within the best-five out of 20 amino acids is regarded as satisfactory and the ratio of the satisfactory sites over all the sites of all the proteins examined is indicative of the efficiency of the pseudo-energy potential employed. We applied the test to our potential function consisting of four terms; side-chain packing, hydration, backbone hydrogen-bonding and local conformation, by setting various kinds of definitions for each term. Through this test, the validity of the minus average operation is confirmed, where the energy level of potential functions is adjusted by referring to the random- environmental state of the proteins. Especially in the side-chain packing function, the success ratio increases from about 30 to 50% with this operation. Failure without the operation is ascribed to bulky hydrophobic residues, which almost always occupy higher ranking positions in the 3D profile table. A maximum success ratio of 55.6% was attained with the final potential set consisting of the above four terms. The efficiency of the final set was further checked in the fold- recognition test for distantly related proteins. The best-five test is a new use of the 3D profile table for assessing the ability of the pseudo-energy potentials.      

The role of Asn14 in the stability and conformation of the reactive-site loop of winged bean chymotrypsin inhibitor: crystal structures of two point mutants Asn14{->}Lys and Asn14{->}Asp

A double-headed chymotrypsin inhibitor, WCI, from winged beanseeds was cloned for structural and biochemical studies. Theinhibitor was subjected to two point mutations at a conservedposition, Asn14. This residue, known to have a pivotal rolein stabilizing the first reactive-site loop (Gln63–Phe68)of the inhibitor, is highly conserved in the sequences of theother members of Kunitz (STI) family as well as in the sequencesof Kazal family of serine protease inhibitors. The mutants,N14K and N14D, were subjected to biochemical assay and theircharacteristics were compared with those of the recombinantinhibitor (rWCI). Crystallographic studies of the recombinantand the mutant proteins are discussed. These studies were primarilyaimed at understanding the importance of the protein scaffoldingtowards the conformational rigidity of the reactive-site loop.Our analysis reveals that, as the Lys14 side chain takes anunusual fold in N14K and the Asp14 side chain in N14D interactswith the loop residues by water-mediated hydrogen bonds, thecanonical conformation of the loop has remained effectivelyintact in both the mutant structures. However, minor alterationssuch as a 2-fold increase in the inhibitory affinity towardsthe cognate enzyme were observed.     

Knowledge-based potential defined for a rotamer library to design protein sequences

A knowledge-based potential for a rotamer library was developedto design protein sequences. Protein side-chain conformationsare represented by 56 templates. Each of their fitness to agiven structural site-environment is evaluated by a combinedfunction of the three knowledge-based terms, i.e. two-body side-chainpacking, one-body hydration and local conformation. The numberof matches between the native sequence and the structural site-environmentin the database and that of the virtually settled mismatches,counted in advance, were transformed into the energy scores.In the best-14 test (assessment for the reproduction abilityof the native rotamer on its structural site within a quarterof 56 fitness rank positions), the structural stability analysison mutants of human and T4 lysozymes and the inverse-foldingsearch by a structure profile against the sequence database,this function performs better than the function deduced withthe conventional normalization and our previously developedfunction. Targeting various structural motifs, de novo sequencedesign was conducted with the function. The sequences thus obtainedexhibit reasonable molecular masses and hydrophobic/hydrophilicpatterns similar to the native sequences of the target and actas if they were the homologs to the target proteins in BLASTPsearch. This significant improvement is discussed in terms ofthe reference state for normalization and the crucial role ofshort-range repulsion to prohibit residue bumps.     

Engineering the Ligand Specificity of the Human Galectin-1 by Incorporation of Tryptophan Analogues

Galectin-1 is a β-galactoside-binding lectin with manifold biological functions. A single tryptophan residue (W68) in its carbohydrate binding site plays a major role in ligand binding and is highly conserved among galectins. To fine tune galectin-1 specificity, we introduced several non-canonical tryptophan analogues at this position of human galectin-1 and analyzed the resulting variants using glycan microarrays. Two variants containing 7-azatryptophan and 7-fluorotryptophan showed a reduced affinity for 3’-sulfated oligosaccharides. Their interaction with different ligands was further analyzed by fluorescence polarization competition assay. Using molecular modeling we provide structural clues that the change in affinities comes from modulated interactions and solvation patterns. Thus, we show that the introduction of subtle atomic mutations in the ligand binding site of galectin-1 is an attractive approach for fine-tuning its interactions with different ligands.     

The molecular basis for A-site mutations conferring aminoglycoside resistance: relationship between ribosomal susceptibility and X-ray crystal structures

Aminoglycoside antibiotics target the 16S ribosomal RNA (rRNA) bacterial A site and induce misreading of the genetic code. Point mutations of the ribosomal A site may confer resistance to aminoglycoside antibiotics. The influence of bacterial mutations (introduced by site-directed mutagenesis) on ribosomal drug susceptibility was investigated in vivo by determination of minimal inhibitory concentrations. To determine the origin of the various resistance phenotypes at a molecular level, the in vivo results were compared with the previously published crystal structures of paromomycin, tobramycin, and geneticin bound to oligonucleotides containing the minimal A site. Two regions appear crucial for binding in the A site: the single adenine residue at position 1408 and the non-Watson-Crick U1406.U1495 pair. The effects of mutations at those positions are modulated by the nature of the substituent at position 6' (either hydroxy or ammonium group) on ring I, by the number of positive charges on the antibiotic, and by the linkage between rings I and III (either 4,5 or 4,6). In particular, the analysis demonstrates: 1) that the C1409-G1491 to A1409-U1491 polymorphism (observed in 15 % of bacteria) is not associated with resistance, which indicates that it does not affect the stacking of ring I on residue 1491, 2) that the high-level resistance to 6'-NH3+ aminoglycosides exhibited by the A1408G mutation most probably results from the inability of ring I forming a pseudo base pair with G1408, which prevents its insertion inside the A site helix, and 3) that mutations of the uracil residues forming the U1406.U1495 pair either to cytosine or to adenine residues mostly confer low to moderate levels of drug resistance, whereas the U1406C/U1495A double mutation confers high-level resistance (except for neomycin), which suggests that aminoglycoside binding to the wild-type A site and its functional consequences strongly depend on a particular geometry of the U1406.U1495 pair. The relationships between the resistance phenotypes observed in vivo and the interactions described at the molecular level define the biological importance of the different structural interactions observed by X-ray crystallography studies.     

Improving Binding Affinity and Stability of Peptide Ligands by Substituting Glycines with D‐Amino Acids

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer‐related protease urokinase‐type plasminogen activator (uPA), we observed a glycine residue that has a positive ? dihedral angle when bound to the target. We hypothesized that replacing it with a D ‐amino acid, which favors positive ? angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D ‐serine in the bicyclic peptide indeed improved inhibitory activity (1.75‐fold) and stability (fourfold). X‐ray‐structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ? angles are found in many protein‐bound peptides. These results suggest that the glycine‐to‐D ‐amino acid mutagenesis strategy could be broadly applied.     

Mechanism of binding of fluoroquinolones to the quinolone resistance-determining region of DNA gyrase: towards an understanding of the molecular basis of quinolone resistance

We have studied the bacterial resistance to fluoroquinolones that arises as a result of mutations in the DNA gyrase target protein. Although it is known that DNA gyrase is a target of quinolone antibacterial agents, the molecular details of the quinolone-gyrase interaction remain unclear. The mode of binding of ciprofloxacin, levofloxacin, and moxifloxacin to DNA gyrase was analyzed by means of docking calculations over the surface of the QRDR of GyrA. The analysis of these binding models allows study of the resistance mechanism associated with gyrA mutations more commonly found in E. coli fluoroquinolone-resistant strains at the atomic level. Asp87 was found to be critical in the binding of these fluoroquinolones because it interacts with the positively charged nitrogens in these bactericidal drugs. The role of the other most common mutations at amino acid codon Ser83 can be explained through the contacts that the side chain of this residue establishes with fluoroquinolone molecules. Finally, our results strongly suggest that, although Arg121 has never been found to be associated with fluoroquinolone resistance, this residue makes a pivotal contribution to the binding of the antibiotic to GyrA and to defining its position in the QRDR of the enzyme.     

A 2.3 A resolution structure of chymosin complexed with a reduced bond inhibitor shows that the active site beta-hairpin flap is rearranged when compared with the native crystal structure

In the crystal structure of uncomplexed native chymosin, the beta- hairpin at the active site, known as 'the flap', adopts a different conformation from that of other aspartic proteinases. This conformation would prevent the mode of binding of substrates/inhibitors generally found in other aspartic proteinase complexes. We now report the X-ray analysis of chymosin complexed with a reduced bond inhibitor CP-113972 (2R,3S)-isopropyl 3-[(L-prolyl-p-iodo-L-phenylalanyl-S-methyl- cysteinyl)amino-4]-cyclohexy l-2-hydroxybutanoate at 2.3 A resolution in a novel crystal form of spacegroup R32. The structure has been refined by restrained least-squares methods to a final R-factor of 0.19 for a total of 11 988 independent reflections in the resolution range 10 to 2.3 A. The extended beta-strand conformation of the inhibitor allows hydrogen bonds within the active site, while its sidechains make both electrostatic and hydrophobic interactions with residues lining the specificity pockets S4-->S1. The flap closes over the active site cleft in a way that closely resembles that of other previously determined aspartic proteinase inhibitor complexes. We conclude that the usual position and conformation of the flap found in other aspartic proteinases is available to native chymosin. The conformation observed in the native crystal form may result from intermolecular interactions between symmetry-related molecules in the crystal lattice.      

In silico mutations and molecular dynamics studies on a winged bean chymotrypsin inhibitor protein

Winged bean chymotrypsin inhibitor (WCI) has an intruding residueAsn14 that plays a crucial role in stabilizing the reactivesite loop conformation. This residue is found to be conservedin the Kunitz (STI) family of serine protease inhibitors. Tounderstand the contribution of this scaffolding residue on thestability of the reactive site loop, it was mutated in silicoto Gly, Ala, Ser, Thr, Leu and Val and molecular dynamics (MD)simulations were carried out on the mutants. The results ofMD simulations reveal the conformational variability and rangeof motions possible for the reactive site loop of differentmutants. The N-terminus side of the scissile bond, which isclose to a ß-barrel, is conformationally less variable,while the C-terminus side, which is relatively far from anysuch secondary structural element, is more variable and needsstability through hydrogen-bonding interactions. The simulatedstructures of WCI and the mutants were docked in the peptide-bindinggroove of the cognate enzyme chymotrypsin and the ability toform standard hydrogen-bonding interactions at P3, P1 and P2'residues were compared. The results of the MD simulations coupledwith docking studies indicate that hydrophobic residues likeLeu and Val at the 14th position are disruptive for the integrityof the reactive site loop, whereas a residue like Thr, whichcan stabilize the C-terminus side of the scissile bond, canbe predicted at this position. However, the size and chargeof the Asn residue made it most suitable for the best maintenanceof the integrity of the reactive site loop, explaining its conservednature in the family. Received October 17, 2002; revised June 6, 2003; accepted June 19, 2003.     

IGMH-based research on the intramolecular weak interaction of TKX-50

To further comprehensively study the intramolecular weak interaction of the gaseous TKX-50 molecule, the two conformations of the TKX-50 molecule were analyzed via the Independent Gradient Model based on the Hirshfeld Partition (IGMH) method based on the B3LYP/6-311 g (d,p) level for geometry optimization and for single point energy. The conclusions manifest that the weak interactions between these fragments are mainly composed of hydrogen bonds and van der Waals interactions. From the strength of inter-fragment interactions formed by contributing atomic pairs and their percentage contributions, these H bonds, together with dispersion-dominated weak interactions provided by non-direct facing atomic pairs near these H bonds, dominate the inter-fragment interaction resulting in the stability of the molecular structure. Meanwhile, the weak interactions enclosed by end-atoms of two fragments not only include the contributions provided by inter-fragment atomic pairs of two fragments but also include the contributions provided by intra-fragment atomic pairs of fragment 1. For conformation II, due to the H transfer between fragments, a pair of symmetric H bonds at the corresponding regions are extremely strong to reach the level of covalent bond while the two groups −OH at the other end become looser, resulting in conformation II own lower energy. Differences in inter-fragment interactions between two conformations were essentially brought by the stronger electron-withdrawing ability of atom O than that of atom N.     

Molecular Dynamic Simulation Insights into the Normal State and Restoration of p53 Function

As a tumor suppressor protein, p53 plays a crucial role in the cell cycle and in cancer prevention. Almost 50 percent of all human malignant tumors are closely related to a deletion or mutation in p53. The activity of p53 is inhibited by over-active celluar antagonists, especially by the over-expression of the negative regulators MDM2 and MDMX. Protein-protein interactions, or post-translational modifications of the C-terminal negative regulatory domain of p53, also regulate its tumor suppressor activity. Restoration of p53 function through peptide and small molecular inhibitors has become a promising strategy for novel anti-cancer drug design and development. Molecular dynamics simulations have been extensively applied to investigate the conformation changes of p53 induced by protein-protein interactions and protein-ligand interactions, including peptide and small molecular inhibitors. This review focuses on the latest MD simulation research, to provide an overview of the current understanding of interactions between p53 and its partners at an atomic level.     

Assessing the Effect of Loop Mutations in the Folding Space of β2-Microglobulin with Molecular Dynamics Simulations

We use molecular dynamics simulations of a full atomistic Gō model to explore the impact of selected DE-loop mutations (D59P and W60C) on the folding space of protein human β₂-microglobulin (Hβ₂m), the causing agent of dialysis-related amyloidosis, a conformational disorder characterized by the deposition of insoluble amyloid fibrils in the osteoarticular system. Our simulations replicate the effect of mutations on the thermal stability that is observed in experiments in vitro. Furthermore, they predict the population of a partially folded state, with 60% of native internal free energy, which is akin to a molten globule. In the intermediate state, the solvent accessible surface area increases up to 40 times relative to the native state in 38% of the hydrophobic core residues, indicating that the identified species has aggregation potential. The intermediate state preserves the disulfide bond established between residue Cys25 and residue Cys80, which helps maintain the integrity of the core region, and is characterized by having two unstructured termini. The movements of the termini dominate the essential modes of the intermediate state, and exhibit the largest displacements in the D59P mutant, which is the most aggregation prone variant. PROPKA predictions of pK_a suggest that the population of the intermediate state may be enhanced at acidic pH explaining the larger amyloidogenic potential observed in vitro at low pH for the WT protein and mutant forms.