Rational design of monomeric IL37 variants guided by stability and dynamical analyses of IL37 dimers.

IL37 plays important roles in the regulation of innate immunity and its oligomeric status is critical to these roles. In its monomeric state, IL37 can effectively inhibit the inflammatory response of IL18 by binding to IL18Rα, a capacity lost in its dimeric form, underlining the pivotal role of the oligomeric status of IL37 in its anti-inflammatory action. Until now, two IL37 dimer structures have been deposited in PDB, reflecting a substantial difference in their dimer interfaces. Given this discrepancy, we analyzed the PDB structures of the IL37 dimer (PDB IDs: 6ncu, 5hn1) along with a AF2-multimer prediction by molecular dynamics (MD) simulations. Results showed that the 5hn1 and AF2-predicted dimers have the same interface and stably maintained their conformations throughout simulations, while the recent IL37 dimer (PDB ID: 6ncu) with a different interface did not, proposing a possible issue with the recent IL37 dimer structure (6ncu). Next, focusing on the stable dimer structures, we have identified five critical positions of V71/Y85/I86/E89/S114, three new positions compared to the literature, that would reduce dimer stability without affecting the monomer structure. Two quintuple mutants were tested by MD simulations and showed partial or complete dissociation of the dimer. Overall, the insights gained from this study reinforce the validity of the 5hn1 and AF2 multimer structures, while also advancing our understanding of the IL37 dimer interface through the generation of monomer-locked IL37 variants.

Computational completion of the Aurora interaction region of N-Myc in the Aurora a kinase complex.

Inhibiting protein-protein interactions of the Myc family is a viable pharmacological strategy for modulation of the levels of Myc oncoproteins in cancer. Aurora A kinase (AurA) and N-Myc interaction is one of the most attractive targets of this strategy because formation of this complex blocks proteasomal degradation of N-Myc in neuroblastoma. Two crystallization studies have captured this complex (PDB IDs: 5g1x, 7ztl), partially resolving the AurA interaction region (AIR) of N-Myc. Prompted by the missing N-Myc fragment in these crystal structures, we modeled the complete structure between AurA and N-Myc, and comprehensively analyzed how the incomplete and complete N-Myc behave in complex by molecular dynamics simulations. Molecular dynamics simulations of the incomplete PDB complex (5g1x) repeatedly showed partial dissociation of the short N-Myc fragment (61-89) from the kinase. The missing N-Myc (19-60) fragment was modeled utilizing the N-terminal lobe of AurA as the protein-protein interaction surface, wherein TPX2, a well-known partner of AurA, also binds. Binding free energy calculations along with flexibility analysis confirmed that the complete AIR of N-Myc stabilizes the complex, accentuating the N-terminal lobe of AurA as a binding site for the missing N-Myc fragment (19-60). We further generated additional models consisting of only the missing N-Myc (19-60), and the fused form of TPX2 (7-43) and N-Myc (61-89). These partners also formed more stable interactions with the N-terminal lobe of AurA than did the incomplete N-Myc fragment (61-89) in the 5g1x complex. Altogether, this study provides structural insights into the involvement of the N-terminus of the AIR of N-Myc and the N-terminal lobe of AurA in formation of a stable complex, reflecting its potential for effective targeting of N-Myc.

Effect of the switch status of Helicobacter pylori outer inflammatory protein A on gastric diseases.

Helicobacter pylori OipA (Outer Inflammatory Protein A) is an outer membrane protein that takes role in the adherence and colonization to the stomach. oipA gene expression is regulated by the slipped-strand mispairing mechanism through a hypermutable CT dinucleotide repeat motif in the 5΄ region. Alterations in the CT number repeats cause frame-shift mutations to result in phase variation of oipA expression. While a functional "On" status has been recognized as a risk factor for peptic ulcer diseases and gastric cancer in many studies, some controversial findings still exist. To this end, this study compiled the sequence data of oipA from 10 different studies between 2000-2019 and 50 oipA DNA sequences from our own research that examined the relationship between the phase On/Off status of oipA and gastric diseases based on CT repeat number. Overall, we have reached 536 oipA DNA sequences from patients. This large collection of oipA sequences first clarified the absolute conservation of the peptide-pentamer of FWLHA for phase ''On'' status, suggesting this pentamer as a superior marker for the determination of oipA status than counting the number of CT repeats. Combining the sequence and patient data, we have re-analyzed the association between the ''On'' status of oipA and gastric diseases. Our results showed a strong association between oipA ''On'' status and gastric cancer supporting previous findings. We also investigated the AlphaFold2 computed structure of OipA that adopts a beta-barrel fold closely resembling to the autotransporter family of H. pylori. Altogether, this study confirms a strong association between oipA ''On'' statuses and severe gastrointestinal diseases like cancer and provides useful insights into the FWLHA pentamer as an indicator of "On" status of oipA putative autotransporter function rather than CT repeats number.

A small non-interface surface epitope in human IL18 mediates the dynamics and self-assembly of IL18-IL18BP heterodimers.

Interleukin 18 (IL18) is a pro-inflammatory cytokine that modulates innate and adaptive immune responses. IL18 activity is tightly controlled by the constitutively secreted IL18 binding protein (IL18BP). PDB structures of human IL18 showed that a short stretch of amino acids between 68 and 81 adopted a disordered conformation in all IL18-IL18BP complexes while adopting a 310 helical structure in other IL18 structures, including the receptor complexes. The C74 of human IL18, which was reported to form a novel intermolecular disulfide bond in the human tetrameric assembly, is also located in this short epitope. These observations reflected the importance of this short surface epitope for the structure and dynamics of the IL18-IL18BP heterodimers. We have analyzed all known IL18-IL18BP complexes in the PDB by all-atom MD simulations. The analysis also included two computed complex models adopting a helical structure for the surface epitope. Heterodimer simulations showed a stabilizing impact of the small surface region at the helical form by reducing flexibility of the complex backbone. Analysis of the symmetry-related human IL18-IL18BP tetramer showed that the unfolding of this small surface region also contributed to the IL18-IL18BP stability through a completely exposed C74 sidechain to form an intermolecular disulfide bond in the self-assembled human IL18-IL18BP dimer. Our findings showed how the conformation of the short IL18 epitope between amino acids 68 and 81 would affect IL18 activity by mediating the intermolecular interactions of IL18.

Follicular homocysteine as a marker of oocyte quality in PCOS and the role of micronutrients.

PURPOSE: Does follicular homocysteine predict the reproductive potential of oocytes following FSH stimulation in PCOS women? Can it be modulated by dietary interventions? METHODS: This was a prospective, randomized, interventional clinical study. Forty-eight PCOS women undergoing in vitro fertilization at a private fertility clinic were randomized for a dietary supplementation providing micronutrients involved in homocysteine clearance or no treatment. The supplement was assumed 2 months before stimulation until pick-up day. Monofollicular fluids were collected and frozen. After embryo transfer, the fluids from the follicles generating the transferred embryos were thawed and analyzed. RESULTS: Follicular homocysteine showed a negative correlation with clinical pregnancy both in the whole population (r = - 0.298; p = 0.041) and in controls (r = - 0.447, p = 0.053). The support achieved a non-significantly lower concentration of follicular homocysteine (median [IQR]-7.6 [13.2] vs 24.3 [22.9]). Supplemented patients required far less FSH for stimulation (1650 [325] vs 2250 [337], p = 0.00002) with no differences in the number of oocytes collected, MII rate, and fertilization rate. Supplemented patients enjoyed higher blastocyst rate (55% [20.5] vs 32% [16.5]; p = 0.0009) and a trend for improved implantation rate (64% vs 32%; p = 0.0606). Clinical pregnancy rates were 58% vs 33% in controls (p = ns). CONCLUSION: Follicular homocysteine is a suitable reporter that might be investigated as a tool for oocyte-embryo selection. A diet enriched with methyl donors may be useful in PCOS and supplements may also help. These findings may be also true for non-PCOS women, which warrants investigation. The study was approved by the Acibadem University Research Ethics Committee (2017-3-42). Clinical trial retrospective registration number ISRCTN55983518.

Evaluation of AlphaFold structure-based protein stability prediction on missense variations in cancer.

Identifying pathogenic missense variants in hereditary cancer is critical to the efforts of patient surveillance and risk-reduction strategies. For this purpose, many different gene panels consisting of different number and/or set of genes are available and we are particularly interested in a panel of 26 genes with a varying degree of hereditary cancer risk consisting of ABRAXAS1, ATM, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MEN1, MLH1, MRE11, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, and XRCC2. In this study, we have compiled a collection of the missense variations reported in any of these 26 genes. More than a thousand missense variants were collected from ClinVar and the targeted screen of a breast cancer cohort of 355 patients which contributed to this set with 160 novel missense variations. We analyzed the impact of the missense variations on protein stability by five different predictors including both sequence- (SAAF2EC and MUpro) and structure-based (Maestro, mCSM, CUPSAT) predictors. For the structure-based tools, we have utilized the AlphaFold (AF2) protein structures which comprise the first structural analysis of this hereditary cancer proteins. Our results agreed with the recent benchmarks that computed the power of stability predictors in discriminating the pathogenic variants. Overall, we reported a low-to-medium-level performance for the stability predictors in discriminating pathogenic variants, except MUpro which had an AUROC of 0.534 (95% CI [0.499-0.570]). The AUROC values ranged between 0.614-0.719 for the total set and 0.596-0.682 for the set with high AF2 confidence regions. Furthermore, our findings revealed that the confidence score for a given variant in the AF2 structure could alone predict pathogenicity more robustly than any of the tested stability predictors with an AUROC of 0.852. Altogether, this study represents the first structural analysis of the 26 hereditary cancer genes underscoring 1) the thermodynamic stability predicted from AF2 structures as a moderate and 2) the confidence score of AF2 as a strong descriptor for variant pathogenicity.

Brave new surfactant world revisited by thermoalkalophilic lipases: computational insights into the role of SDS as a substrate analog.

Non-ionic surfactants were shown to stabilize the active conformation of thermoalkalophilic lipases by mimicking the lipid substrate while the catalytic interactions formed by anionic surfactants have not been well characterized. In this study, we combined µs-scale molecular dynamics (MD) simulations and lipase activity assays to analyze the effect of ionic surfactant, sodium dodecyl sulfate (SDS), on the structure and activity of thermoalkalophilic lipases. Both the open and closed lipase conformations that differ in geometry were recruited to the MD analysis to provide a broader understanding of the molecular effect of SDS on the lipase structure. Simulations at 298 K showed the potential of SDS for maintaining the active lipase through binding to the sn-1 acyl-chain binding pocket in the open conformation or transforming the closed conformation to an open-like state. Consistent with MD findings, experimental analysis showed increased lipase activity upon SDS incubation at ambient temperature. Notably, the lipase cores stayed intact throughout 2 µs regardless of an increase in the simulation temperature or SDS concentration. However, the surface structures were unfolded in the presence of SDS and at elevated temperature for both conformations. Simulations of the dimeric lipase were also carried out and showed reduced flexibility of the surface structures which were unfolded in the monomer, indicating the insulating role of dimer interactions against SDS. Taken together, this study provides insights into the possible substrate mimicry by the ionic surfactant SDS for the thermoalkalophilic lipases without temperature elevation, underscoring SDS's potential for interfacial activation at ambient temperatures.

Modeling and dynamical analysis of the full-length structure of factor XII with zinc.

Zinc (II), the second most abundant transition metal in blood, binds to the initiator of the contact pathway, factor XII (FXII). This binding induces conformational changes in the structure of FXII eventually leading to its activation. Despite many in vitro and in vivo studies on zinc-mediated activation of FXII, its molecular mechanism remains elusive mainly due to absence of a full-length structural model of FXII. To this end, this study investigated the role of zinc in the structure and dynamics of the full-length structure FXII that was obtained through molecular modeling. We have used four structural templates covering more than 70% of the FXII sequence and the remaining interconnecting regions were built by loop modeling. The resulting full-length structure of FXII contained disordered regions, but in comparison to the AlphaFold (AF) prediction, our full-length model represented a more realistic structure because of the disordered regions which were modeled to yield a more compact full-length structure in our model than the AF structure. Other than the disordered regions, our model and AF prediction were highly similar. The resulting full-length FXII structure was used to generate different systems representing the zinc-bound form (holo). Further to assess the contribution of the disulfide bridges, we also analyzed the apo and holo FXII structures with oxidized or reduced cysteine side-chains. Simulations suggested zinc binding conferred rigidity to the structure, particularly to the light chain of FXII. Zinc binding alone was sufficient to limit the backbone flexibility while 15 disulfide bonds, which were scattered throughout the structure, made a less significant contribution to the backbone rigidity. Altogether our results provide insights into the first realistic full-length structure of FXII focusing on the impact of structural zinc and disulfide bridges in the dynamics of this structure.

Toward Compilation of Balanced Protein Stability Data Sets: Flattening the ΔΔG Curve through Systematic Enrichment.

Often studies analyzing stability data sets and/or predictors ignore neutral mutations and use a binary classification scheme labeling only destabilizing and stabilizing mutations. Recognizing that highly concentrated neutral mutations interfere with data set quality, we have explored three protein stability data sets: S2648, PON-tstab, and the symmetric Ssym that differ in size and quality. A characteristic leptokurtic shape in the ΔΔG distributions of all three data sets including the curated and symmetric ones was reported due to concentrated neutral mutations. To further investigate the impact of neutral mutations on ΔΔG predictions, we have comprehensively assessed the performance of 11 predictors on the PON-tstab data set. Correlation and error analyses showed that all of the predictors performed the best on the neutral mutations, while their performance became gradually worse as the ΔΔG of the mutations departed further from the neutral zone regardless of the direction, implying a bias toward dense mutations. To this end, after unraveling the role of concentrated neutral mutations in biases of stability data sets, we described a systematic enrichment approach to balance the ΔΔG distributions. Before enrichment, mutations were clustered based on their biochemical and/or structural features, and then three mutations were selected from every 2 kcal/mol of each cluster. Upon implementation of this approach by distinct clustering schemes, we generated five subsets varying in size and ΔΔG distributions. All subsets showed improved ΔΔG and frequency distributions. We ultimately reported that the errors toward enriched subsets were higher than those toward the parent data sets, confirming the enrichment of difficult-to-predict mutations in the subsets. In summary, we elaborated the prediction bias toward a concentrated neutral zone and also implemented a rational strategy to tackle this and other forms of biases. Ultimately, this study equipping us with an extended view of shortcomings of stability data sets is a step taken toward development of an unbiased predictor.

Molecular modelling of the FOXO4-TP53 interaction to design senolytic peptides for the elimination of senescent cancer cells.

BACKGROUND: Senescent cells accumulate in tissues over time as part of the natural ageing process and the removal of senescent cells has shown promise for alleviating many different age-related diseases in mice. Cancer is an age-associated disease and there are numerous mechanisms driving cellular senescence in cancer that can be detrimental to recovery. Thus, it would be beneficial to develop a senolytic that acts not only on ageing cells but also senescent cancer cells to prevent cancer recurrence or progression. METHODS: We used molecular modelling to develop a series of rationally designed peptides to mimic and target FOXO4 disrupting the FOXO4-TP53 interaction and releasing TP53 to induce apoptosis. We then tested these peptides as senolytic agents for the elimination of senescent cells both in cell culture and in vivo. FINDINGS: Here we show that these peptides can act as senolytics for eliminating senescent human cancer cells both in cell culture and in orthotopic mouse models. We then further characterized one peptide, ES2, showing that it disrupts FOXO4-TP53 foci, activates TP53 mediated apoptosis and preferentially binds FOXO4 compared to TP53. Next, we show that intratumoural delivery of ES2 plus a BRAF inhibitor results in a significant increase in apoptosis and a survival advantage in mouse models of melanoma. Finally, we show that repeated systemic delivery of ES2 to older mice results in reduced senescent cell numbers in the liver with minimal toxicity. INTERPRETATION: Taken together, our results reveal that peptides can be generated to specifically target and eliminate FOXO4+ senescent cancer cells, which has implications for eradicating residual disease and as a combination therapy for frontline treatment of cancer. FUNDING: This work was supported by the Cancer Early Detection Advanced Research Center at Oregon Health & Science University.

Bridging the Bridging Imidazolate in the Bimetallic Center of the Cu/Zn SOD1 and ALS.

Metallation status of human Cu/Zn superoxide dismutase 1 (SOD1) plays a pivotal role in the pathogenesis of amyotrophic lateral sclerosis (ALS). All of the amino acids found in the bimetallic center have been associated with ALS except for two positions. H63 which forms the bridging imidazolate ion in the bimetallic center and K136 which is not directly involved in coordination but located in the bimetallic center were not reported to be mutated in any of the identified ALS cases. In this study, we investigated the structure and flexibility of five SOD1 variants by using classical molecular dynamics simulations. These variants include three substitutions on the non-ALS-linked positions; H63A, H63R, K136A and ALS-linked positions; G37R, H46R/H48D. We have generated four systems for each variant differing in metallation and presence of the intramolecular disulfide bond. Overall, a total of 24 different dimers including the wild-type were generated and simulated at two temperatures, 298 and 400 K. We have monitored backbone mobility, fluctuations and compactness of the dimer structures to assess whether the hypothetical mutations would behave similar to the ALS-linked variants. Results showed that particularly two mutants, H63R and K136A, drastically affected the dimer dynamics by increasing the fluctuations of the metal binding loops compared with the control mutations. Further, these variants resulted in demetallation of the dimers, highlighting probable ALS toxicity that could be elicited by the SOD1 variants of H63R and K136A. Overall, this study bridges two putative SOD1 positions in the metallic center and ALS, underlining the potential use of atomistic simulations for studying disease variants.

Lipase and Water in a Deep Eutectic Solvent: Molecular Dynamics and Experimental Studies of the Effects of Water-In-Deep Eutectic Solvents on Lipase Stability.

Given the accumulated evidence on the effects of water-in-deep eutectic solvents (DESs) on the solvent nanostructure and the yield of lipase reactions, here we have used molecular dynamics (MD) simulations to delineate the structure and dynamics of thermoalkalophilic lipases in choline chloride/urea-based DES (reline) with varying hydration levels. Results indicated that pure reline almost froze the lipase backbone, while hydrated reline that showed a less ordered nanostructure than the pure form introduced some fluctuations to lipase structures, particularly to the lid domain. Although none of the solvents led to unfolding, solvation by 8 M urea or water when accompanied with elevated temperature caused the most significant loss of secondary structure. Experimental results indicated that lipase incubation in slightly hydrated reline [5% (v/v)] led to the highest level of residual activity, implying interfacial activation. Overall, we report that slightly hydrated reline activates thermoalkalophilic lipases, consistent with the particular MD observation showing enhanced mobility of the lid domain. These insights provided by this study contribute to designing efficient lipase applications in DES-based reaction media, giving cues for customizing water-in-DESs for optimal enzyme stability and activity.

Understanding thermal and organic solvent stability of thermoalkalophilic lipases: insights from computational predictions and experiments.

Bacillus thermocatenulatus lipase (BTL2), a member of the isolated lipase family known as thermoalkalophilic lipases, carries potential for industrial applications owing to its ability to catalyze versatile reactions under extreme conditions. This study investigates the molecular effects of distinct solvents on the stability of BTL2 at different temperatures, aiming to contribute to lipase use in industrial applications. Initially, molecular dynamic (MD) simulations were carried out to address for the molecular impacts of distinct solvents on the structural stability of BTL2 at different temperatures. Two lipase conformations representing the active and inactive forms were simulated in 5 solvents including water, ethanol, methanol, cyclohexane, and toluene. Low temperature simulations showed that polar solvents led to enhanced lid fluctuations compared with non-polar solvents reflecting a more dynamic equilibrium between active and inactive lipase conformations in polar solvents including water, while the overall structure of the lipase in both forms became more rigid in non-polar solvents than they were in polar solvent. Notably, the native lipase fold was maintained in non-polar solvents even at high temperatures, indicating an enhancement of lipase's thermostability in non-polar organic solvents. Next, we conducted experiments for which BTL2 was expressed in a heterologous host and purified to homogeneity, and its thermostability in different solvents was assessed. Parallel to the computational findings, experimental results suggested that non-polar organic solvents contributed to BTL2's thermostability at concentrations as high as 70% (v/v). Altogether, this study provides beneficial insights to the lipase use under extreme conditions. Graphical Abstract.

Comprehensive evaluation of the MM-GBSA method on bromodomain-inhibitor sets.

MM-PB/GBSA methods represent a higher-level scoring theory than docking. This study reports an extensive testing of different MM-GBSA scoring schemes on two bromodomain (BRD) datasets. The first set is composed of 24 BRPF1 complexes, and the second one is a nonredundant set constructed from the PDBbind and composed of 28 diverse BRD complexes. A variety of MM-GBSA schemes were analyzed to evaluate the performance of four protocols with different numbers of minimization and MD steps, 10 different force fields and three different water models. Results showed that neither additional MD steps nor unfixing the receptor atoms improved scoring or ranking power. On the contrary, our results underscore the advantage of fixing receptor atoms or limiting the number of MD steps not only for a reduction in the computational costs but also for boosting the prediction accuracy. Among Amber force fields tested, ff14SB and its derivatives rather than ff94 or polarized force fields provided the most accurate scoring and ranking results. The TIP3P water model yielded the highest scoring and ranking power compared to the others. Posing power was further evaluated for the BRPF1 set. A slightly better posing power for the protocol which uses both minimization and MD steps with a fixed receptor than the one which uses only minimization with a fully flexible receptor-ligand system was observed. Overall, this study provides insights into the usage of the MM-GBSA methods for screening of BRD inhibitors, substantiating the benefits of shorter protocols and latest force fields and maintaining the crystal waters for accuracy.

Translation and cross-cultural adaptation of the extended version of the Nordic musculoskeletal questionnaire into Turkish.

OBJECTIVES: The purpose of this study was to translate and culturally adapt the Extended Version of the Nordic Musculoskeletal Questionnaire (NMQ-E) for use in Turkey. METHODS: The cross-cultural adaptation was achieved by translating the items from the original version, with back-translation performed by independent mother-tongue translators, followed by committee review. Reliability (internal consistency and test-retest) was examined for 132 students (97 females, 35 males; mean±SD age: 19.91±1.24 years, mean±SD body mass index: 21.77±3.31 kg/m2) who completed the NMQ-E twice (with a 1 week interval). Construct validity was analyzed with the Cornell Musculoskeletal Discomfort questionnaire. Cronbach alpha was used to assess internal consistency. Intraclass correlation coefficient (ICC) and prevalence-adjusted bias-adjusted kappa (PABAK) were used to estimate the test-retest reliability. All of the statistical analyses were performed by using SPSS (version 22.0). RESULTS: The Turkish version of the NMQ-E showed adequate internal consistency (Cronbach coefficient α=.78). The test-retest reliability was examined with PABAK and all items showed moderate to almost perfect reliability (PABAK=0.610-0.955), excellent ICC= 0.88 and good construct validity (p<0.001). CONCLUSIONS: The Turkish version of the NMQ-E has applicable psychometric properties, including good test-retest reliability, internal consistency and construct validity.

Comparative Assessment of Seven Docking Programs on a Nonredundant Metalloprotein Subset of the PDBbind Refined.

Extensive usage of molecular docking for computer-aided drug discovery resulted in development of numerous programs with versatile scoring and posing algorithms. Selection of the docking program among these vast number of options is central to the outcome of drug discovery. To this end, comparative assessment studies of docking offer valuable insights into the selection of the optimal tool. Despite the availability of various docking assessment studies, the performance difference of docking programs has not been well addressed on metalloproteins which comprise a substantial portion of the human proteome and have been increasingly targeted for treatment of a wide variety of diseases. This study reports comparative assessment of seven docking programs on a diverse metalloprotein set which was compiled for this study. The refined set of the PDBbind (2017) was screened to gather 710 complexes with metal ion(s) closely located to the ligands (<4 Å). The redundancy was eliminated by clustering and overall 213 complexes were compiled as the nonredundant metalloprotein subset of the PDBbind refined. The scoring, ranking, and posing powers of seven noncommercial docking programs, namely, AutoDock4, AutoDock4Zn, AutoDock Vina, Quick Vina 2, LeDock, PLANTS, and UCSF DOCK6, were comprehensively evaluated on this nonredundant set. Results indicated that PLANTS (80%) followed by LeDock (77%), QVina (76%), and Vina (73%) had the most accurate posing algorithms while AutoDock4 (48%) and DOCK6 (56%) were the least successful in posing. Contrary to their moderate-to-high level of posing success, none of the programs was successful in scoring or ranking of the binding affinities (r2 ≈ 0). Screening power was further evaluated by using active-decoy ligand sets for a large compilation of metalloprotein targets. PLANTS stood out among other programs to be able to enrich the active ligand for every target, underscoring its robustness for screening of metalloprotein inhibitors. This study provides useful information for drug discovery studies targeting metalloproteins.

Insights into an alternative benzofuran binding mode and novel scaffolds of polyketide synthase 13 inhibitors.

Small molecules targeting biosynthesis of mycolic acids in the tuberculosis causing bacterium carry high potential for anti-tuberculosis drug discovery. Hitherto, benzofuran containing molecules were identified to target the thioesterase domain of polyketide synthase 13 (Pks13), one of the crucial constituents of this pathway. Among these, TAM16 was also reported to be highly potent in vivo. Here we performed a multi-stage virtual screening methodology recruiting both ligand- and structure-based screening tools for identification of novel Pks13 inhibitors. The large ZINC15 database comprising more than 1 billion ligands was reduced to 21,277 ligands with benzofuran rings and similarity to TAM16. This collection was screened by docking and the 20 top scoring ligands were further analyzed by molecular dynamics simulations. Molecular mechanics/generalized Born surface area (MM-GBSA) based binding free energy predictions confirmed five molecules in the ZINC15 database lead to remarkable increases in the binding free energy of TAM16. Essentially, the most potent ligand ZINC840169872, which carries a distinct scaffold with a cyclohexane group fused to the furan ring, produced a twofold change in the ligand efficiency of TAM16. Further, assessments of the structure-based tools on five different Pks13-TAM complex structures suggested a high-level agreement with the experiments, substantiating the validity of our methodology for screening Pks13 inhibitors. Overall, these in silico insights into a low energy benzofuran-based scaffold and an alternative binding mode for the benzofuran ring unravel viable strategies to generate potent anti-tuberculosis drugs, accentuating the applications of virtual screening approaches for exploring large compound libraries that cannot be easily accessed by experimentation. Graphical abstract In silico virtual screening of ZINC15 database for the identification of potential inhibitors targeting PKS13.

In silico identification of inhibitors targeting N-Terminal domain of human Replication Protein A.

Replication Protein A (RPA) mediates DNA Damage Response (DDR) pathways through protein-protein interactions (PPIs). Targeting the PPIs formed between RPA and other DNA Damage Response (DDR) mediators has become an intriguing area of research for cancer drug discovery. A number of studies applied different methods ranging from high throughput screening approaches to fragment-based drug design tools to discover RPA inhibitors. Although these methods are robust, virtual screening approaches may be allocated as an alternative to such experimental methods, especially for screening of large libraries. Here we report the comprehensive screening of the large database, ZINC15 composed of ∼750 M compounds and the comparison of the identified ligands with the previously known inhibitors by means of binding affinity and drug-likeness. Initially, a ligand library sharing similarity with a promising inhibitor of the N-terminal domain of the RPA70 subunit (RPA70N) was generated by screening of the ZINC15 library. 46,999 ligands were collected and screened by LeDock which produced a satisfactory correlation with the experimental values (R2 = 0.77). 10 of the top-scoring ligands in LeDock were directly progressed to molecular dynamics (MD) simulations, while 10 additional ligands were also selected based on their LeDock scores and the presence of a functional group that could interact with the key amino acids in the RPA70N cleft. MD simulations were used to predict the binding free energy of the ligands by the MM-PBSA method which produced a high level of agreement with the experiments (R2 = 0.85). Binding free energy predictions pointed out 2 ligands with higher binding affinity than any of the reference inhibitors. Particularly the ligand ZINC000753854163 exhibited superior drug-likeness features than any of the known inhibitors. Overall, this study reports ZINC000753854163 as a possible inhibitor of RPA70N, reflecting its possible use in RPA70N targeted cancer therapy.

Thermostability of the PYL-PP2C Heterodimer Is Dependent on Magnesium: In Silico Insights into the Link between Heat Stress Response and Magnesium Deficiency in Plants.

Magnesium deficiency increases the susceptibility of plants toward heat stress. The correlation between magnesium levels and stress response has been studied at the physiological level; albeit, the molecular explanation to this relationship remains elusive. Among diverse pathways implicated in the heat stress, the abscisic acid (ABA) signal modulates the heat stress response by magnesium dependent phosphatases (PP2Cs). Exclusively, sequestration of PP2Cs by ABA receptors (PYLs) in the heterodimer form activates the stress response through ABA responsive transcription factors. In this study, the molecular interplay between magnesium levels and ABA related heat stress response was investigated. Molecular dynamics simulations have been applied to two different PYL-PP2C heterodimer systems representing normal and magnesium deficient conditions. The heterodimer conformation and stability were delineated at high temperatures mimicking heat stress. Results showed that the thermostability of the heat stress response heterodimer was significantly dependent on the magnesium. Furthermore, a conserved aromatic cluster at the dimer interface acted synergistically with the metal to confer thermostability to the heterodimer structure. These structural insights into one of the possible links between magnesium levels and stress highlight the importance of metal micronutrients for tuning the stability of the stress-related proteins and optimizing tolerance.

RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy.

Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double- or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways.