The Ba/F3 cell line is a widely used model in kinase drug development. Such cells are transformed to depend on a certain kinase for proliferation, and the use of an inhibitor of the kinase thus prevents their growth. We used Ba/F3 cells that expressed mutated FLT3 (FLT3-ITD), a known drug target in acute myeloid leukaemia (AML), to study drug resistance against two potent and selective inhibitors (gilteritinib and FF-10101). The cells could be made resistant to the drugs in concentrations that are similar to those in the plasma of patients, but this often required multiple secondary mutations. Several novel inhibitors, designed to be active against FLT3 mutants were tested but could not inhibit the growth of the resistant Ba/F3 cells. Several hitherto unidentified mutations in FLT3 were discovered that lead to drug resistance. These mutations were further studied using computational tools in order to understand how they lead to drug resistance. The discovery of novel mutations is significant since few patients were tested upon relapse due to lack of therapeutic options. Finally, we discuss the pros and cons of the Ba/F3 cell lines in the context of AML where patients express FLT3-ITD mutations in comparison with other cell lines, when the aim is development of drugs that overcome resistance.

Despite continuous strides forward in drug development, resistance to treatment looms large in the battle against cancer as well as communicable diseases. Chronic myeloid leukemia (CML) is treated with targeted therapy and treatment is personalized when resistance arises. It has been extensively studied and is used as a model for targeted therapy. In this study, we examine combination treatments of type II Abl1 inhibitors and asciminib (an allosteric regulator) through a computational model at patient relevant concentrations. Due to the separate binding sites of type II inhibitors and asciminib, we propose their combination treatment as potentially robust to resistance. We find that the simultaneous cobinding of type II inhibitors and asciminib is high in synergetic combinations. As an aid to designing and comparing combination treatments, we put forward an equation that expands on the previously published effective ratio of IC50 (ERIC). Unlike usual comparisons of IC50 values, ERIC takes patient plasma concentrations into account. This study shows that the product of two ERIC values (ERIC_combo) creates comparable approximations of the effectiveness of combination treatments with low levels of synergy or antagonism at different concentrations. Its simple formulation is done without experiments and requires less computation and input data than the current standard of ZIP values. As such, the new scheme is a useful complement to experiments that deal with synergy in drug use.

Molecular interactions within the electrolyte are important for the ultimate understanding and better design of batteries. Simulation studies can complement experimental findings and give atom-scale details of the intricate solvent-ion and ion-ion interactions. However, compromising between accuracy and calculation speed is a challenge. Here, fully atomistic, quantum mechanics-based simulations of NaClO4 and NaPF6 salts in ethylene carbonate are performed by use of divide-and-conquer DFTB molecular dynamics (DC-DFTB-MD) simulations. The simulations show a clear-cut difference between the solutions, with a much higher tendency for the formation of contact-ion pairs with NaClO4, which reduces the diffusion coefficient of the Na+ ions and the activity coefficient of the solution. Density functional theory and energy decomposition analysis calculations shed light on the reason for the association of Na+ and ClO4 - in contrast to PF6 -, explaining recent experimental findings. The results demonstrate the potential of DC-DFTB-MD for simulations of electrolytes that are difficult to study by force-field-based MD simulations.

Several types of radioactive isotopes are used for cancer treatment. While most are embedded in chelating agents, 223Ra is given as RaCl2 salt and 90Y in microspherical particles. If ionic radionuclides are free, they have the potential to bind to proteins instead of their endogenous ions, interfere with their activity and be transported by them. In this study, a computational approach was used to estimate the binding affinities of Y3+, Ra2+ and Pb2+ (207Pb is the decay product of 223Ra) to proteins, instead of their native cofactors Ca2+ and Mn2+. Y3+ was found to bind strongly to proteins with the ability to replace Ca2+ and to some degree also Mn2+. Ra2+ does not bind to the studied proteins but Pb2+ can replace Ca2+ in Ca2+ binding proteins. A recently identified coordination compound was found to be highly selective for 223Ra.

The Gibbs energy of binding (absolute binding free energy, ABFE) of a drug to proteins in the body determines the drug's affinity to its molecular target and its selectivity. ABFE is challenging to measure, and experimental values are not available for many proteins together with potential drugs and other molecules that bind them. Accurate means of calculating such values are, therefore, highly in demand. Realizing that toxicity and side effects are closely related to off-target binding, here we calculate the ABFE of two drugs, each to multiple proteins, in order to examine whether it is possible to carry out such calculations and achieve the required accuracy. The methods that were used were free energy perturbation with replica exchange molecular dynamics (FEP/REMD) and density functional theory (DFT) with a cluster approach and a simplified model. DFT calculations were supplemented with energy decomposition analysis (EDA). The accuracy of each method is discussed, and suggestions are made for the approach toward better ABFE calculations.

Mutations in FLT3 make this receptor tyrosine kinase overactive. Such mutations found in similar to 30 % of the patients who suffer from acute myeloid leukaemia (AML). FLT3 mediates signalling networks that lead to cell proliferation and survival. FLT3 inhibitors are used to treat AML but patients who are treated with them typically become resistant. Such resistance often emerges through secondary mutations which either restore the activity of FLT3 in the presence of drugs or activate a key player in a signalling network such as NRAS. We had developed AML-specific cell lines resistant to two advanced FLT3 inhibitors: gilteritinib and FF-10101. Resistance in these cell lines proceeds though different mechanisms. In this study, we followed on the efficacy of five FLT3 inhibitors (gilteritinib, FF-10101 and three promising inhibitors that are being developed), two pan-PI3K inhibitors (one of which also inhibits mTOR) and two c-KIT inhibitors in order to examine the significance of different signalling cascades in FLT3+-AML. In addition, we used molecular modelling and quantum chemistry calculations to explain why specific FLT3 mutations affect some inhibitors more than others. Two novel FLT3 inhibitors were found to be only weakly affected by resistance mutations against gilteritinib and FF-10101. The efficacy of most FLT3 inhibitors was only weakly (or not at all) affected by the NRAS/G12C activating mutation. Finally, no nonFLT3 inhibitor has shown sufficient efficacy in the cells, suggesting the central role of FLT3 in FLT3-mutated AML.

Microbial alpha-galactosidases (AGals) are widely used in agriculture and food industries for degrading raffinose family oligosaccharides and synthesizing galactooligosaccharides (GOSs). While rational engineering of AGals is ongoing, limited understanding of substrate specificity and the determinants of hydrolysis and transglycosylation hinders progress. Here, we apply quantum mechanics/molecular mechanics (QM/MM) simulations to investigate the catalytic mechanism and substrate specificity of Saccharomyces cerevisiae glycoside hydrolase family 27 (GH27) AGal. The enzyme catalyzes hydrolysis via a Koshland double-displacement mechanism and cleaves linear galactomannans in an exo-mode. Free-energy calculations indicate glycosylation is the rate-determining step with a barrier (Delta G‡) of 17.8 kcalmol^-1, consistent with experimental data. A key 4-OHnucleophile interaction stabilizes the transition state, particularly for deglycosylation. Machine learning identified Trp188 and Phe235 at positive subsites as mutational hotspots. Six AGal variants were evaluated for in silico transglycosylation activity. Aromatic substitutions at Phe235 (F235Y and F235W) favored nucleophilic attack (NA) with sucrose, while W188A, W188R, and F235S showed low reaction barriers for lactose. The W188A variant showed improvement with a 10 kcalmol^-1 decrease in Delta G‡, a pronounced 0.3 Ų; shortening of NA distance, and an increased solvent exposure of similar to ∼500-600 Ų. These results highlight the potential of computer-aided subsite engineering to enhance AGal performance in GOS production.

The rapid evolution of microorganisms and cancer cells makes it difficult to treat tumours and infectious diseases, because resistance to drugs is the rule rather than the exception. Structures or models of protein-drug complexes help to understand how mutations lead to resistance and to design better drugs. However, it is difficult to reason how small changes in the structure lead to drug resistance. Thus, protein and drug dynamics need to be considered. Strategies to increase drug residence are sought after to increase the efficacy of drugs. Computational methods to calculate the effect of mutations on drug binding and residence times are being developed and improved, but are challenging. A priori prediction of a mutation's effect on drug binding is an even greater challenge. On the other hand, knowledge about protein-drug complexes has led to the development of multiple design strategies that aim to reduce mutation-driven drug resistance.

Acute myeloid leukaemia (AML) is a hard to treat blood cancer. Mutations in FLT3 are common among the genetic aberrations that characterise the cancer. Patients initially react to FLT3 inhibitors but drug resistance is a hinder to successful therapy. To better understand the mechanisms leading to drug resistance, we generated four AML cell lines resistant to the inhibitors gilteritinib or FF-10101, and explored their resistance mechanisms. We further tested whether the novel inhibitor Chen-9u could be used to limit cell growth. The results showed that each of the four resistant cell lines became resistant through a different mechanism. Resistant cells showed decreased FLT3 and increased NRAS pathway activity and reduced DNA synthesis due to decrease in CDK4 activity. Resistance mechanisms included resistance mutations in FLT3 (C695F and N701K), and a novel mutation in NRAS (G12C). In a fourth line, resistance might have developed through a MYCN mutation. Cell growth was inhibited by Chen-9u and resistant clones could not be obtained with this inhibitor. The results highlight opportunities and limitations. On the one hand, resistant cells were produced due to different mechanisms, showing the versatility of tumour cells. Furthermore, resistance developed to the most advanced inhibitors, one of which is covalent and the other non-covalent but highly specific. On the other hand, it is shown that DNA synthesis is reduced, which means that resistance has evolutionary consequences. Finally, the novel drug-resistant cell lines may serve as useful models for better understanding of the cellular events associated with inherent and acquired drug resistance.

Abl1, a nonreceptor tyrosine kinase closely related to Src kinase, regulates critical cellular processes like proliferation, differentiation, cytoskeletal dynamics, and response to environmental cues through phosphorylation-driven activation. Dysregulation places it centrally in the oncogenic pathway leading to blood cancers. making it an ideal drug target for small molecule inhibitors. We sought to understand the underlying mechanism of the phosphoryl-transfer step from the ATP molecule to the substrate tyrosine, as carried out by the Abl1 enzyme. By calculating free energy profiles for the reaction using the empirical valence bond representation of the reacting fragments paired with molecular dynamics and free energy perturbation calculations, a combination of several plausible reaction pathways, ATP conformations, and the number of magnesium ion cofactors have been investigated. For the best-catalyzed pathway, which proceeds through a dissociative mechanism with a single magnesium ion situated in Site I, a close agreement was reached with the experimentally determined catalytic rates. We conclude that the catalytic mechanism in Abl1 requires one magnesium ion for efficient catalysis, unlike other kinases, where two ions are utilized. A better overall understanding of the phosphoryl-transfer reactions in Abl1 can be used for type-I inhibitor development and generally contributes to a comprehensive overview of the mechanism for ATP-driven reactions.