Structure-based Molecular Design Research Articles

Discovery and Identification of Novel 5-Hydroxy-4H-benzo[1,4]oxazin-3-one Derivatives as Potent β2-Adrenoceptor Agonists through Structure-Based Design, Synthesis, and Biological Evaluation.

Although β2-agonists are crucial for treatment of chronic respiratory diseases, optimizing β2-agonistic activity and selectivity remains essential for achieving favorable therapeutic outcomes. A structure-based molecular design workflow was employed to discover a novel class of β2 agonists featuring a 5-hydroxy-4H-benzo[1,4]oxazin-3-one scaffold, which potently stimulated β2 adrenoceptors (β2-ARs). Screening for the β2-agonistic activity and selectivity led to the identification of compound A19 (EC50 = 3.7 pM), which functioned as a partial β2-agonist in HEK-293 cells containing endogenous β2-ARs. Compound A19 exhibited significant relaxant effects, rapid onset time (Ot50 = 2.14 min), and long duration of action (>12 h) on isolated guinea pig tracheal strips, as well as advantageous pharmacokinetic characteristics in vivo, rendering A19 suitable for inhalation administration. Moreover, A19 suppressed the upregulation of inflammatory cytokines and leukocytes and improved lung function in a rat model of COPD, thereby indicating that A19 is a potential β2 agonist candidate for further study.

SiteMine: Large-scale binding site similarity searching in protein structure databases.

Drug discovery and design challenges, such as drug repurposing, analyzing protein-ligand and protein-protein complexes, ligand promiscuity studies, or function prediction, can be addressed by protein binding site similarity analysis. Although numerous tools exist, they all have individual strengths and drawbacks with regard to run time, provision of structure superpositions, and applicability to diverse application domains. Here, we introduce SiteMine, an all-in-one database-driven, alignment-providing binding site similarity search tool to tackle the most pressing challenges of binding site comparison. The performance of SiteMine is evaluated on the ProSPECCTs benchmark, showing a promising performance on most of the data sets. The method performs convincingly regarding all quality criteria for reliable binding site comparison, offering a novel state-of-the-art approach for structure-based molecular design based on binding site comparisons. In a SiteMine showcase, we discuss the high structural similarity between cathepsin L and calpain 1 binding sites and give an outlook on the impact of this finding on structure-based drug design. SiteMine is available at https://uhh.de/naomi.

Design, Synthesis, and Antifungal Activity of N-(alkoxy)-Diphenyl Ether Carboxamide Derivates as Novel Succinate Dehydrogenase Inhibitors.

Succinate dehydrogenase (SDH, EC 1.3.5.1) is one of the most promising targets for fungicide development and has attracted great attention worldwide. However, existing commercial fungicides targeting SDH have led to the increasingly prominent problem of pathogen resistance, so it is necessary to develop new fungicides. Herein, we used a structure-based molecular design strategy to design and synthesize a series of novel SDHI fungicides containing an N-(alkoxy)diphenyl ether carboxamide skeleton. The mycelial growth inhibition experiment showed that compound M15 exhibited a very good control effect against four plant pathogens, with inhibition rates of more than 60% at a dose of 50 μg/mL. A structure-activity relationship study found that N-O-benzyl-substituted derivatives showed better antifungal activity than others, especially the introduction of a halogen on the benzyl. Furthermore, the molecular docking results suggested that π-π interactions with Trp35 and hydrogen bonds with Tyr33 and Trp173 were crucial interaction sites when inhibitors bound to SDH. Morphological observation of mycelium revealed that M15 could inhibit the growth of mycelia. Moreover, in vivo and in vitro tests showed that M15 not only inhibited the enzyme activity of SDH but also effectively protected rice from damage due to R. solani infection, with a result close to that of the control at a concentration of 200 μg/mL. Thus, the N-(alkoxy)diphenyl ether carboxamide skeleton is a new starting point for the discovery of new SDH inhibitors and is worthy of further investigation.

Probing the origins of programmed death ligand-1 inhibition by implementing machine learning-assisted sequential virtual screening techniques.

PD-L1 is a key immunotarget involved in binding to its receptor PD-1. PD-L1/PD-1 interface blocking using antibodies (or small molecules) is the central area of interest for tumor suppression in various cancers. Blocking the PD-L1/PD-1 pathway in the tumor cells results in its immune activation and destruction, and thereby restoring the T-cell proliferation and cytokine production. The active binding site interface residues of PD-L1/PD-1 were experimentally known and proven by structural biology and site-directed mutagenesis studies. Structure-based molecular design technique was employed to identify the inhibitors for blocking the PD-L1/PD-1 interface. Nine hits to leads were identified from the SPECS small molecule database by machine learning, molecular docking, and molecular dynamics simulation techniques. Following this, a machine learning-assisted QSAR modeling approach was implemented using ChEMBL database to gain insights into the inhibitory potential of PD-L1 inhibitors and predict the activity of our previously screened nine hit molecules. The best leads identified in the present study bind strongly with the active sites of PD-L1/PD-1 interface residues, which include A121, M115, I116, S117, I54, Y56, D122, and Y123. These computational leads are considered promising molecules for furtherin vitroandin vivoanalysis to be developed as potential PD-L1 checkpoint inhibitors to cure different types of cancers.

Computer-aided Design of New Hydroxamic Acid Derivatives Targeting the Plasmodium falciparum M17 Metallo-aminopeptidase with Favorable Pharmacokinetic Profile

Through structure-based molecular design, we virtually design new subnanomolar range antimalarial, inhibitors of Plasmodium falciparum M17 aminopeptidase (PfA-M17). We developed the complexation QSAR models from hydroxamic acid derivatives (HDA). A linear correlation was established between the computed Gibbs free energies of binding (GFE: ΔΔGcom) and observed enzyme inhibition constants (Ki exp) for each training set pKi exp = , R2 = 0.97. The predictive power of the QSAR model was validated with 3D-QSAR pharmacophore generation (PH4): pKi exp = 0.707×pKi pred − 2.5182, R2 = 0.89. We then conducted a study on catalytic residues to exploit the different interactions (enzyme: inhibitor). Structural information from the models guided us in designing a virtual combinatorial library (VCL) of more than 56 thousand HDAs. The PH4 screening retained 48 new and potent HDAs with predicted inhibitory potencies pKi pre up to 73 times lower than that of HDA1 (pKi exp = 2.5 nM). Combining molecular modeling and PH4 in-silico screening of the VCL resulted in the proposed novel potent antimalarial agent candidates with favorable pharmacokinetic profiles.

Computer-Assisted Design of Hydroxamic Acid Derivatives Inhibitors of M1 Metallo Aminopeptidase of Plasmodium falciparum with Favorable Pharmacokinetic Profile

We virtually design here new subnanomolar range antimalarial, inhibitors of plasmodium falciparum M1 Aminopeptidase (PfA-M1), by means of structure-based molecular design. We developed the complexation QSAR models from Hydroxamic Acid derivatives (AHO). A linear correlation was established between the computed Gibbs free energies of binding (GFE: ∆∆Gcom) and observed enzyme inhibition constants (Kiexp) for each training set pKiexp = −0.063×∆∆Gcom+ 8.003, R2 = 0.92. The predictive power of the QSAR model was validated with 3D-QSAR pharmacophore generation (PH4): pKiexp = 1.0289×pKipred − 0.155, R2 = 0.90. We then conducted a study on catalytic residues to exploit the different interactions (enzyme: inhibitor). Structural information from the models guided us in designing of a virtual combinatorial library (VCL) of more than 44 thousands AHOs. The PH4 screening retained 51 new and potent AHOs with predicted inhibitory potencies pKipre up to 13 times lower than that of AHO1 (pKiexp = 700 nM). Combining molecular modeling and PH4 in silico screening of the VCL resulted in the proposed novel potent antimalarial agent candidates with favorable pharmacokinetic profiles.

Development of a First-in-Class Small-Molecule Inhibitor of the C-Terminal Hsp90 Dimerization.

Heat shock proteins 90 (Hsp90) are promising therapeutic targets due to their involvement in stabilizing several aberrantly expressed oncoproteins. In cancerous cells, Hsp90 expression is elevated, thereby exerting antiapoptotic effects, which is essential for the malignant transformation and tumor progression. Most of the Hsp90 inhibitors (Hsp90i) under investigation target the ATP binding site in the N-terminal domain of Hsp90. However, adverse effects, including induction of the prosurvival resistance mechanism (heat shock response or HSR) and associated dose-limiting toxicity, have so far precluded their clinical approval. In contrast, modulators that interfere with the C-terminal domain (CTD) of Hsp90 do not inflict HSR. Since the CTD dimerization of Hsp90 is essential for its chaperone activity, interfering with the dimerization process by small-molecule protein–protein interaction inhibitors is a promising strategy for anticancer drug research. We have developed a first-in-class small-molecule inhibitor (5b) targeting the Hsp90 CTD dimerization interface, based on a tripyrimidonamide scaffold through structure-based molecular design, chemical synthesis, binding mode model prediction, assessment of the biochemical affinity, and efficacy against therapy-resistant leukemia cells. 5b reduces xenotransplantation of leukemia cells in zebrafish models and induces apoptosis in BCR-ABL1+ (T315I) tyrosine kinase inhibitor-resistant leukemia cells, without inducing HSR.

Exploring the structural determinants for inhibitor selectivity towards the BACE protein family using structure-based molecular design

Alzheimer’s disease (AD) and type 2 diabetes (T2D) are the 6th and 7th leading causes of death in the United States. Both AD and T2D share a common pathology of protein misfolding. The two enzymes in the BACE protein family, BACE1 and BACE2, are responsible for the recognition and cleavage of two proteins that either aggregate into amyloid plaques, a hallmark of AD or, reduce β-cell heath which contributes to T2D. Despite being discovered over 20 years ago, to date there are no FDA-approved therapeutics that target either BACE1 or BACE2.

Computer-Aided Virtual Design of 3-Dehydroquinic Acid Analogues Inhibitors of Dehydroquinase Type II of Mycobacterium tuberculosis with Favorable Pharmacokinetics Profile

New potent 3-dehydroquinic acid (AQs) inhibitors of 3-dehydroquinate dehydratase (dehydroquinase type II) of Mycobacterium tuberculosis (MtDHQ2) were obtained by using structure-based molecular design via the in situ modification of the template inhibitor AQ1 within the MtDHQ2-AQ1 crystal structure (PDB ID: 2XB8), in order to describe the interactions upon the formation of the complex MtDHQ2-inhibitor. A training set of 14 AQs with known inhibition constants (Kiexp) was used to establish a quantitative structure-activity relationship (QSAR) model for correlating the pKiexp (pKiexp = ‑log10 (Kiexp)) to the computed Gibbs free energies of formation (ΔΔGcom) of MtDHQ2-AQs complexes (pKiexp= -0.278 ΔΔGcom + 7.653 , R² = 0.98) is derived. This accounts for the solvent effect and the loss of inhibitor entropy upon enzyme binding. Validation of this QSAR model was performed with 3D- QSAR pharmacophore generation (PH4). The structural information derived from the 3D model and breakdown of computed MtDHQ2-AQs interaction energies up to individual active site residue contribution led to the design of a virtual combinatorial library of 1600 AQs that was screened through the PH4 filter to retain about forty molecules. The absorption, distribution, metabolism and excretion parameters of these designed molecules were computed with the QikProp program while the inhibition constants were predicted by the generated QSAR model. Our computational approach, which combines molecular modeling, pharmacophore generation and analysis of MtDHQ2-AQ interaction energies, resulted in the proposed novel predicted potent antitubercular agents with favorable pharmacokinetic profile, of which the best candidate predicted inhibition constant (Kipre) value within the picomolar range.

Structure-based molecular hybridization design of Keap1-Nrf2 inhibitors as novel protective agents of acute lung injury.

Blocking the Kelch-like epichlorohydrin-related protein 1 (Keap1)-nuclear factor-erythroid 2 related factor 2 (Nrf2) pathway represents as a promising strategy to reduce oxidative stress and related-inflammation, including acute lung injury (ALI). NXPZ-2, a naphthalensulfonamide derivative, was previously reported to effectively inhibit the Keap1-Nrf2 protein-protein interaction (PPI) by our group. In the present work, a series of novel isothiocyanate-containing naphthalensulfonamides with the thioether, sulfoxide and sulfone moieties were designed by a structure-based molecular hybridization strategy using NXPZ-2 and the Nrf2 activator sulforaphane. They possessed good Keap1-Nrf2 PPI inhibitory activity and low cytotoxicity. The molecular docking study was performed to further explain the different activity of the thioether-, sulfoxide- and sulfone-containing naphthalensulfonamides. Among these new derivatives, 2-((N-(4-((N-(2-amino-2-oxoethyl)-4-((3-isothiocyanatopropyl)sulfinyl)phenyl)sulfonamido) naphthalen-1-yl)-4-methoxyphenyl)sulfonamido)acetamide (SCN-16) showed a good KD2 value of 0.455μM to disrupt the PPI. In an LPS-induced peritoneal macrophage cell model, this compound could cause a significant increase in the nuclear Nrf2 protein, decrease in the cytosolic Nrf2 protein, and further elevate the downstream protective enzymes HO-1 and NQO-1, which were better than the lead compound NXPZ-2 and sulforaphane. What's more, the production of ROS and NO and the expression of pro-inflammatory cytokine TNF-α were also suppressed. In the LPS-induced ALI model, SCN-16 could significantly reduce LPS-induced inflammations and alleviate lung injuries by triggering Nrf2 nuclear translocation. Collectively, our results suggested that SCN-16 could be a novel lead compound targeting Keap1-Nrf2 protective pathway for clinical treatment of ALI.

Computer-Aided Design of Peptidomimetic Inhibitors of Falcipain-3: QSAR and Pharmacophore Models

In this work, antiparasitic peptidomimetics inhibitors (PEP) of falcipain-3 (FP3) of Plasmodium falciparum (Pf) are proposed using structure-based and computer-aided molecular design. Beginning with the crystal structure of PfFP3-K11017 complex (PDB ID: 3BWK), three-dimensional (3D) models of FP3-PEPx complexes with known activities ( IC50exp) were prepared by in situ modification, based on molecular mechanics and implicit solvation to compute Gibbs free energies (GFE) of inhibitor-FP3 complex formation. This resulted in a quantitative structure–activity relationships (QSAR) model based on a linear correlation between computed GFE (ΔΔGcom) and the experimentally measured IC50exp. Apart from the structure-based relationship, a ligand-based quantitative pharmacophore model (PH4) of novel PEP analogues where substitutions were directed by comparative analysis of the active site interactions was derived using the proposed bound conformations of the PEPx. This provided structural information useful for the design of virtual combinatorial libraries (VL), which was virtually screened based on the 3D-QSAR PH4. The end results were predictive inhibitory activities falling within the low nanomolar concentration range.

In silico design of novel diamino-quinoline-5,8‑dione derivatives as putative inhibitors of NAD(P)H:Quinone oxidoreductase 1 based on docking studies and molecular dynamics simulations

NAD(P)H:Quinone Oxidoreductase 1 has been proved as a promising therapeutic target in anticancer drug discovery because of its overexpression in various cancerous cells. In this study, we designed a series of diamino-quinoline-5,8‑dione derivatives as potential inhibitors of NQO1 using in silico structure-based molecular design approach. Initially, molecular docking was used to explore key amino acid residues and binding energies at the active site of enzyme. Based on obtained results, compounds A21, A22, C22 and A26, bearing appreciable enzyme binding affinity (ΔG binding between -11.7 and -11.4 kcal. mol−1 and -15.4 and -15.3 kcal. mol−1 by Vina and Vinardo scoring functions, respectively) and orientation towards the active site of NQO1, were selected for 100 ns molecular dynamics (MD) simulation. MD simulation analyses confirmed that NH of His162A and C2-carbonyl of isoalloxazine ring of the flavin adenine dinucleotide (FAD) cofactor formed important hydrogen bonds, and residues Trp106A and Tyr129B played a key role in the binding affinity via π-π stacking interactions. Eventually, designed compounds’ binding orientation was verified and confirmed by comparison with a set of previously reported NQO1 inhibitors and bioreductive substrates. According to the results of MM-PBSA calculations, these four selected compounds showed lower free binding energies compared to dicoumarol as a well-known native ligand of enzyme indicating their potential inhibitory effect against NQO1. Designed molecules also obeyed drug-likeness rules and exhibited acceptable predictive ADMET properties. These results provide valuable insight for the discovery of new NQO1 inhibitors.

Structure-Based Design of Tetrahydroisoquinoline-Based Hydroxamic Acid Derivatives Inhibiting Human Histone Deacetylase 8

We have designed new human histone deacetylase 8 (HDAC8) inhibitors using structure-based molecular design. 3D models of HDAC8–inhibitor complexes were prepared by in situ modification of the crystal structure of HDAC8 co-crystallized with the hydroxamic acid suberoylanilide (SAHA) and a training set (TS) of tetrahydroisoquinoline-based hydroxamic acid derivatives (DAHTs) with known inhibitory potencies. A QSAR model was elaborated for the TS yielding a linear correlation between the computed Gibbs free energies (GFE) of HDAC8–DAHTs complexation (∆∆Gcom) and observed half-maximal enzyme inhibitory concentrations (IC50exp). From this QSAR model a 3D-QSAR pharmacophore (PH4) was generated. Structural information derived from the 3D model and breakdown of computed HDAC8–DAHTs interaction energies up to individual active site residue contributions helped us to design new more potent HDAC8 inhibitors. We obtained a reasonable agreement ∆∆Gcom and values: (pIC50exp = – 0.0376 × ∆∆Gcom + 7.4605, R2 = 0.89). Similar agreement was established for the PH4 model (pIC50exp = 0.8769 × + 0.7854, R2 = 0.87). A comparative analysis of the contributions of active site residues guided the choice of fragments used in designing a virtual combinatorial library (VCL) of DAHT analogs. The VCL of more than 17 thousand DAHTs was screened by the PH4 and furnished 229 new DAHTs. The best-designed analog displayed predicted inhibitory potency up to 110 times higher than that of DAHT1 (IC50exp = 0.047 µM). Predicted pharmacokinetic profiles of the new analogs were compared to current per oral anticancer compounds. This computational approach, which combines molecular modelling, pharmacophore generation, analysis of HDAC8–DAHTs interaction energies and virtual screening of a combinatorial library of DAHTs resulted in a set of proposed new HDAC8 inhibitors. It can thus direct medicinal chemists in their search for new anticancer agents.

Teaching of Biopharmaceutics in a Drug Design Course: Use of GastroPlus as Educational Software

Drug design and development requires collaboration among scientists with diverse expertise. In early-stage drug design, establishment of promising drug candidates requires integration of structure-based molecular design with biopharmaceutics and ADMET (absorption, distribution, metabolism, excretion, and toxicity) principles. Much of this work is increasingly computational. Thus, students in a drug design course should learn the basis for and effects of candidate small molecules binding to therapeutic targets and the importance of simultaneous understanding of ADMET characteristics that will largely determine if the molecule can achieve a therapeutically relevant concentration at the target site and become an orally deliverable drug. This second aspect of education in drug design has been relatively overlooked compared to structure-based design. Here, we describe a course using sophisticated simulation and modeling software, GastroPlus and ADMET Predictor, which are commonly used in the pharmaceutical industry. Our teaching approach has evolved over a decade in early graduate and advanced undergraduate settings. We use a combination of short lectures, software demonstration, and hands-on use, which allows students to “design a drug” with incorporation of structural and ADMET considerations. Student feedback indicated that the use of the software and the course design were helpful in enhancing their understanding of the importance of biopharmaceutics properties and ADMET in drug design. GastroPlus and ADMET Predictor have recently become more accessible to educators, and our experience using this software in an educational setting may be helpful for instructors who wish to develop a similar course.

Design, Synthesis, Biological Evaluation and Inhibition Mechanism of 3-/4-Alkoxy Phenylethylidenethiosemicarbazides as New, Potent and Safe Tyrosinase Inhibitors.

Tyrosinase plays important roles in many different disease related processes, and the development of its inhibitors is particularly important in biotechnology. In this study, thirty-nine 3-/4-alkoxyphenylethylidenethiosemicarbazides were synthesized as novel tyrosinase inhibitors based on structure-based molecular design. Our experimental results demonstrated that thirty-one of them possess remarkable tyrosinase inhibitory activities with IC50 value below 1 µM, and 5a, 6e, 6g and 6t did not display any toxicity to 293T cell line at the concentration of 1000 µmol/L. According to the inhibitory activities, several compounds were selected for detail investigation on the structure-activity relationships (SARs), mechanisms of enzyme inhibition, inhibitory kinetics and cytotoxicity. In particular, the interaction between the selected inhibitors and the active center of tyrosinase was considered and discussed in detail based on their structural characteristics. Taken together, the results presented here demonstrated that the newly designed compounds are promising candidates for the treatment of tyrosinase-related disorders and further development of them may have significant contribution in biomedical science.

Rational modulator design by exploitation of protein-protein complex structures.

The horizon of drug discovery is currently expanding to target and modulate protein-protein interactions (PPIs) in globular proteins and intrinsically disordered proteins that are involved in various diseases. To either interrupt or stabilize PPIs, the 3D structure of target protein-protein (or protein-peptide) complexes can be exploited to rationally design PPI modulators (inhibitors or stabilizers) through structure-based molecular design. In this review, we present an overview of experimental and computational methods that can be used to determine 3D structures of protein-protein complexes. Several approaches including rational and in silico methods that can be applied to design peptides, peptidomimetics and small compounds by utilization of determined 3D protein-protein/peptide complexes are summarized and illustrated.

Therapeutic Targeting of HSP90 in AML and ALL

Abstract Background: Even with the prevalent usage of specified treatment protocols, treatment gap remains and besides that the conventional therapies used routinely inflict significant toxicity due to low specificity. Therefore, the development of novel targeted therapies which are active against resistant leukemia subtypes and at the same time offer low toxicity in patients is of high importance. We, therefore, aimed to develop and characterize novel precision compounds, which target oncogene stabilization via HSP90 axis. Aims: We have previously developed a novel peptidomimetic HSP90 inhibitor (AX) which was active as a pan-leukemia inhibitor against LSCs without inducing any Heat Shock Resoponse (HSR). However due to peptidomimetic nature and high molecular weight, the clinical implication of AX was limited. Therefore, using the previous knowledge we focused on developing second generation of small molecule inhibitor against the C-terminal dimerization of HSP90 with better efficacy and clinical potential. Methods: We have generated the library of small molecule inhibitor targeting HSP90 C-terminus and selected the most potent analogue (VWK147) depending upon its higher potency against leukemic/cancerous cells. The specificity of VWK147 was further evaluated by microscale thermophoresis (MST), cell-based luciferase refolding assay, 2D NMR spectroscopy, analytical ultracentrifugation and molecular dynamics simulations. Results: HSP90 act as molecular chaperone and is highly expressed in several therapy-resistant leukemia subtypes thereby ensuring correct protein folding of several oncogenic proteins such as BCR-ABL1, FLT3-ITD and AKT. Therefore, targeting HSP90 could be a promising option in the treatment of therapy-refractory leukemia. Majority of available HSP90 inhibitors target the N-terminal domain thereby induce a protective mechanism called heat shock response (HSR), which potentially weakens the cytotoxic effect of HSP90 inhibitors and induce toxicity. We have now developed first in class small molecule HSP90 C-terminal dimerization inhibitor 'VWK147' through structure-based molecular design and chemical synthesis which specifically targets C-terminal dimerization of HSP90. Like AX, VWK147 destabilizes BCR-ABL oncoprotein and its related pro-oncogenic cellular response (involving proliferation, apoptosis and differentiation), effective in preclinical AML and TKI (2nd and 3rd generation) resistant cell line models in vitro and induces apoptosis in primary AML and BCR-ABL1+ BCP-ALL patient derived leukemic cell, without inducing any HSR. The next step would be to to evaluate its in vivo activity and pharmacodynamic profiling. Conclusion: Taken together, VWK147 represents a promising next step for future efforts towards the development of novel targeted HSP90 inhibitors to overcome drug resistance and reduce toxicity, especially for the treatment of relapsed/refractory ALL.

Targeting HSP90 dimerization via the C terminus is effective in imatinib-resistant CML and lacks the heat shock response

AbstractHeat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38−) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)–resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.

Affinity purification of the avidin protein family, based on crystal structures of avidin-HABA complexes

Since the importance of the high affinity between avidin and biotin, Kd = 3 × 10−16 M, gained universal recognition, numerous chemical, biological and medical avidin-biotin based applications have been developed. However, in some cases the high affinity may be a disadvantage, as this interaction is irreversible under physiological conditions. The dye, 4′-hydroxyazobenzene-2-carboxylic acid (HABA), binds avidin, at the biotin binding site, as determined by X-ray, at a much lower affinity constant, Kd = 6 × 10−6 M. We prepared a HABA affinity column (amber colored). Avidin bound to the column at a pH between 4 and 8.5, causing a change of color to red, and it could be eluted at mild conditions with buffers containing biotin, HABA, 1.5 M potassium chloride or a pH lower than 4.0 or higher than 8.5. Avidin eluted with HABA, created a red avidin-HABA complex, which was visualized. HABA free avidin was obtained by dialysis, which was followed by the loss of red coloration. The novel and easy to use HABA-affinity column was employed in our lab to prepare pure, fully glycosylated avidin from egg white. Most importantly, it may serve as an ideal tool for educational purposes, illuminating concepts of molecular recognition, reversible molecular binding, structure-based molecular design and solid phase chemical synthesis, as it is a reliable and visible reagent.

Hydrodynamic radius coincides with the slip plane position in the electrokinetic behavior of lysozyme.

The zeta potential (ζ) is the effective charge energy of a solvated protein, describing the magnitude of electrostatic interactions in solution. It is commonly used in the assessment of adsorption processes and dispersion stability. Predicting ζ from molecular structure would be useful to the structure-based molecular design of drugs, proteins, and other molecules that hold charge-dependent function while remaining suspended in solution. One challenge in predicting ζ is identifying the location of the slip plane (XSP ), a distance from the protein surface where ζ is theoretically defined. This study tests the hypothesis that the XSP can be estimated by the Stokes-Einstein hydrodynamic radius (Rh ), using globular hen egg white lysozyme as a model system. Although the XSP and Rh differ in their theoretical definitions, with the XSP being the position of the ζ during electrokinetic phenomena (e.g., electrophoresis) and the Rh being a radius pertaining to the edge of solvation during diffusion, they both represent the point where water and ions no longer adhere to a molecule. This work identifies the limited range of ionic strengths in which the XSP can be determined using diffusivity measurements and the Stokes-Einstein equation. In addition, a computational protocol is developed for determining the ζ from a protein crystal structure. At low ionic strengths, a hyperdiffusivity regime exists, requiring direct measurement of electrophoretic mobility to determine ζ. This work, therefore, supports a basic tenant of EDL theory that the electric double layer during diffusion and electrophoresis are equivalent in the Stokes-Einstein regime.