A Modular Synthesis of Teraryl-Based α-Helix Mimetics, Part†5: A Complete Set of Pyridine Boronic Acid Pinacol Esters Featuring Side Chains of Proteinogenic Amino Acids.

Teraryl-based α-helix mimetics have proven to be useful compounds for the inhibition of protein-protein interactions (PPI). We have developed a modular and flexible approach for the synthesis of teraryl-based α-helix mimetics using pyridine containing boronic acid building blocks to increase the water solubility. Following our initial publication in which we have introduced the methodology in combination with sequential Pd-catalyzed cross-coupling for teraryl assembly, we can now report a complete set of pyridine based boronic acid building blocks decorated with side chains of all proteinogenic amino acids relevant for PPI (Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val) to complement the core fragment set. For a representative set of teraryls we have studied the influence of the pyridine rings on the solubility of the assembled oligoarenes.

Gas-phase aluminium acetylacetonate decomposition: revision of the current mechanism by VUV synchrotron radiation.

Although aluminium acetylacetonate, Al(C5H7O2)3, is a common precursor for chemical vapor deposition (CVD) of aluminium oxide, its gas-phase decomposition is not well-known. Here, we studied its thermal decomposition in a microreactor by double imaging photoelectron photoion coincidence spectroscopy (i2PEPICO) between 325 and 1273 K. The reactor flow field was characterized by CFD. Quantum chemical calculations were used for the assignment of certain species. The dissociative ionization of the room temperature precursor molecule starts at a photon energy of 8.5 eV by the rupture of the bond to an acetylacetonate ligand leading to the formation of the Al(C5H7O2)2+ ion. In pyrolysis experiments, up to 49 species were detected and identified in the gas-phase, including reactive intermediates and isomeric/isobaric hydrocarbons, oxygenated species as well as aluminium containing molecules. We detected aluminium bis(diketo)acetylacetonate-H, Al(C5H7O2)C5H6O2, at m/z 224 together with acetylacetone (C5H8O2) as the major initial products formed at temperatures above 600 K. A second decomposition channel affords Al(OH)2(C5H7O2) along with the formation of a substituted pentalene ring species (C10H12O2) as assigned by Franck-Condon simulations and quantum chemical calculations. Acetylallene (C5H6O), acetone (C3H6O) and ketene (C2H2O) were major secondary decomposition products, formed upon decomposition of the primary products. Three gas-phase aromatic hydrocarbons were also detected and partially assigned for the first time: m/z 210, m/z 186 (C14H18 or C12H10O2) and m/z 146 (C11H14 or C9H6O2) and their formation mechanism is discussed. Finally, Arrhenius parameters are presented on the gas-phase decomposition kinetics of Al(C5H7O2)3, aided by numerical simulation of the flow field.

Drug Discovery Maps, a Machine Learning Model That Visualizes and Predicts Kinome-Inhibitor Interaction Landscapes.

The interpretation of high-dimensional structure-activity data sets in drug discovery to predict ligand-protein interaction landscapes is a challenging task. Here we present Drug Discovery Maps (DDM), a machine learning model that maps the activity profile of compounds across an entire protein family, as illustrated here for the kinase family. DDM is based on the t-distributed stochastic neighbor embedding (t-SNE) algorithm to generate a visualization of molecular and biological similarity. DDM maps chemical and target space and predicts the activities of novel kinase inhibitors across the kinome. The model was validated using independent data sets and in a prospective experimental setting, where DDM predicted new inhibitors for FMS-like tyrosine kinase 3 (FLT3), a therapeutic target for the treatment of acute myeloid leukemia. Compounds were resynthesized, yielding highly potent, cellularly active FLT3 inhibitors. Biochemical assays confirmed most of the predicted off-targets. DDM is further unique in that it is completely open-source and available as a ready-to-use executable to facilitate broad and easy adoption.

Comprehensive structure-activity-relationship of azaindoles as highly potent FLT3 inhibitors.

Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown emergence of drug resistant mutants, emphasizing the ongoing need for new chemical matter to enable the treatment of this disease. Here, we present the discovery and topological structure-activity relationship (SAR) study of analogs of isoquinolinesulfonamide H-89, a well-known PKA inhibitor, as FLT3 inhibitors. Surprisingly, we found that the SAR was not consistent with the observed binding mode of H-89 in PKA. Matched molecular pair analysis resulted in the identification of highly active sub-nanomolar azaindoles as novel FLT3-inhibitors. Structure based modelling using the FLT3 crystal structure suggested an alternative, flipped binding orientation of the new inhibitors.

The CD33xCD123xCD70 Multispecific CD3-Engaging DARPin MP0533 Induces Selective T Cell-Mediated Killing of AML Leukemic Stem Cells.

The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging designed ankyrin repeat protein (DARPin) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell-mediated killing of AML cells coexpressing at least two of the antigens. In vitro, MP0533 induced selective T cell-mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen-targeting T-cell engagers. In xenograft AML mice models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity toward LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with a high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057).

Unexpected T-cell recognition of an altered peptide ligand is driven by reversed thermodynamics.

The molecular basis underlying T-cell recognition of MHC molecules presenting altered peptide ligands is still not well-established. A hierarchy of T-cell activation by MHC class I-restricted altered peptide ligands has been defined using the T-cell receptor P14 specific for H-2D(b) in complex with the immunodominant lymphocytic choriomeningitis virus peptide gp33 (KAVYNFATM). While substitution of tyrosine to phenylalanine (Y4F) or serine (Y4S) abolished recognition by P14, the TCR unexpectedly recognized H-2D(b) in complex with the alanine-substituted semiagonist Y4A, which displayed the most significant structural modification. The observed functional hierarchy gp33 > Y4A > Y4S = Y4F was neither due to higher stabilization capacity nor to differences in structural conformation. However, thermodynamic analysis demonstrated that while recognition of the full agonist H-2D(b) /gp33 was strictly enthalpy driven, recognition of the weak agonist H-2D(b) /Y4A was instead entropy driven with a large reduction in the favorable enthalpy term. The fourfold larger negative heat capacity derived for the interaction of P14 with H-2D(b) /gp33 compared with H-2D(b) /Y4A can possibly be explained by higher water entrapment at the TCR/MHC interface, which is also consistent with the measured opposite entropy contributions for the interactions of P14 with both MHCs. In conclusion, this study demonstrates that P14 makes use of different strategies to adapt to structural modifications in the MHC/peptide complex.

Origin of the conformational heterogeneity of cardiolipin-bound cytochrome C.

Interactions of cytochrome c (cyt c) with cardiolipin (CL) partially unfold the protein, activating its peroxidase function, a critical event in the execution of apoptosis. However, structural features of the altered protein species in the heterogeneous ensemble are difficult to probe with ensemble averaging. Analyses of the dye-to-heme distance distributions P(r) from time-resolved FRET (TR-FRET) have uncovered two distinct types of CL-bound cyt c conformations, extended and compact. We have combined TR-FRET, fluorescence correlation spectroscopy (FCS), and biolayer interferometry to develop a systematic understanding of the functional partitioning between the two conformations. The two subpopulations are in equilibrium with each other, with a submillisecond rate of conformational exchange reflecting the protein folding into a compact non-native state, as well as protein interactions with the lipid surface. Electrostatic interactions with the negatively charged lipid surface that correlate with physiologically relevant changes in CL concentrations strongly affect the kinetics of cyt c binding and conformational exchange. A predominantly peripheral binding mechanism, rather than deep protein insertion into the membrane, provides a rationale for the general denaturing effect of the CL surface and the large-scale protein unfolding. These findings closely relate to cyt c folding dynamics and suggest a general strategy for extending the time window in monitoring the kinetics of folding.

Detection of antigens using a protein-DNA chimera developed by enzymatic covalent bonding with phiX gene A*.

The chemical reactions used to make antibody-DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of φX174 gene A* protein and Z(mab25) (A*-Zmab). The φX174 gene A* protein is an enzyme that can covalently link with DNA, while the Z(mab25) protein moiety can bind to specific species of antibodies. The DNA in an A*-Zmab conjugate was attached to the A* protein at a site chosen to not interfere with protein function, as determined by enzyme-linked immunosorbent assay (ELISA) and gel mobility shift analysis. The novel A*-Zmab-DNA conjugate retained its binding capabilities to a specific class of murine immunoglobulin γ1 (IgG1) but not to rabbit IgG. This indicates the generality of the A*-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic covalent method dramatically increased the yields of A*-Zmab-DNA conjugates up to 80% after a 15 min reaction. Finally, sensitive detection of human interferon-γ (IFN-γ) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-specific enzymatic DNA conjugation to proteins should be applicable to fabrication of novel immunoassays for biosensing.

Activity-Based Protein Profiling Identifies α-Ketoamides as Inhibitors for Phospholipase A2 Group XVI.

Phospholipase A2, group XVI (PLA2G16) is a thiol hydrolase from the HRASLS family that regulates lipolysis in adipose tissue and has been identified as a host factor enabling the cellular entry of picornaviruses. Chemical tools are essential to visualize and control PLA2G16 activity, but they have not been reported to date. Here, we show that MB064, which is a fluorescent lipase probe, also labels recombinant and endogenously expressed PLA2G16. Competitive activity-based protein profiling (ABPP) using MB064 enabled the discovery of α-ketoamides as the first selective PLA2G16 inhibitors. LEI110 was identified as a potent PLA2G16 inhibitor ( Ki = 20 nM) that reduces cellular arachidonic acid levels and oleic acid-induced lipolysis in human HepG2 cells. Gel-based ABPP and chemical proteomics showed that LEI110 is a selective pan-inhibitor of the HRASLS family of thiol hydrolases (i.e., PLA2G16, HRASLS2, RARRES3 and iNAT). Molecular dynamic simulations of LEI110 in the reported crystal structure of PLA2G16 provided insight in the potential ligand-protein interactions to explain its binding mode. In conclusion, we have developed the first selective inhibitor that can be used to study the cellular role of PLA2G16.

Development of a Multiplexed Activity-Based Protein Profiling Assay to Evaluate Activity of Endocannabinoid Hydrolase Inhibitors.

Endocannabinoids, an important class of signaling lipids involved in health and disease, are predominantly synthesized and metabolized by enzymes of the serine hydrolase superfamily. Activity-based protein profiling (ABPP) using fluorescent probes, such as fluorophosphonate (FP)-TAMRA and ß-lactone-based MB064, enables drug discovery activities for serine hydrolases. FP-TAMRA and MB064 have distinct, albeit partially overlapping, target profiles but cannot be used in conjunction due to overlapping excitation/emission spectra. We therefore synthesized a novel FP-probe with a green BODIPY as a fluorescent tag and studied its labeling profile in mouse proteomes. Surprisingly, we found that the reporter tag plays an important role in the binding potency and selectivity of the probe. A multiplexed ABPP assay was developed in which a probe cocktail of FP-BODIPY and MB064 visualized most endocannabinoid serine hydrolases in mouse brain proteomes in a single experiment. The multiplexed ABPP assay was employed to profile endocannabinoid hydrolase inhibitor activity and selectivity in the mouse brain.

Molecular design of radiocopper-labelled Affibody molecules.

The use of long-lived positron emitters 64Cu or 61Cu for labelling of Affibody molecules may improve breast cancer patients' stratification for HER-targeted therapy. Previous animal studies have shown that the use of triaza chelators for 64Cu labelling of synthetic Affibody molecules is suboptimal. In this study, we tested a hypothesis that the use of cross-bridged chelator, CB-TE2A, in combination with Gly-Glu-Glu-Glu spacer for labelling of Affibody molecules with radiocopper would improve imaging contrast. CB-TE2A was coupled to the N-terminus of synthetic Affibody molecules extended either with a glycine (designation CB-TE2A-G-ZHER2:342) or Gly-Glu-Glu-Glu spacer (CB-TE2A-GEEE-ZHER2:342). Biodistribution and targeting properties of 64Cu-CB-TE2A-G-ZHER2:342 and 64Cu-CB-TE2A-GEEE-ZHER2:342 were compared in tumor-bearing mice with the properties of 64Cu-NODAGA-ZHER2:S1, which had the best targeting properties in the previous study. 64Cu-CB-TE2A-GEEE-ZHER2:342 provided appreciably lower uptake in normal tissues and higher tumor-to-organ ratios than 64Cu-CB-TE2A-G-ZHER2:342 and 64Cu-NODAGA-ZHER2:S1. The most pronounced was a several-fold difference in the hepatic uptake. At the optimal time point, 6 h after injection, the tumor uptake of 64Cu-CB-TE2A-GEEE-ZHER2:342 was 16 ± 6%ID/g and tumor-to-blood ratio was 181 ± 52. In conclusion, a combination of the cross-bridged CB-TE2A chelator and Gly-Glu-Glu-Glu spacer is preferable for radiocopper labelling of Affibody molecules and, possibly, other scaffold proteins having high renal re-absorption.

Fine epitope signature of antibody neutralization breadth at the HIV-1 envelope CD4-binding site.

Major advances in donor identification, antigen probe design, and experimental methods to clone pathogen-specific antibodies have led to an exponential growth in the number of newly characterized broadly neutralizing antibodies (bnAbs) that recognize the HIV-1 envelope glycoprotein. Characterization of these bnAbs has defined new epitopes and novel modes of recognition that can result in potent neutralization of HIV-1. However, the translation of envelope recognition profiles in biophysical assays into an understanding of in vivo activity has lagged behind, and identification of subjects and mAbs with potent antiviral activity has remained reliant on empirical evaluation of neutralization potency and breadth. To begin to address this discrepancy between recombinant protein recognition and virus neutralization, we studied the fine epitope specificity of a panel of CD4-binding site (CD4bs) antibodies to define the molecular recognition features of functionally potent humoral responses targeting the HIV-1 envelope site bound by CD4. Whereas previous studies have used neutralization data and machine-learning methods to provide epitope maps, here, this approach was reversed, demonstrating that simple binding assays of fine epitope specificity can prospectively identify broadly neutralizing CD4bs-specific mAbs. Building on this result, we show that epitope mapping and prediction of neutralization breadth can also be accomplished in the assessment of polyclonal serum responses. Thus, this study identifies a set of CD4bs bnAb signature amino acid residues and demonstrates that sensitivity to mutations at signature positions is sufficient to predict neutralization breadth of polyclonal sera with a high degree of accuracy across cohorts and across clades.

A system for concomitant overexpression of four periplasmic folding catalysts to improve secretory protein production in Escherichia coli.

Although Escherichia coli is in wide use for preparative protein expression, problems with the folding of the recombinant gene product and protein aggregation are frequently encountered. Apart from cytoplasmic expression, this is also true for secretion into the bacterial periplasm, the method of choice for the production of proteins that carry structural disulfide bonds. Here we report the construction of the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC that catalyze the formation and isomerization of disulfide bridges and the peptidyl-prolyl cis/trans-isomerases with chaperone activity, FkpA and SurA. pTUM4 carries a p15a origin of replication and a chloramphenicol resistance gene and, thus, it is compatible with many conventional expression vectors that use the ColEI origin and an ampicillin resistance. Its positive effects on the yield of soluble recombinant protein and the homogeneity of disulfide pattern are illustrated here using the human plasma retinol-binding protein as well as the extracellular carbohydrate recognition domain of the dendritic cell membrane receptor DC-SIGN. Hence, pTUM4 represents a novel helper vector which complements existing cytosolic chaperone coexpression plasmids and should be useful for the functional secretion of various recombinant proteins with hampered folding efficiency.

Directed evolution of a yeast-displayed HIV-1 SOSIP gp140 spike protein toward improved expression and affinity for conformational antibodies.

Design of an envelope-based immunogen capable of inducing a broadly neutralizing antibody response is thought to be key to the development of a protective HIV-1 vaccine. However, the broad diversity of viral variants and a limited ability to produce native envelope have hampered such design efforts. Here we describe adaptation of the yeast display system and use of a combinatorial protein engineering approach to permit directed evolution of HIV envelope variants. Because the intrinsic instability and complexity of this trimeric glycoprotein has greatly impeded the development of immunogens that properly represent the structure of native envelope, this platform addresses an essential need for methodologies with the capacity to rapidly engineer HIV spike proteins towards improved homogeneity, stability, and presentation of neutralizing epitopes. We report for the first time the display of a designed SOSIP gp140 on yeast, and the in vitro evolution of derivatives with greatly improved expression and binding to conformation-dependent antibodies. These efforts represent an initial and critical step toward the ability to rapidly engineer HIV-1 envelope immunogens via directed evolution.

An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1.

Broadly HIV-1-neutralizing antibodies (BnAbs) display one or more unusual traits, including a long heavy chain complementarity-determining region 3 (HCDR3), polyreactivity, and high levels of somatic mutations. These shared characteristics suggest that BnAb development might be limited by immune tolerance controls. It has been postulated that HIV-1-infected individuals with autoimmune disease and defective immune tolerance mechanisms may produce BnAbs more readily than those without autoimmune diseases. In this study, we identified an HIV-1-infected individual with SLE who exhibited controlled viral load (<5,000 copies/ml) in the absence of controlling HLA phenotypes and developed plasma HIV-1 neutralization breadth. We collected memory B cells from this individual and isolated a BnAb, CH98, that targets the CD4 binding site (CD4bs) of HIV-1 envelope glycoprotein 120 (gp120). CH98 bound to human antigens including dsDNA, which is specifically associated with SLE. Anti-dsDNA reactivity was also present in the patient's plasma. CH98 had a mutation frequency of 25% and 15% nt somatic mutations in the heavy and light chain variable domains, respectively, a long HCDR3, and a deletion in the light chain CDR1. The occurrence of anti-dsDNA reactivity by a HIV-1 CD4bs BnAb in an individual with SLE raises the possibility that some BnAbs and SLE-associated autoantibodies arise from similar pools of B cells.

Vaccine design: emerging concepts and renewed optimism.

Arguably, vaccination represents the single most effective medical intervention ever developed. Yet, vaccines have failed to provide any or adequate protection against some of the most significant global diseases. The pathogens responsible for these vaccine-recalcitrant diseases have properties that allow them to evade immune surveillance and misdirect or eliminate the immune response. However, genomic and systems biology tools, novel adjuvants and delivery systems, and refined molecular insight into protective immunity have started to redefine the landscape, and results from recent efficacy trials of HIV and malaria vaccines have instilled hope that another golden age of vaccines may be on the horizon.

Monitored whole gene in vitro evolution of an anti-hRaf-1 affibody molecule towards increased binding affinity.

The use of library technologies for the generation of affinity proteins often includes an affinity maturation step, based on the construction of secondary libraries from which second generation variants with improved affinities are selected. Here, we describe for the first time the affinity maturation of affibody molecules based on step-wise in vitro molecular evolution, involving cycles of error-prone PCR (epPCR) amplification for the introduction of diversity over the entire 58-residue three-helix bundle structure and ribosome display (RD) for the selection of improved variants. The model affibody molecule for the process was Z(RAF322), binding with a 1.9µm equilibrium dissociation constant (K(D)) to human Raf-1 (hRaf-1), a protein kinase of central importance in the MAPK/ERK proliferation pathway. The molecular evolution process was followed on both gene and protein levels via DNA sequencing and a biosensor-based binding analysis of pools of selected variants. After two cycles of diversification and selection, a significant increase in binding response of selected pools was seen. DNA sequencing showed that a dominant alanine to valine substitution had been effectively enriched, and was found in 83% of all selected clones, either alone or in combination with other enriched substitutions. The evolution procedure resulted in variants showing up to 26-fold increases in affinity to the hRaf-1 target. Noteworthy, for the two variants showing the highest affinities, substitutions were also found in affibody framework positions, corresponding to regions of the protein domain not addressed by traditional affibody molecule affinity maturation strategies. Interestingly, thermal melting point (T(m)) analyses showed that an increased affinity could be associated with both higher and lower T(m) values. All investigated variants showed excellent refolding properties and selective binding to hRaf-1, as analysed using a multiplexed bead-based binding assay, making them potentially valuable affinity reagents for cell biology studies.

Ribosome display selection of a murine IgG1 Fab binding affibody molecule allowing species selective recovery of monoclonal antibodies.

Affinity reagents recognizing constant parts of antibody molecules are invaluable tools in immunotechnology applications, including purification, immobilization, and detection of immunoglobulins. In this article, murine IgG1, the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 10¹¹ affibody molecules. Four rounds of selection using three different mouse IgG1 mAbs as alternating targets resulted in the identification of binders with broad mIgG1 recognition and dissociation constants (K(D)) in the low nanomolar to low micromolar range. For one of the binders, denoted Z(mab25), competition in binding to full length mIgG1 by a streptococcal protein G (SPG) fragment and selective affinity capture of mouse IgG1 Fab fragments after papain cleavage of a full mAb suggest that an epitope functionally overlapping with the SPG-binding site in the CH1 domain of mouse IgG1 had been addressed. Interestingly, biosensor-based binding experiments showed that neither human IgG1 nor bovine Ig, the latter present in fetal bovine serum (FBS) was recognized by Z(mab25). This selective binding profile towards murine IgG1 was successfully exploited in species selective recovery of two different mouse mAbs from complex samples containing FBS, resembling a hybridoma culture supernatant.

Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro.

Development of molecules with the ability to selectively inhibit particular protein-protein interactions is important in providing tools for understanding cell biology. In this work, we describe efforts to select small Ras- and Raf-specific three-helix bundle affibody binding proteins capable of inhibiting the interaction between H-Ras and Raf-1, from a combinatorial library displayed on bacteriophage. Target-specific variants with typically high nanomolar or low micromolar affinities (K(D)) could be selected successfully against both proteins, as shown by dot blot, ELISA and real-time biospecific interaction analyses. Affibody molecule variants selected against H-Ras were shown to bind epitopes overlapping each other at a site that differed from that at which H-Ras interacts with Raf-1. In contrast, an affibody molecule isolated during selection against Raf-1 was shown to effectively inhibit the interaction between H-Ras and Raf-1 in a dose-dependent manner. Possible intracellular applications of the selected affibody molecules are discussed.