Drug Design of “Undruggable” Targets

Drug innovation is not only reflected in the discovery of new chemotypes of active compounds against existing targets but also more dependent on the innovation of drug targets. Currently, a number of attractive and validated targets could not be targeted pharmacologically. Some have been described as “undruggable”. In this review, we summarized the current situation of “undruggable” targets, and the design strategies for “undruggable” targets, hoping to provide references for the development of innovative drugs.     

“Head‐to‐Middle” and “Head‐to‐Tail” cis‐Prenyl Transferases: Structure of Isosesquilavandulyl Diphosphate Synthase

We report the first X‐ray crystallographic structure of the “head‐to‐middle” prenyltransferase, isosesquilavandulyl diphosphate synthase, involved in biosynthesis of the merochlorin class of antibiotics. The protein adopts the ζ or cis‐prenyl transferase fold but remarkably, unlike tuberculosinol adenosine synthase and other cis‐prenyl transferases (e.g. cis‐farnesyl, decaprenyl, undecaprenyl diphosphate synthases), the large, hydrophobic side chain does not occupy a central hydrophobic tunnel. Instead, it occupies a surface pocket oriented at 90° to the hydrophobic tunnel. Product chain‐length control is achieved by squeezing out the ligand from the conventional allylic S1 binding site, with proton abstraction being achieved using a diphosphate‐Asn‐Ser relay. The structures revise and unify our thinking as to the mechanism of action of many other prenyl transferases and may also be of use in engineering new merochlorin‐class antibiotics.     

Substitution of an Internal Disulfide Bridge with a Diselenide Enhances both Foldability and Stability of Human Insulin

Insulin analogues, mainstays in the modern treatment of diabetes mellitus, exemplify the utility of protein engineering in molecular pharmacology. Whereas chemical syntheses of the individual A and B chains were accomplished in the early 1960s, their combination to form native insulin remains inefficient because of competing disulfide pairing and aggregation. To overcome these limitations, we envisioned an alternative approach: pairwise substitution of cysteine residues with selenocysteine (Sec, U). To this end, Cys^A6 and Cys^A11 (which form the internal intrachain A6–A11 disulfide bridge) were each replaced with Sec. The A chain[C6U, C11U] variant was prepared by solid-phase peptide synthesis; while sulfitolysis of biosynthetic human insulin provided wild-type B chain-di-S-sulfonate. The presence of selenium atoms at these sites markedly enhanced the rate and fidelity of chain combination, thus solving a long-standing challenge in chemical insulin synthesis. The affinity of the Se-insulin analogue for the lectin-purified insulin receptor was indistinguishable from that of WT-insulin. Remarkably, the thermodynamic stability of the analogue at 25 °C, as inferred from guanidine denaturation studies, was augmented (ΔΔG_u ≈0.8 kcal mol⁻¹). In accordance with such enhanced stability, reductive unfolding of the Se-insulin analogue and resistance to enzymatic cleavage by Glu-C protease occurred four times more slowly than that of WT-insulin. 2D-NMR and X-ray crystallographic studies demonstrated a native-like three-dimensional structure in which the diselenide bridge was accommodated in the hydrophobic core without steric clash.     

“First‐Principles” kinetic monte carlo simulations revisited: CO oxidation over RuO2(110)

First principles‐based kinetic Monte Carlo (kMC) simulations are performed for the CO oxidation on RuO₂(110) under steady‐state reaction conditions. The simulations include a set of elementary reaction steps with activation energies taken from three different ab initio density functional theory studies. Critical comparison of the simulation results reveals that already small variations in the activation energies lead to distinctly different reaction scenarios on the surface, even to the point where the dominating elementary reaction step is substituted by another one. For a critical assessment of the chosen energy parameters, it is not sufficient to compare kMC simulations only to experimental turnover frequency (TOF) as a function of the reactant feed ratio. More appropriate benchmarks for kMC simulations are the actual distribution of reactants on the catalyst's surface during steady‐state reaction, as determined by in situ infrared spectroscopy and in situ scanning tunneling microscopy, and the temperature dependence of TOF in the from of Arrhenius plots. © 2012 Wiley Periodicals, Inc.     

Chemical/Light‐Powered Hybrid Micromotors with “On‐the‐Fly” Optical Brakes

Hybrid micromotors capable of both chemically powered propulsion and fuel‐free light‐driven actuation and offering built‐in optical brakes for chemical propulsion are described. The new hybrid micromotors are designed by combining photocatalytic TiO₂ and catalytic Pt surfaces into a Janus microparticle. The chemical reactions on the different surfaces of the Janus particle hybrid micromotor can be tailored by using chemical or light stimuli that generate counteracting propulsion forces on the catalytic Pt and photocatalytic TiO₂ sides. Such modulation of the surface chemistry on a single micromotor leads to switchable propulsion modes and reversal of the direction of motion that reflect the tuning of the local ion concentration and hence the dominant propulsion force. An intermediate Au layer (under the Pt surface) plays an important role in determining the propulsion mechanism and operation of the hybrid motor. The built‐in optical braking system allows “on‐the‐fly” control of the chemical propulsion through a photocatalytic reaction on the TiO₂ side to counterbalance the chemical propulsion force generated on the Pt side. The adaptive dual operation of these chemical/light hybrid micromotors, associated with such control of the surface chemistry, holds considerable promise for designing smart nanomachines that autonomously reconfigure their propulsion mode for various on‐demand operations.     

“Click” on Tubes: a Versatile Approach towards Multimodal Functionalization of SWCNTs

Organic functionalization of carbon nanotube sidewalls is a tool of primary importance in material science and nanotechnology, equally from a fundamental and an applicative point of view. 1 , 2 Here, an efficient and versatile approach for the organic/organometallic functionalization of single‐walled carbon nanotubes (SWCNTs) capable of imparting multimodality to these fundamental nanostructures, is described. Our strategy takes advantage of well‐established Cu‐mediated acetylene‐azide coupling (CuAAC) reactions applied to phenylazido‐functionalized SWCNTs for their convenient homo‐/heterodecoration with a number of organic/organometallic frameworks, or mixtures thereof, bearing terminal acetylene pendant arms. Phenylazido‐decorated SWCNTs were prepared by chemoselective arylation of the CNT sidewalls with diazonium salts under mild conditions, and subsequently used for the copper‐mediated cycloaddition protocol in the presence of terminal acetylenes. The latter reaction was performed in one step by using either single acetylene derivatives or equimolar mixtures of terminal alkynes bearing either similar functional groups (masked with orthogonally cleavable protecting groups) or easily distinguishable functionalities (on the basis of complementary analytical/spectroscopic techniques). All materials and intermediates were characterized with respect to their most relevant aspects/properties by TEM microscopy, thermogravimetric analysis coupled with MS analysis of volatiles (TG‐MS), elemental analysis, cyclic voltammetry (CV), Raman and UV/Vis spectroscopy. The functional loading and related chemical grafting of both primary amino‐ and ferrocene‐decorated SWCNTs were spectroscopically (UV/Vis, Kaiser test) and electrochemically (CV) determined, respectively.     

An Antibody with a Variable‐Region Coiled‐Coil “Knob” Domain

The X‐ray crystal structure of a bovine antibody (BLV1H12) revealed a unique structure in its ultralong heavy chain complementarity determining region 3 (CDR3H) that folds into a solvent‐exposed β‐strand “stalk” fused to a disulfide crosslinked “knob” domain. We have substituted an antiparallel heterodimeric coiled‐coil motif for the β‐strand stalk in this antibody. The resulting antibody (Ab‐coil) expresses in mammalian cells and has a stability similar to that of the parent bovine antibody. MS analysis of H–D exchange supports the coiled‐coil structure of the substituted peptides. Substitution of the knob‐domain of Ab‐coil with bovine granulocyte colony‐stimulating factor (bGCSF) results in a stably expressed chimeric antibody, which proliferates mouse NFS‐60 cells with a potency comparable to that of bGCSF. This work demonstrates the utility of this novel coiled‐coil CDR3 motif as a means for generating stable, potent antibody fusion proteins with useful pharmacological properties.     

Graphene Quantum Dots Assembled with Metalloporphyrins for “Turn on” Sensing of Hydrogen Peroxide and Glucose

Noncovalent and multifunctional hybrids have been generated via π–π stacking and electrostatic interactions by combining the nanometer‐scale graphene structure of graphene quantum dots (GQDs) with Fe^III 5,10,15,20‐tetrakis(1‐methyl‐4‐pyridyl)porphine (FeTMPyP). The inner filter effect (IFE) of FeTMPyP on the GQDs results in substantial PL quenching of the GQDs. The quenched PL of GQDs by the FeTMPyP can be switched back “on” in response to the reaction between FeTMPyP and H₂O₂, which causes rupture of the cyclic tetrapyrrolic nucleus with consequential loss of iron from FeTMPyP, and then proceeds further to produce colorless dipyrroles and monopyrroles. This “turn on” system can be applied for simple and convenient H₂O₂ sensing and can be further extended to the detection of glucose in combination with the specific catalytic effect of glucose oxidase (GOx) through the oxidation of glucose and formation of H₂O₂. Because of the inherent synthetic control available for the design of metalloporphyrins, the GQDs‐based optical sensing approach described here has the potential to be highly versatile for other target analytes.     

Reassessment of an Innovative Insulin Analogue Excludes Protracted Action yet Highlights the Distinction between External and Internal Diselenide Bridges

Long-acting insulin analogues represent the most prescribed class of therapeutic proteins. An innovative design strategy was recently proposed: diselenide substitution of an external disulfide bridge. This approach exploited the distinctive physicochemical properties of selenocysteine (U). Relative to wild type (WT), Se-insulin[C7U^A, C7U^B] was reported to be protected from proteolysis by insulin-degrading enzyme (IDE), predicting prolonged activity. Because of this strategy's novelty and potential clinical importance, we sought to validate these findings and test their therapeutic utility in an animal model of diabetes mellitus. Surprisingly, the analogue did not exhibit enhanced stability, and its susceptibility to cleavage by either IDE or a canonical serine protease (glutamyl endopeptidase Glu-C) was similar to WT. Moreover, the analogue's pharmacodynamic profile in rats was not prolonged relative to a rapid-acting clinical analogue (insulin lispro). Although [C7U^A, C7U^B] does not confer protracted action, nonetheless its comparison to internal diselenide bridges promises to provide broad biophysical insight.     

Array‐based “Chemical Nose” Sensing in Diagnostics and Drug Discovery

Array‐based sensor “chemical nose/tongue” platforms are inspired by the mammalian olfactory system. Multiple sensor elements in these devices selectively interact with target analytes, producing a distinct pattern of response and enabling analyte identification. This approach offers unique opportunities relative to “traditional” highly specific sensor elements such as antibodies. Array‐based sensors excel at distinguishing small changes in complex mixtures, and this capability is being leveraged for chemical biology studies and clinical pathology, enabled by a diverse toolkit of new molecular, bioconjugate and nanomaterial technologies. Innovation in the design and analysis of arrays provides a robust set of tools for advancing biomedical goals, including precision medicine.     

“Stainomics”: Identification of mitotracker labeled proteins in mammalian cells

Organelle‐specific cell‐permeable fluorescent dyes are invaluable tools in cell biology as they reveal intracellular dynamics in living cells. Mitrotracker is a family of dyes that strongly label the mitochondrion, a key organelle associated with many crucial cellular functions. Despite the popularity of these dyes, little is known about the molecular mechanism behind their staining specificity. Here, we aimed to identify the protein targets of one member of this dye family, mitotracker red (MTR), by 2DE and MS. MTR bound to cellular proteins covalently, and its fluorescence persisted even after cell lysis, protein solubilization, denaturation, and electrophoresis. This enabled us to display MTR‐labeled proteins by 2DE. The MTR‐specific fluorescent signals on the gel revealed the spots that contained MTR‐conjugated proteins. These spots were analyzed by MS, resulting into the identification of ten proteins. We discovered that one major target is the mitochondrial protein HSP60 and that MTR staining could induce production of HSP60, predisposing cells to heat shock‐like responses. The identification of the molecular targets of biological dyes, or “stainomics,” can help correlate their intracellular staining properties with biochemical affinities. We believe this approach can be applied to a wide range of fluorescent probes.     

Structural Transformation with “Negative Volume Expansion”: Chemical Bonding and Physical Behavior of TiGePt

The synthesis and a joint experimental and theoretical study of the crystal structure and physical properties of the new ternary intermetallic compound TiGePt are presented. Upon heating, TiGePt exhibits an unusual structural phase transition with a huge volume contraction of about 10 %. The transformation is characterized by a strong change in the physical properties, in particular, by an insulator–metal transition. At temperatures below 885 °C TiGePt crystallizes in the cubic MgAgAs (half‐Heusler) type (LT phase, space group F$\bar 4$ 3m, a=5.9349(2) Å). At elevated temperatures, the crystal structure of TiGePt transforms into the TiNiSi structure type (HT phase, space group Pnma, a=6.38134(9) Å, b=3.89081(5) Å, c=7.5034(1) Å). The reversible, temperature‐dependent structural transition was investigated by in‐situ neutron powder diffraction and dilatometry measurements. The insulator–metal transition, indicated by resistivity measurements, is in accord with band structure calculations yielding a gap of about 0.9 eV for the LT phase and a metallic HT phase. Detailed analysis of the chemical bonding in both modifications revealed an essential change of the Ti–Pt and Ti–Ge interactions as the origin of the dramatic changes in the physical properties.