Andreas Brunschweiger

Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28

Lin28 inhibits the biogenesis of let-7 miRNAs through a direct interaction with the terminal loop of pre-let-7. This interaction requires the zinc-knuckle domains of Lin28. We show that the zinc knuckle domains of Lin28 are sufficient to provide binding selectivity for pre-let-7 miRNAs and present the NMR structure of human Lin28 zinc knuckles bound to the short sequence 5′-AGGAGAU-3′. The structure reveals that each zinc knuckle recognizes an AG dinucleotide separated by a single nucleotide spacer. This defines a new 5′-NGNNG-3′ consensus motif that explains how Lin28 selectively recognizes pre-let-7 family members. Binding assays in cell lysates and functional assays in cultured cells demonstrate that the interactions observed in the solution structure also occur between the full-length protein and members of the pre-let-7 family. The consensus sequence explains several seemingly disparate previously published observations on the binding properties of Lin28.

A Decade of the Human Genome Sequence—How Does the Medicinal Chemist Benefit?

Many have claimed that the sequencing of the human genome has failed to deliver the promised new era of drug discovery and development. Here, we argue that in fact, the availability of the human genome sequence and the genomics technologies that resulted from those research efforts have had a major impact on drug discovery. Medicinal chemists are actively using the data gleaned from structural genomics projects over the past decade to design more selective and more effective drug candidates. For example, large superfamilies of related enzymes, such as the kinome, proteome, proteasome, transportome, identified because of the sequencing of the human genome represent a huge number of potential drug targets. Ten years on, we′re able to design multitarget drugs where the selectivity for a certain subgroup of receptors can lead to increased efficacy rather than the side effects traditionally associated with “off‐targets”. New trends and discoveries in biomedical research are notoriously slow to show their value, and this is also true for genomics technologies. However, the examples we′ve selected show that these are firmly set in the drug‐discovery process, and without the human genome sequence, a number of current clinical candidates and promising drug leads would not have been possible.

Chemical Synthesis of Mono- and Bis-Labeled Pre-MicroRNAs†

and other related fields. Most applicationsinvolve relatively short DNAs and RNAs; however thedemand for labeled long RNA (>60 nt) has increased inline with our growing appreciation of a rich, only partiallyunderstood biology of structured non-coding RNAs.A common secondary structure motif in RNA is thehairpin, which is composed of a terminal loop region anda double-stranded RNA stem that often bears mismatchesandbulges.Thismotifiscentraltoseveralbiologicalfunctionsincluding RNA maturation, RNA folding, ribozyme function,and protection of RNA from degradation.

1,3-Dialkyl-substituted tetrahydropyrimido[1,2-f]purine-2,4-diones as multiple target drugs for the potential treatment of neurodegenerative diseases.

Adenosine receptors and monoamine oxidases are drug targets for neurodegenerative diseases such as Parkinsons and Alzheimers disease. In the present study we prepared a library of 55 mostly novel tetrahydropyrimido[2,1-f]purinediones with various substituents in the 1- and 3-position (1,3-dimethyl, 1,3-diethyl, 1,3-dipropyl, 1-methyl-3-propargyl) and broad variation in the 9-position. A synthetic strategy to obtain 3-propargyl-substituted tetrahydropyrimido[2,1-f]purinedione derivatives was developed. The new compounds were evaluated for their interaction with all four adenosine receptor subtypes and for their ability to inhibit monoamine oxidases (MAO). Introduction of mono- or di-chloro-substituted phenyl, benzyl or phenethyl residues at N9 of the 1,3-dimethyl series led to the discovery of a novel class of potent MAO-B inhibitors, the most potent compound being 9-(3,4-dichlorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[1,2-f]purine-2,4(1H,3H)-dione (21g, IC(50) human MAO-B: 0.0629 μM), which displayed high selectivity versus the other investigated targets. Potent dually active A1/A2A adenosine receptor antagonists were identified, for example, 9-benzyl-1-methyl-3-propargyl-6,7,8,9-tetrahydropyrimido[1,2-f]purine-2,4(1H,3H)dione (19f, Ki, human receptors, A1: 0.249 μM, A2A: 0.253 μM). Several compounds showed triple-target inhibition, the best compound being 9-(2-methoxybenzyl)-1-methyl-3-(prop-2-ynyl)-6,7,8,9-tetrahydro pyrimido [1,2-f]purine-2,4(1H,3H)-dione (19g, Ki A1: 0.605 μM, Ki A2A: 0.417 μM, IC(50) MAO-B: 1.80 μM). Compounds inhibiting several different targets involved in neurodegeneration may exhibit additive or even synergistic effects in vivo.

8‐Benzyltetrahydropyrazino[2,1‐f]purinediones: Water‐Soluble Tricyclic Xanthine Derivatives as Multitarget Drugs for Neurodegenerative Diseases

8‐Benzyl‐substituted tetrahydropyrazino[2,1‐f]purinediones were designed as tricyclic xanthine derivatives containing a basic nitrogen atom in the tetrahydropyrazine ring to improve water solubility. A library of 69 derivatives was prepared and evaluated in radioligand binding studies at adenosine receptor (AR) subtypes and for their ability to inhibit monoamine oxidases (MAO). Potent dual‐target‐directed A1/A2A adenosine receptor antagonists were identified. Several compounds showed triple‐target inhibition; one of the best compounds was 8‐(2,4‐dichloro‐5‐fluorobenzyl)‐1,3‐dimethyl‐6,7,8,9‐tetrahydropyrazino[2,1‐f]purine‐2,4(1H,3H)‐dione (72) (human AR: Ki A1 217 nM, A2A 233 nM; IC50 MAO‐B: 508 nM). Dichlorinated compound 36 [8‐(3,4‐dichlorobenzyl)‐1,3‐dimethyl‐6,7,8,9‐tetrahydropyrazino[2,1‐f]purine‐2,4(1H,3H)‐dione] was found to be the best triple‐target drug in rat (Ki A1 351 nM, A2A 322 nm; IC50 MAO‐B: 260 nM), and may serve as a useful tool for preclinical proof‐of‐principle studies. Compounds that act at multiple targets relevant for symptomatic as well as disease‐modifying treatment of neurodegenerative diseases are expected to show advantages over single‐target therapeutics.

Design and synthesis of DNA-encoded libraries based on a benzodiazepine and a pyrazolopyrimidine scaffold

Selection-based screening of large DNA-encoded libraries of drug-like small molecules is a validated method to identify bioactive compounds. Among the chemical space of bioactive compounds certain scaffold structures are well represented. These are commonly called “privileged scaffolds”. We have synthesized DNA-encoded libraries based on two representatives of these scaffolds, a benzodiazepine and a pyrazolopyrimidine, and additionally a third library based on propargyl glycine. All three core structures possess a carboxylic acid to couple them to aminolinker-modified DNA. For subsequent library synthesis they contained an amino function to which a set of carboxylic acid building blocks were coupled, and a terminal alkyne that was reacted with a set of azides to furnish triazoles. The two sets of building blocks, 114 carboxylic acids and 104 azides, were selected with the help of chemoinformatic methods in order to control the physicochemical properties of the final libraries, remove unwanted substructures, and maximize diversity. The set of building blocks contained desthiobiotin allowing for validation of library synthesis. The DNA-encoded libraries were synthesized by split-and-pool combinatorial chemistry yielding three libraries that contain 28.254 compounds together. For DNA barcoding, 5′-phosphorylated double-stranded coding DNA sequences with four base overhangs were ligated with T4 ligase. The resulting DNA-encoded libraries were compared to bioactivity databases and, though being based on core structures well-established in medicinal chemistry, showed novelty with respect to the known bioactive chemical space.

8-Substituted 1,3-dimethyltetrahydropyrazino[2,1-f]purinediones: Water-soluble adenosine receptor antagonists and monoamine oxidase B inhibitors

Multitarget approaches, i.e., addressing two or more targets simultaneously with a therapeutic agent, are hypothesized to offer additive therapeutic benefit for the treatment of neurodegenerative diseases. Validated targets for the treatment of Parkinsons disease are, among others, the A2A adenosine receptor (AR) and the enzyme monoamine oxidase B (MAO-B). Additional blockade of brain A1 ARs may also be beneficial. We recently described 8-benzyl-substituted tetrahydropyrazino[2,1-f]purinediones as a new lead structure for the development of such multi-target drugs. We have now designed a new series of tetrahydropyrazino[2,1-f]purinediones to extensively explore their structure-activity-relationships. Several compounds blocked human and rat A1 and A2AARs at similar concentrations representing dual A1/A2A antagonists with high selectivity versus the other AR subtypes. Among the best dual A1/A2AAR antagonists were 8-(3-(4-chlorophenyl)propyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (41, Ki human A1: 65.5nM, A2A: 230nM; Ki rat A1: 352nM, A2A: 316nM) and 1,3-dimethyl-8-((2-(thiophen-2-yl)thiazol-4-yl)methyl)-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (57, Ki human A1: 642nM, A2A: 203nM; Ki rat A1: 166nM, A2A: 121nM). Compound 57 was found to be well water-soluble (0.7mg/mL) at a physiological pH value of 7.4. One of the new compounds showed triple-target inhibition: (R)-1,3-dimethyl-8-(2,1,3,4-tetrahydronaphthalen-1-yl)-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (49) was about equipotent at A1 and A2AARs and at MAO-B (Ki human A1: 393nM, human A2A: 595nM, IC50 human MAO-B: 210nM) thus allowing future in vivo explorations of the intended multi-target approach.

Measurement by SPR of very low dissociation rates: oxidation-mediated loss of biotin-streptavidin affinity.

Long-term relationship: biotin labels on RNAs, and possibly other biomacromolecules, are easily oxidized causing a dramatic loss of affinity for streptavidin and adversely affecting the measurement of high-affinity interactions. A new SPR method has been developed for measuring the very low rate-dissociation constants of biotin- and biotin oxide-conjugated RNAs with streptavidin.

Two approaches to α,α-bis-Mannich salts of N-monosubstituted amides

Abstract Two generally applicable syntheses of 2-bis-(dimethylaminomethyl)acetamides 9 are described. The first one involves reaction of N -mono substituted acetamides 3 with dimethyl(methylene)ammonium chloride in the presence of phosphorus oxychloride using diethyl ether as a solvent. Starting from ketoximes, the amines 9 are obtained under the same reaction conditions via Beckmann rearrangement. C -Aminomethylation observed in all cases may be the result of the formation of imidoyl chlorides 5 as intermediates.

Towards improved oligonucleotide therapeutics through faster target binding kinetics

When used as inhibitors of gene expression in vivo, oligonucleotides require modification of their structures to boost their binding affinity for complementary target RNAs. To date, hundreds of modifications have been designed and tested but few have proven to be useful. Among those investigated are mono- and polyamino-groups. These are positively charged at physiological pH and have been appended to oligonucleotides in an effort to reduce electrostatic repulsion during hybridization to RNAs, but have generally shown relatively minor benefits to binding. We conjugated spermine to uracils in oligonucleotides via a triazole linker so that the polyamine fits in the major groove of a subsequently formed RNA-duplex. The modifications produced large increases in target-binding affinity of the oligonucleotides. Using surface plasmon resonance-based assays, we showed that the increases derived mainly from faster annealing (kon ). We propose that the spermine fragments play a similar role to that of natural polyamines during oligonucleotide-target interactions in cells, and may be advantageous for oligonucleotides that operate catalytic mechanisms.