Structure-guided evolution of potent and selective CHK1 inhibitors through scaffold morphing.

Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice.

RNA as scaffold for pyrene excited complexes.

Synthesis and spectral properties of 1-ethynylpyrene base modified RNA are reported. The fluorophore attached to the 2-position of adenosine is directed into the easily accessible minor groove in RNA. Through an intermolecular interaction of the pyrene residues in twofold labelled RNA, single and double strands can be distinguished by their fluorescence maxima around 450 and 480 nm, respectively. This behaviour allows the kinetic investigation of RNA hybridisation and folding by fluorescence spectroscopy.

Synthesis of pyrene labeled RNA for fluorescence measurements.

The fluorophores 1-ethynylpyrene and 1-(p-ethynyl-phenylethynyl)-pyrene were attached to RNA through a Sonogashira cross-coupling with 5-iodocytidine either in solution through phosphoamidite synthesis or via on-column conjugation during solid-phase oligonucleotide synthesis. Six probes with the sequence 5'-CUU UUC UUU CUU-3' were derivatized with both fluorophores, whereby the position of the modified cytidine was varied. Fluorescence measurements showed sensitivity of the pyrene group to its environment in the single strands and corresponding duplexes.

Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances.

In this protocol, we describe the facile synthesis of the nitroxide spin-label 2,2,5,5-tetramethyl-pyrrolin-1-oxyl-3-acetylene (TPA) and then its coupling to DNA/RNA through Sonogashira cross-coupling during automated solid-phase synthesis. Subsequently, we explain how to perform distance measurements between two such spin-labels on RNA/DNA using the pulsed electron paramagnetic resonance method pulsed electron double resonance (PELDOR). This combination of methods can be used to study global structure elements of oligonucleotides in frozen solution at RNA/DNA amounts of approximately 10 nmol. We especially focus on the Sonogashira cross-coupling step, the advantages of the ACE chemistry together with the appropriate parameters for the RNA synthesizer and on the PELDOR data analysis. This procedure is applicable to RNA/DNA strands of up to approximately 80 bases in length and PELDOR yields reliably spin-spin distances up to approximately 6.5 nm. The synthesis of TPA takes approximately 5 days and spin labeling together with purification approximately 4 days. The PELDOR measurements usually take approximately 16 h and data analysis from an hour up to several days depending on the extent of analysis.

Base-specific spin-labeling of RNA for structure determination.

To facilitate the measurement of intramolecular distances in solvated RNA systems, a combination of spin-labeling, electron paramagnetic resonance (EPR), and molecular dynamics (MD) simulation is presented. The fairly rigid spin label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA) was base and site specifically introduced into RNA through a Sonogashira palladium catalyzed cross-coupling on column. For this purpose 5-iodo-uridine, 5-iodo-cytidine and 2-iodo-adenosine phosphoramidites were synthesized and incorporated into RNA-sequences. Application of the recently developed ACE chemistry presented the main advantage to limit the reduction of the nitroxide to an amine during the oligonucleotide automated synthesis and thus to increase substantially the reliability of the synthesis and the yield of labeled oligonucleotides. 4-Pulse Electron Double Resonance (PELDOR) was then successfully used to measure the intramolecular spin-spin distances in six doubly labeled RNA-duplexes. Comparison of these results with our previous work on DNA showed that A- and B-Form can be differentiated. Using an all-atom force field with explicit solvent, MD simulations gave results in good agreement with the measured distances and indicated that the RNA A-Form was conserved despite a local destabilization effect of the nitroxide label. The applicability of the method to more complex biological systems is discussed.

Synthesis of spin-labeled RNAs for long range distance measurements by peldor.

Site directed spin labeled RNA duplexes with different interspin distances were synthesized. The radical 2,2,5,5-tetramethyl-pyrrolin- 1-yloxyl-3-acetylene (TPA) was introduced during the solid-phase synthesis through a Sonogashira cross-coupling with 5-iodo-uridine. Tm and CD studies showed that the spin label does not to disturb significantly the A-form of these duplexes. 4-Pulse Electron Double Resonance (PELDOR) was then used to measure intramolecular spin-spin distances of 19.3, 33.0 and 40.9 A, which are in very good agreement with the calculated values of 17.6, 32.1 and 39.1 A, obtained from Molecular Dynamics (MD) simulations.

A PELDOR-based nanometer distance ruler for oligonucleotides.

A pulsed electron paramagnetic resonance (EPR) spectroscopic ruler for oligonucleotides was developed using a series of duplex DNAs. The spin-labeling is accomplished during solid-phase synthesis of the oligonucleotides utilizing a palladium-catalyzed cross-coupling reaction between 5-iodo-2'-deoxyuridine and the rigid spin-label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA). 4-Pulse electron double resonance (PELDOR) was then used to measure the intramolecular spin-spin distances via the dipolar coupling, yielding spin-spin distances of 19.2, 23.3, 34.7, 44.8, and 52.5 A. Employing a full-atom force field with explicit water, molecular dynamic (MD) simulations on the same spin-labeled oligonucleotides in their duplex B-form gave spin-spin distances of 19.6, 21.4, 33.0, 43.3, and 52.5 A, respectively, in very good agreement with the measured distances. This shows that the oligonucleotides adopt a B-form duplex structure also in frozen aqueous buffer solution. It also demonstrates that the combined use of site-directed spin-labeling, PELDOR experiments, and MD simulations can yield a microscopic picture about the overall structure of oligonucleotides. The technique is also applicable to more complex systems, like ribozymes or DNA/RNA-protein complexes, which are difficult to access by NMR or X-ray crystallography.