No abstract

Through fragment-based drug design focused on engaging the active site of IRAK4 and leveraging three-dimensional topology in a ligand-efficient manner, a micromolar hit identified from a screen of a Pfizer fragment library was optimized to afford IRAK4 inhibitors with nanomolar potency in cellular assays. The medicinal chemistry effort featured the judicious placement of lipophilicity, informed by co-crystal structures with IRAK4 and optimization of ADME properties to deliver clinical candidate PF-06650833 (compound 40). This compound displays a 5-unit increase in lipophilic efficiency from the fragment hit, excellent kinase selectivity, and pharmacokinetic properties suitable for oral administration.

A supercritical fluid chromatography method was developed for the detection of intramolecular hydrogen bonds in pharmaceutically relevant molecules. The identification of compounds likely to form intramolecular hydrogen bonds is an important drug design consideration given the correlation of intramolecular hydrogen bonding with increased membrane permeability. The technique described here correlates chromatographic retention with the exposed polarity of a molecule. Molecules that can form an intramolecular hydrogen bond can hide their polarity and therefore exhibit lower retention than similar compounds that cannot. By use of a pairwise analysis strategy, intramolecular hydrogen bonds are identified within a test set of compounds with diverse topologies. The chromatographic results are confirmed by NMR chemical shift and temperature coefficient studies.

A novel method for the determination of the relative stereochemistry of six-membered chairlike ring molecules by residual dipolar couplings is presented. C-H residual dipolar couplings were used to investigate the relative stereochemistry of 4,6-O-ethylidene-d-glucopyranose. For this and similar systems it is not necessary to acquire redundant dipolar couplings and to calculate the orientation order tensor. The presented methodology is a paradigmatic leap for the determination of the relative stereochemistry or remote stereochemistry in this kind of fused ring system. Residual dipolar coupling data were collected by 1D and 2D direct-measurement heteronuclear multiple quantum coherence (HMQC) spectroscopy. It was demonstrated that direct measurement of HMQC was quick and accurate for small molecules at natural abundance.

No abstract

Residual dipolar couplings (RDCs), in combination with molecular order matrix calculations, were used to unambiguously determine the complete relative stereochemistry of an organic compound with five stereocenters. Three simple one-dimensional experiments were utilized for the measurements of (13)C-(1)H, (13)C-(19)F, (19)F-(1)H, and (1)H-(1)H RDCs. The order matrix calculation was performed on each chiral isomer independently. The fits were evaluated by the comparison of the root-mean-square deviation (rmsd) of calculated and measured RDCs. The order tensor simulations based on two different sets of RDC data collected with phage and bicelles are consistent. The resulting stereochemical assignments of the stereocenters obtained from using only RDCs are in perfect agreement with those obtained from the single-crystal X-ray structure. Six RDCs are found to be necessary to run the simulation, and seven are the minimum to get an acceptable result for the investigated compound. It was also shown that (13)C-(1)H and (1)H-(1)H RDCs, which are the easiest to measure, are also the most important and information-rich data for the order matrix calculation. The effect of each RDC on the calculation depends on the location of the corresponding vector in the structure. The direct RDC of a stereocenter is important to the configuration determination, but the configuration of stereocenters devoid of protons can also be obtained from analysis of nearby RDCs.

A novel method based on diffusion NMR for the epitope mapping of ligand binding is presented. The intermolecular NOE builds up during a long diffusion period and creates a deviation from the linearity. The ligand proton nearest the protein generates the strongest NOE from protein during the diffusion period and has the largest deviation. Therefore, this diffusion artifact can be used to characterize the ligand binding epitope. The concept was investigated using dihydrofolate reductase (DHFR) and its ligand trimethoprim (TMP), and the epitope map of TMP on DHFR generated with this method is in excellent agreement with the structural and dynamic studies by crystallography and NMR, as well as the medicinal chemistry results.

Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in hematopoiesis and in the development of bone, stomach epithelium, and proprioceptive neurons. Mutations in CBF genes are found in leukemias, bone disorders, and gastric cancer. CBFs consist of a DNA-binding CBF␣ subunit and a non-DNA-binding CBF␤ subunit. DNA binding and heterodimerization with CBF␤ are mediated by the Runt domain in CBF␣. Here we report an alanine-scanning mutagenesis study of the Runt domain that targeted amino acids identified by structural studies to reside at the DNA or CBF␤ interface, as well as amino acids mutated in human disease. We determined the energy contributed by each of the DNA-contacting residues in the Runt domain to DNA binding both in the absence and presence of CBF␤. We propose mechanisms by which mutations in the Runt domain found in hematopoietic and bone disorders affect its affinity for DNA.The core-binding factors (CBFs) 1 are a small family of heterodimeric transcription factors that play critical roles both in mammalian development and in human disease. CBF␣ subunits are encoded by three related genes, RUNX1, RUNX2, and RUNX3 (1-3), whereas the common CBF␤ subunit is encoded by a single gene called CBFB (4, 5). Genetic loss-and gain-of-function studies in mice documented roles for the CBF proteins in at least four developmental processes. Runx1 and Cbfb are required for definitive hematopoiesis (6 -9), and both Runx2 and Cbfb are required for bone formation (10 -14). The Runx3 gene is required for proper development of the stomach epithelium (15), for the development of a subset of sensory neurons (16,17), and for epigenetic silencing of the CD4 gene in T lymphocytes (18). Lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer (15).RUNX1 and CBFB are frequent targets of mutations in human leukemias (19,20). RUNX1 is disrupted by the t(8; 21)(q22;q22) in 15% of de novo acute myeloid leukemia (AML M 2 subtype) (2, 21), the t(12;21)(p13;q22) in 30% of pediatric de novo acute lymphoblastic leukemia (22-25), and by the relatively rare t(1;21)(p36;q22), t(3;21)(q26;q22), t(5;21)(q13;q22), t(12;21)(q24;q22), t(14;21)(q22;q22), t(15;21)(q22;q22), t(16; 21)(q24;q22), and t(17;21)(q11.2;q22) in the therapy-related leukemias and myelodysplasias (MDS) (2, 22, 23, 26 -29). Biallelic point mutations in RUNX1 are found in AML of the M 0 subtype (30 -32). RUNX1 mutations in AML M 0 include both nonsense mutations that introduce premature termination codons and missense mutations. Monoallelic point mutations in RUNX1 were identified in AML M 0 , MDS, pediatric acute lymphoblastic leukemia, and chronic myelogenous leukemia in blast crisis (30 -33). In addition, haploinsufficiency of the RUNX1 gene is responsible for a rare familial platelet disorder with propensity for acute myelogenous leukemia (FPD/AML) (34 -36).The RUNX2 gene is also the target of mutations that are responsible for an inherited human skeletal disorder called c...