Novel Potent Selective Orally Active S1P5 Receptor Antagonists.

S1P5 is one of the five sphingosine-1-phosphate (S1P) receptors which play important roles in immune and CNS cell homeostasis, growth, and differentiation. Little is known about the effect of modulation of S1P5 due to the lack of S1P5 specific modulators with suitable druglike properties. Here we describe the discovery and optimization of a novel series of potent selective S1P5 antagonists and the identification of an orally active brain-penetrant tool compound 15.

Potent PDZ-Domain PICK1 Inhibitors that Modulate Amyloid Beta-Mediated Synaptic Dysfunction.

Protein interacting with C kinase (PICK1) is a scaffolding protein that is present in dendritic spines and interacts with a wide array of proteins through its PDZ domain. The best understood function of PICK1 is regulation of trafficking of AMPA receptors at neuronal synapses via its specific interaction with the AMPA GluA2 subunit. Disrupting the PICK1-GluA2 interaction has been shown to alter synaptic plasticity, a molecular mechanism of learning and memory. Lack of potent, selective inhibitors of the PICK1 PDZ domain has hindered efforts at exploring the PICK1-GluA2 interaction as a therapeutic target for neurological diseases. Here, we report the discovery of PICK1 small molecule inhibitors using a structure-based drug design strategy. The inhibitors stabilized surface GluA2, reduced Aß-induced rise in intracellular calcium concentrations in cultured neurons, and blocked long term depression in brain slices. These findings demonstrate that it is possible to identify potent, selective PICK1-GluA2 inhibitors which may prove useful for treatment of neurodegenerative disorders.

Lock and chop: A novel method for the generation of a PICK1 PDZ domain and piperidine-based inhibitor co-crystal structure.

The membrane protein interacting with kinase C1 (PICK1) plays a trafficking role in the internalization of neuron receptors such as the amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor. Reduction of surface AMPA type receptors on neurons reduces synaptic communication leading to cognitive impairment in progressive neurodegenerative diseases such as Alzheimer disease. The internalization of AMPA receptors is mediated by the PDZ domain of PICK1 which binds to the GluA2 subunit of AMPA receptors and targets the receptor for internalization through endocytosis, reducing synaptic communication. We planned to block the PICK1-GluA2 protein-protein interaction with a small molecule inhibitor to stabilize surface AMPA receptors as a therapeutic possibility for neurodegenerative diseases. Using a fluorescence polarization assay, we identified compound BIO124 as a modest inhibitor of the PICK1-GluA2 interaction. We further tried to improve the binding affinity of BIO124 using structure-aided drug design but were unsuccessful in producing a co-crystal structure using previously reported crystallography methods for PICK1. Here, we present a novel method through which we generated a co-crystal structure of the PDZ domain of PICK1 bound to BIO124.

Pericyte MyD88 and IRAK4 control inflammatory and fibrotic responses to tissue injury.

Fibrotic disease is associated with matrix deposition that results in the loss of organ function. Pericytes, the precursors of myofibroblasts, are a source of pathological matrix collagens and may be promising targets for treating fibrogenesis. Here, we have shown that pericytes activate a TLR2/4- and MyD88-dependent proinflammatory program in response to tissue injury. Similarly to classic immune cells, pericytes activate the NLRP3 inflammasome, leading to IL-1ß and IL-18 secretion. Released IL-1ß signals through pericyte MyD88 to amplify this response. Unexpectedly, we found that MyD88 and its downstream effector kinase IRAK4 intrinsically control pericyte migration and conversion to myofibroblasts. Specific ablation of MyD88 in pericytes or pharmacological inhibition of MyD88 signaling by an IRAK4 inhibitor in vivo protected against kidney injury by profoundly attenuating tissue injury, activation, and differentiation of myofibroblasts. Our data show that in pericytes, MyD88 and IRAK4 are key regulators of 2 major injury responses: inflammatory and fibrogenic. Moreover, these findings suggest that disruption of this MyD88-dependent pathway in pericytes might be a potential therapeutic approach to inhibit fibrogenesis and promote regeneration.

Synapto-depressive effects of amyloid beta require PICK1.

Amyloid beta (Aß), a key component in the pathophysiology of Alzheimer's disease, is thought to target excitatory synapses early in the disease. However, the mechanism by which Aß weakens synapses is not well understood. Here we showed that the PDZ domain protein, protein interacting with C kinase 1 (PICK1), was required for Aß to weaken synapses. In mice lacking PICK1, elevations of Aß failed to depress synaptic transmission in cultured brain slices. In dissociated cultured neurons, Aß failed to reduce surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit 2, a subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors that binds with PICK1 through a PDZ ligand-domain interaction. Lastly, a novel small molecule (BIO922) discovered through structure-based drug design that targets the specific interactions between GluA2 and PICK1 blocked the effects of Aß on synapses and surface receptors. We concluded that GluA2-PICK1 interactions are a key component of the effects of Aß on synapses.

Stereochemistry-activity relationship of orally active tetralin S1P agonist prodrugs.

Modifying FTY720, an immunosuppressant modulator, led to a new series of well phosphorylated tetralin analogs as potent S1P1 receptor agonists. The stereochemistry effect of tetralin ring was probed, and (-)-(R)-2-amino-2-((S)-6-octyl-1,2,3,4-tetrahydronaphthalen-2-yl)propan-1-ol was identified as a good SphK2 substrate and potent S1P1 agonist with good oral bioavailability.

Design and synthesis of a series of meta aniline-based LFA-1 ICAM inhibitors.

A series of meta-substituted anilines were designed and synthesized to inhibit the interaction of LFA-1 with ICAM for the treatment of autoimmune disease. Design of these molecules was performed by utilizing a co-crystal structure for structure-based drug design. The resulting molecules were found to be potent and to possess favorable pharmaceutical properties.

Structure-activity relationship of ortho- and meta-phenol based LFA-1 ICAM inhibitors.

LFA-1 ICAM inhibitors based on ortho- and meta-phenol templates were designed and synthesized by Mitsunobu chemistry. The selection of targets was guided by X-ray co-crystal data, and led to compounds which showed an up to 30-fold increase in potency over reference compound 1 in the LFA-1/ICAM1-Ig assay. The most active compound exploited a new hydrogen bond to the I-domain and exhibited subnanomolar potency.

DNA detection and signal amplification via an engineered allosteric enzyme.

Rapid, sensitive, and sequence-specific DNA detection can be achieved in one step using an engineered intrasterically regulated enzyme. The semi-synthetic inhibitor-DNA-enzyme (IDE) construct (left) rests in the inactive state but upon exposure to a complementary DNA sequence undergoes a DNA hybridization-triggered allosteric enzyme activation (right). The ensuing rapid substrate turnover provides the built-in signal amplification mechanism for detecting approximately 10 fmol DNA in less than 3 min under physiological conditions.

DNA-based photonic logic gates: AND, NAND, and INHIBIT.

Conventional microprocessors use elementary logic gates to perform complex computational tasks. Mimicking such computational processes using purely molecular systems has been limited in most cases by the lack of design generality or potential addressability of existing molecular logic gates. Herein we report that by employing the universal recognition properties of DNA simple photonic logic gates can be created that are capable of AND, NAND, and INHIBIT logic operations.

Factors Contributing to Aromatic Stacking in Water: Evaluation in the Context of DNA.

We report the use of thermodynamic measurements in a self-complementary DNA duplex (5'-dXCGCGCG)(2), where X is an unpaired natural or nonnatural deoxynucleoside, to study the forces that stabilize aqueous aromatic stacking in the context of DNA. Thermal denaturation experiments show that the core duplex (lacking X) is formed with a free energy (37 °C) of -8.1 kcal·mol(-1) in a pH 7.0 buffer containing 1 M Na(+). We studied the effects of adding single dangling nucleosides (X) where the aromatic "base" is adenine, guanine, thymine, cytosine, pyrrole, benzene, 4-methylindole, 5-nitroindole, trimethylbenzene, difluorotoluene, naphthalene, phenanthrene, and pyrene. Adding these dangling residues is found to stabilize the duplex by an additional -0.8 to -3.4 kcal·mol(-1). At 5 µM DNA concentration, T(m) values range from 41.7 °C (core sequence) to 64.1 °C (with dangling pyrene residues). For the four natural bases, the order of stacking ability is A > G ≥ T = C. The nonpolar analogues stack more strongly in general than the more polar natural bases. The stacking geometry was confirmed in two cases (X = adenine and pyrene) by 2-D NOESY experiments. Also studied is the effect of ethanol cosolvent on the stacking of natural bases and pyrene. Stacking abilities were compared to calculated values for hydrophobicity, dipole moment, polarizability, and surface area. In general, hydrophobic effects are found to be larger than other effects stabilizing stacking (electrostatic effects, dispersion forces); however, the natural DNA bases are found to be less dependent on hydrophobic effects than are the more nonpolar compounds. The results also point out strategies for the design nucleoside analogues that stack considerably more strongly than the natural bases; such compounds may be useful in stabilizing designed DNA structures and complexes.

Solution Structure of a Nonpolar, Non-Hydrogen-Bonded Base Pair Surrogate in DNA.

We describe the structure in aqueous solution of a DNA duplex containing a base pair that is structurally analogous to A-T but which lacks hydrogen bonds. Base analogues F (a nonpolar isostere of thymine) and Z (a nonpolar isostere of adenine) are paired opposite one another in a 12 base pair duplex. The sequence context is the binding site of recently studied transcription factor hSRY. The Z-F pair has been shown to be replicated surprisingly well and selectively by DNA polymerase enzymes, considering that it is destabilizing and lacks Watson-Crick hydrogen bonds. The enzymatic studies led to the suggestion that part of the functional activity arises because the pair resembles a natural one in geometry. The present results show that, despite the absence of Watson-Crick hydrogen bonds, the Z-F pair structurally resembles an A-T pair in the same context. This lends support to the proposal that shape matching is an important component in replication, and suggests the general utility of using Z-F as a nonpolar replacement for A-T in probing protein-DNA interactions.

Structure and Base Pairing Properties of a Replicable Nonpolar Isostere for Deoxyadenosine.

We report the synthesis, structure, and pairing properties in DNA of an isostere for deoxyadenosine which lacks all hydrogen-bonding functionality on the Watson-Crick pairing edge. A deoxyribo-nucleoside derivative of 4-methylbenzimidazole (1), which was recently shown to be inserted into DNA by Klenow DNA polymerase (Morales, J. C.; Kool, E. T. Nature Struct. Biol.1998, 5, 950), is prepared from 1-chloro-2-deoxy-3,5-bis-O-p-toluoyl-α-D-erythro-pentofuranose. The X-ray crystal structure of the nucleoside confirms that the compound is a close steric match for deoxyadenosine (2), although the methylbenzimidazole base is in the syn glycosidic orientation in the crystal. In D(2)O solution, 1H NMR studies show that 1 and 2 have similar (60% vs 70% S) sugar conformations and anti glycosidic orientations. Compound 1 is incorporated into a 12mer oligodeoxynucleotide and its base pairing properties in duplexes assessed by thermal denaturation. The results show that 1 has low affinity for the four natural bases but displays a stronger preference for being situated opposite a nonpolar difluorotoluene nucleoside analogue of thymine (3). The structural similarities of 1 and 2, combined with recent polymerase studies, add support to the hypothesis that steric complementarity plays an important role in base pair replication by polymerase enzymes and that Watson-Crick hydrogen bonds are not absolute requirements. Compound 1 should have significant utility as a probe of the importance of electrostatic effects in protein-DNA and protein-nucleotide binding as well as in DNA replication.