Structure-Activity Studies of Novel Di-substituted [1,2,5]oxadiazolo [3,4-b]pyrazine Analogs Targeting the A-loop Regulatory Site of p38 MAP Kinase.

INTRODUCTION: In the quest for novel allosteric inhibitors of the p38 MAP kinase, we recently described the A-loop regulatory site, identified by means of molecular modeling studies together with the disclosure of a small molecule hit with a moderate inhibitory profile. Starting from this structure, we subsequently identified two additional hits with simpler molecular structures from an in silico screening study, using a substructure search in the SciFinder database. After corroboration of their inhibitory profile, analysis of their structures permitted to conclude about the suitability of the [1,2,5]oxadiazolo[3,4-b]pyrazine (furazano[ 3,4-b]pyrazine) scaffold for the development of potent A-loop regulatory site p38 MAP kinase inhibitors. Accordingly, we report the synthesis and pharmacological evaluation of a series of di-substituted analogs with a potent inhibitory profile of p38 MAP kinase, as shown by in vitro assays of their capability to inhibit IL-1ß secretion in human monocyte-derived macrophages. OBJECTIVE: To find small molecule potent inhibitors of the p38 MAP kinase A-loop regulatory site. METHODS: Starting from this structure, we subsequently identified two additional hits with simpler molecular structures from an in silico screening study, using a substructure search in the SciFinder database. After corroboration of their inhibitory profile, we carried out a hit-tolead optimization process guided by molecular modeling using a [1,2,5]oxadiazolo[3,4- b]pyrazine (furazano[3,4-b]pyrazine) scaffold. RESULTS: We report the synthesis and pharmacological evaluation of a series of di-substituted analogs with a potent inhibitory profile of p38 MAP kinase, as shown by in vitro assays of their capability to inhibit IL-1ß secretion in human monocyte-derived macrophages. CONCLUSION: We describe in the present work a series of [1,2,5]oxadiazolo[3,4-b]pyrazine (furazano[3,4-b]pyrazine), which are potent inhibitors of IL-1ß secretion in human monocytederived macrophages allosteric modulators of the p38 MAP kinase A-loop regulatory site.

Inhibition of Sema-3A Promotes Cell Migration, Axonal Growth, and Retinal Ganglion Cell Survival.

Purpose: Semaphorin 3A (Sema-3A) is a secreted protein that deflects axons from inappropriate regions and induces neuronal cell death. Intravitreal application of polyclonal antibodies against Sema-3A prevents loss of retinal ganglion cells ensuing from axotomy of optic nerves. This suggested a therapeutic approach for neuroprotection via inhibition of the Sema-3A pathway. Methods: To develop potent and specific Sema-3A antagonists, we isolated monoclonal anti-Sema-3A antibodies from a human antibody phage display library and optimized low-molecular weight Sema-3A signaling inhibitors. The best inhibitors were identified using in vitro scratch assays and semiquantitative repulsion assays. Results: A therapeutic approach for neuroprotection must have a long duration of action. Therefore, antibodies and low-molecular weight inhibitors were formulated in extruded implants to allow controlled and prolonged release. Following release from the implants, Sema-3A inhibitors antagonized Sema-3A effects in scratch and repulsion assays and protected retinal ganglion cells in animal models of optic nerve injury, retinal ischemia, and glaucoma. Conclusions and Translational Relevance: Collectively, our findings indicate that the identified Sema-3A inhibitors should be further evaluated as therapeutic candidates for the treatment of Sema-3A-driven central nervous system degenerative processes.

Semaphorin 3A-Glycosaminoglycans Interaction as Therapeutic Target for Axonal Regeneration.

Semaphorin 3A (Sema3A) is a cell-secreted protein that participates in the axonal guidance pathways. Sema3A acts as a canonical repulsive axon guidance molecule, inhibiting CNS regenerative axonal growth and propagation. Therefore, interfering with Sema3A signaling is proposed as a therapeutic target for achieving functional recovery after CNS injuries. It has been shown that Sema3A adheres to the proteoglycan component of the extracellular matrix (ECM) and selectively binds to heparin and chondroitin sulfate-E (CS-E) glycosaminoglycans (GAGs). We hypothesize that the biologically relevant interaction between Sema3A and GAGs takes place at Sema3A C-terminal polybasic region (SCT). The aims of this study were to characterize the interaction of the whole Sema3A C-terminal polybasic region (Sema3A 725-771) with GAGs and to investigate the disruption of this interaction by small molecules. Recombinant Sema3A basic domain was produced and we used a combination of biophysical techniques (NMR, SPR, and heparin affinity chromatography) to gain insight into the interaction of the Sema3A C-terminal domain with GAGs. The results demonstrate that SCT is an intrinsically disordered region, which confirms that SCT binds to GAGs and helps to identify the specific residues involved in the interaction. NMR studies, supported by molecular dynamics simulations, show that a new peptoid molecule (CSIC02) may disrupt the interaction between SCT and heparin. Our structural study paves the way toward the design of new molecules targeting these protein-GAG interactions with potential therapeutic applications.

Discovery of novel 2,3,5-trisubstituted pyridine analogs as potent inhibitors of IL-1ß via modulation of the p38 MAPK signaling pathway.

Interleukin-1ß is a central mediator of innate immune responses and inflammation. It plays a key role in a wide variety of pathologies, ranging from autoinflammatory diseases to metabolic syndrome and malignant tumors. It is well established that its inhibition results in a rapid and sustained reduction in disease severity, underlining the importance of having a repertoire of drugs of this class. At present, there are only three interleukin-1ß blockers approved in the clinic. All of them are biologics, requiring parenteral administration and resulting in expensive treatments. In an exercise to identify small molecule allosteric inhibitors of MAP kinases, we discovered a series of compounds that block IL-1ß release produced as a consequence of a stimulus involved in triggering an inflammatory response. The present study reports the hit-to-lead optimization process that permitted the identification of the compound 13b (AIK3-305) an orally available, potent and selective inhibitor of IL-1ß. Furthermore, the study also reports the results of an in vivo efficacy study of 13b in a LPS endotoxic shock model in male BALB/c mice, where IL-1ß inhibition is monitored in different tissues.

Centrally Active Multitarget Anti-Alzheimer Agents Derived from the Antioxidant Lead CR-6.

Oxidative stress is a major pathogenic factor in Alzheimer's disease, but it should not be tackled alone rather together with other key targets to derive effective treatments. The combination of the scaffold of the polar antioxidant lead 7-methoxy-2,2-dimethylchroman-6-ol (CR-6) with that of the lipophilic cholinesterase inhibitor 6-chlorotacrine results in compounds with favorable brain permeability and multiple activities in vitro (acetylcholinesterase, butyrylcholinesterase, ß-site amyloid precursor protein (APP) cleaving enzyme-1 (BACE-1), and Aß42 and tau aggregation inhibition). In in vivo studies on wild-type and APP/presenilin 1 (PS1) mice, two selected compounds were well tolerated and led to positive trends, albeit statistically nonsignificant in some cases, on memory performance, amyloid pathology (reduced amyloid burden and potentiated non-amyloidogenic APP processing), and oxidative stress (reduced cortical oxidized proteins and increased antioxidant enzymes superoxide dismutase 2 (SOD2), catalase, glutathione peroxidase 1 (GPX1), and heme oxygenase 1 (Hmox1) and transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2)). These compounds emerge as interesting brain-permeable multitarget compounds, with a potential as anti-Alzheimer agents beyond that of the original lead CR-6.

Axonal and Myelin Neuroprotection by the Peptoid BN201 in Brain Inflammation.

The development of neuroprotective therapies is a sought-after goal. By screening combinatorial chemical libraries using in vitro assays, we identified the small molecule BN201 that promotes the survival of cultured neural cells when subjected to oxidative stress or when deprived of trophic factors. Moreover, BN201 promotes neuronal differentiation, the differentiation of precursor cells to mature oligodendrocytes in vitro, and the myelination of new axons. BN201 modulates several kinases participating in the insulin growth factor 1 pathway including serum-glucocorticoid kinase and midkine, inducing the phosphorylation of NDRG1 and the translocation of the transcription factor Foxo3 to the cytoplasm. In vivo, BN201 prevents axonal and neuronal loss, and it promotes remyelination in models of multiple sclerosis, chemically induced demyelination, and glaucoma. In summary, we provide a new promising strategy to promote neuroaxonal survival and remyelination, potentially preventing disability in brain diseases.

Dynamic Covalent Identification of an Efficient Heparin Ligand.

Despite heparin being the most widely used macromolecular drug, the design of small-molecule ligands to modulate its effects has been hampered by the structural properties of this polyanionic polysaccharide. Now a dynamic covalent selection approach is used to identify a new ligand for heparin, assembled from extremely simple building blocks. The amplified molecule strongly binds to heparin (KD in the low µm range, ITC) by a combination of electrostatic, hydrogen bonding, and CH-π interactions as shown by NMR and molecular modeling. Moreover, this ligand reverts the inhibitory effect of heparin within an enzymatic cascade reaction related to blood coagulation. This study demonstrates the power of dynamic covalent chemistry for the discovery of new modulators of biologically relevant glycosaminoglycans.

First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseases.

Modulating T cell activation is critical for treating autoimmune diseases but requires avoiding concomitant opportunistic infections. Antigen binding to the T cell receptor (TCR) triggers the recruitment of the cytosolic adaptor protein Nck to a proline-rich sequence in the cytoplasmic tail of the TCR's CD3ε subunit. Through virtual screening and using combinatorial chemistry, we have generated an orally available, low-molecular weight inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activation with an IC50 (median inhibitory concentration) ~1 nM. By modulating TCR signaling, the inhibitor prevented the development of psoriasis and asthma and, furthermore, exerted a long-lasting therapeutic effect in a model of autoimmune encephalomyelitis. However, it did not prevent the generation of a protective memory response against a mouse pathogen, suggesting that the compound might not exert its effects through immunosuppression. These results suggest that inhibiting an immediate TCR signal has promise for treating a broad spectrum of human T cell-mediated autoimmune and inflammatory diseases.

Regioselective Synthesis of a Family of ß-Lactams Bearing a Triazole Moiety as Potential Apoptosis Inhibitors.

Apoptosis is a biological process important to several human diseases; it is strongly regulated through protein-protein interactions and complex formation. We previously reported the synthesis of apoptosis inhibitors bearing an exocyclic triazole amide isoster by using an Ugi four-component coupling reaction (Ugi-4CC), followed by a base-promoted intramolecular cyclization. Depending on the substitution patterns and the reaction conditions, this cyclization forms the six- or four-membered ring. Two compounds bearing the ß-lactam scaffold turned out to be the most potent inhibitors. This encouraged us to optimize the modulation of the cyclization, and prepare a library of 15 ß-lactams with total regioselectivity. Moreover, we aimed to improve the bioavailability of these compounds through the introduction of diversity at different substitution positions. The activity of these compounds as apoptosis inhibitors in cellular extracts has been evaluated, showing an increase in their potency.

Synthesis and in vitro, ex-vivo and in vivo activity of hybrid compounds linking a potent ROS and RNS scavenger activity with diverse substrates addressed to pass across the blood-brain barrier.

The synthesis of a small library of CR-6 (a potent ROS and RNS scavenger agent) derivatives bearing covalent linkage with different endogen nutrients that have specific transport through the blood-brain barrier (BBB) is reported. The synthetic sequence involved the preparation of a common precursor ester 6 derived from CR-6, which was easily converted into the carboxylic acid 7a or the amino derivative 11, for its further coupling with the required substrates through amide bonds. Antioxidant in vitro (DPPH) and cellular assays (CAA) with the SH-S5SY cell line performed on these library members revealed that the couplings did not affect the antioxidant activity elicited by CR-6 itself. More interestingly, results from the intraperitoneal administration of selected library components in rats showed that compounds 2b, 2c and 2d were able to pass across the BBB. In particular, the amino acid compound 2d was the most penetrating derivative (15.8 ± 1.7 nmol/g brain with respect to the 4.0 ± 1.2 nmol/g brain found for the parent CR-6).

Cationic Peptides and Peptidomimetics Bind Glycosaminoglycans as Potential Sema3A Pathway Inhibitors.

Semaphorin3A (Sema3A) is a vertebrate-secreted protein that was initially characterized as a repulsive-guidance cue. Semaphorins have crucial roles in several diseases; therefore, the development of Sema3A inhibitors is of therapeutic interest. Sema3A interacts with glycosaminoglycans (GAGs), presumably through its C-terminal basic region. We used different biophysical techniques (i.e., NMR, surface plasmon resonance, isothermal titration calorimetry, fluorescence, and UV-visible spectroscopy) to characterize the binding of two Sema3A C-terminus-derived basic peptides (FS2 and NFS3) to heparin and chondroitin sulfate A. We found that these peptides bind to both GAGs with affinities in the low-micromolar range. On the other hand, a peptoid named SICHI (semaphorin-induced chemorepulsion inhibitor), which is positively charged at physiological pH, was first identified by our group as being able to block Sema3A chemorepulsion and growth-cone collapse in axons at the extracellular level. To elucidate the direct target for the reported SICHI inhibitory effect in the Sema3A signaling pathway, we looked first to the protein-protein interaction between secreted Sema3A and the Nrp1 receptor. However, our results show that SICHI does not bind directly to the Sema3A sema domain or to Nrp1 extracellular domains. We evaluated a new, to our knowledge, hypothesis, according to which SICHI binds to GAGs, thereby perturbing the Sema3A-GAG interaction. By using the above-mentioned techniques, we observed that SICHI binds to GAGs and competes with Sema3A C-terminus-derived basic peptides for binding to GAGs. These data support the ability of SICHI to block the biologically relevant interaction between Sema3A and GAGs, thus revealing SICHI as a new, to our knowledge, class of inhibitors that target the GAG-protein interaction.

Apaf1 inhibition promotes cell recovery from apoptosis.

The protein apoptotic protease activating factor 1 (Apaf1) is the central component of the apoptosome, a multiprotein complex that activates procaspase-9 after cytochrome c release from the mitochondria in the intrinsic pathway of apoptosis. We have developed a vital method that allows fluorescence-activated cell sorting of cells at different stages of the apoptotic pathway and demonstrated that upon pharmacological inhibition of Apaf1, cells recover from doxorubicin- or hypoxia-induced early apoptosis to normal healthy cell. Inhibiting Apaf1 not only prevents procaspase-9 activation but delays massive mitochondrial damage allowing cell recovery.

Efficient Synthesis of Conformationally Restricted Apoptosis Inhibitors Bearing a Triazole Moiety.

Apoptosis is a biological process relevant to different human diseases that is regulated through protein-protein interactions and complex formation. Peptidomimetic compounds based on linear peptoids and cyclic analogues with different ring sizes have been previously reported as potent apoptotic inhibitors. Among them, the presence of cis/trans conformers of an exocyclic tertiary amide bond in slow exchange has been characterized. This information encouraged us to perform an isosteric replacement of the amide bond by a 1,2,3-triazole moiety, in which different substitution patterns would mimic different amide rotamers. The syntheses of these restricted analogues have been carried out through an Ugi multicomponent reaction followed by an intramolecular cyclization. The unexpected formation of a ß-lactam scaffold prompted us to study the course of the intramolecular cyclization of the Ugi adducts. In order to modulate this cyclization, a small library of compounds bearing both heterocyclic scaffolds has been synthesized and their activities as apoptosis inhibitors have been evaluated.

Inhibitory effect of positively charged triazine antagonists of prokineticin receptors on the transient receptor vanilloid type-1 (TRPV1) channel.

Four positively charged compounds, previously shown to produce analgesic activity by interacting with prokineticin receptor or T-type calcium channels, were tested for their ability to inhibit capsaicin-induced elevation of intracellular Ca(2+) in HEK-293 cells stably transfected with the human recombinant TRPV1, with the goal of identifying novel TRPV1 open-pore inhibitors. KYS-05090 showed the highest potency as a TRPV1 antagonist, even higher than that of the open-pore triazine inhibitor 8aA. The latter showed quite remarkable agonist/desensitizer activity at the rat recombinant TRPM8 channel. The activity of KYS-05090 and the other compounds was selective because none of these compounds was able to modulate the rat TRPA1 channel. Open-pore inhibitors of TRPV1 may be a new class of multi-target analgesics with lesser side effects, such as loss of acute pain sensitivity and hyperthermia, than most TRPV1 antagonists developed so far.

Positional Scanning Synthesis of a Peptoid Library Yields New Inducers of Apoptosis that Target Karyopherins and Tubulin.

We describe the synthesis of a library of 11, 638 N-alkylglycine peptoid trimers in a positional scanning format with adjustment of reaction conditions to account for different reactivities of the monomer building blocks. Evaluation of the library by high-content phenotypic screening for modulators of the cytoskeleton and mitosis resulted in the identification of two apoptosis-inducing peptoids, which, despite their structural similarity, target different proteins and cellular mechanisms. Whereas one peptoid binds to karyopherins, which mediate nuclear transport, the other N-alkylglycine trimer binds tubulin at the vinca alkaloid binding site.

Apaf-1 inhibitors protect from unwanted cell death in in vivo models of kidney ischemia and chemotherapy induced ototoxicity.

BACKGROUND: Excessive apoptosis induces unwanted cell death and promotes pathological conditions. Drug discovery efforts aimed at decreasing apoptotic damage initially targeted the inhibition of effector caspases. Although such inhibitors were effective, safety problems led to slow pharmacological development. Therefore, apoptosis inhibition is still considered an unmet medical need. METHODOLOGY AND PRINCIPAL FINDINGS: The interaction between Apaf-1 and the inhibitors was confirmed by NMR. Target specificity was evaluated in cellular models by siRNa based approaches. Cell recovery was confirmed by MTT, clonogenicity and flow cytometry assays. The efficiency of the compounds as antiapoptotic agents was tested in cellular and in vivo models of protection upon cisplatin induced ototoxicity in a zebrafish model and from hypoxia and reperfusion kidney damage in a rat model of hot ischemia. CONCLUSIONS: Apaf-1 inhibitors decreased Cytc release and apoptosome-mediated activation of procaspase-9 preventing cell and tissue damage in ex vivo experiments and in vivo animal models of apoptotic damage. Our results provide evidence that Apaf-1 pharmacological inhibition has therapeutic potential for the treatment of apoptosis-related diseases.

Effect of triazine derivatives on neuronal nicotinic receptors.

We have characterized the effect of triazine derivatives on neuronal nicotinic receptors expressed in Xenopus oocytes. All triazines investigated inhibit the current of α7 and α3ß4 neuronal nicotinic receptors elicited by acetylcholine. The effect is concentration dependent, reversible, and noncompetitive. In contrast, some derivatives have a dual effect on α4ß2 receptors, by potentiating the currents at intermediate concentration and causing inhibition at higher concentrations. Triazine derivatives also affect the macroscopic kinetics of the heteromeric receptors α3ß4 and α4ß2 accelerating the rise and decay time course of the currents, but have no significant effect on the kinetics of homomeric α7 receptors. Two simple kinetic models are presented. The first reproduces the effects of different concentrations of triazines both on the peak currents and on the macroscopic kinetics of α7 with a simple inhibitory result. The second model describes the behavior of α4ß2 receptors involving a more complex dual action.

15N NMR spectroscopic and theoretical GIAO-DFT studies for the unambiguous characterization of disubstituted 1,2,3-triazoles.

The 1,2,3-triazole ring has recently attracted a renewed interest as a structural scaffold with many applications in different fields. However, very often, the unambiguous assignment of the correct structure is not an easy task, and the development of robust characterization methodologies is needed. Herein, the three possible isomeric forms of disubstituted 1,2,3-triazole (1,4- or 1,5- or 2,4-disubstituted derivatives) have been characterized and distinguished by routine (1)H/(15)N gHMBC experiments at (15)N natural abundance. The calculated (GIAO-B3LYP/6-311++G**) (15)N NMR chemical shifts showed good agreement with the experimental values, further supporting their unambiguous structural characterization.

Vestibulotoxic properties of potential metabolites of allylnitrile.

This study addressed the hypothesis that epoxidation of the double bond in allylnitrile mediates its vestibular toxicity, directly or after subsequent metabolism by epoxide hydrolases. The potential metabolites 3,4-epoxybutyronitrile and 3,4-dihydroxybutyronitrile were synthesized and characterized. In aqueous solutions containing sodium or potassium ions, 3,4-epoxybutyronitrile rearranged to 4-hydroxybut-2-enenitrile, and this compound was also isolated for study. Male adult Long-Evans rats were exposed to allylnitrile or 3,4-epoxybutyronitrile by bilateral transtympanic injection, and vestibular toxicity was assessed using a behavioral test battery and scanning electron microscopy (SEM) observation of the sensory epithelia. Overt vestibular toxicity was caused by 3,4-epoxybutyronitrile at 0.125 mmol/ear and by allylnitrile in some animals at 0.25 mmol/ear. Additional rats were exposed by unilateral transtympanic injection. In these studies, behavioral evidences and SEM observations demonstrated unilateral vestibular toxicity after 0.125 mmol of 3,4-epoxybutyronitrile and bilateral vestibular toxicity after 0.50 mmol of allylnitrile. However, 0.25 mmol of allylnitrile did not cause vestibular toxicity. Unilateral administration of 0.50 mmol of 3,4-dihydroxybutyronitrile or 4-hydroxybut-2-enenitrile caused no vestibular toxicity. The four compounds were also evaluated in the mouse utricle explant culture model. In 8-h exposure experiments, hair cells completely disappeared after 3,4-epoxybutyronitrile at concentrations of 325 or 450µM but not at concentrations of 150µM or lower. In contrast, no difference from controls was recorded in utricles exposed to 450µM or 1.5mM of allylnitrile, 3,4-dihydroxybutyronitrile, or 4-hydroxybut-2-enenitrile. Taken together, the present data support the hypothesis that 3,4-epoxybutyronitrile is the active metabolite of allylnitrile for vestibular toxicity.

Optimizing the control of apoptosis by amide/triazole isosteric substitution in a constrained peptoid.

Apoptosis is a biological process relevant to several human diseases that is strongly regulated through protein-protein complex formation. We have previously reported a peptidomimetic compound as potent apoptotic modulator. Structural studies of this compound showed the presence of cis/trans isomers of the exocyclic tertiary amide bond in slow exchange. This information encouraged us to perform an isosteric replacement of the amide bond by a 1,2,3-triazole moiety, where different substitution patterns would mimic different amide rotamers. The syntheses of these restricted analogs have been carried out using the Ugi multicomponent reaction followed by an intramolecular cyclization. Unexpectedly, for one of the proposed structures, a novel ß -lactam compound was formed. All compounds showed to efficiently inhibit apoptosis, in vitro and in cellular extracts, with slight differences for the corresponding regioisomers. We propose the binding to Apaf-1 as the inhibition mechanism.