Non-competitive Allosteric Inhibitor Research Articles

CXCR2 Is Deregulated in ALS Spinal Cord and Its Activation Triggers Apoptosis in Motor Neuron-Like Cells Overexpressing hSOD1-G93A.

Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterized by progressive depletion of motor neurons (MNs). Recent evidence suggests a role in ALS pathology for the C-X-C motif chemokine receptor 2 (CXCR2), whose expression was found increased at both mRNA and protein level in cortical neurons of sporadic ALS patients. Previous findings also showed that the receptor inhibition is able to prevent iPSC-derived MNs degeneration in vitro and improve neuromuscular function in SOD1-G93A mice. Here, by performing transcriptional analysis and immunofluorescence studies, we detailed the increased expression and localization of CXCR2 and its main ligand CXCL8 in the human lumbar spinal cord of sporadic ALS patients. We further investigated the functional role of CXCR2/ligands axis in NSC-34 motor neuron-like cells expressing human wild-type (WT) or mutant (G93A) SOD1. A significant expression of CXCR2 was found in doxycycline-induced G93A-SOD1-expressing cells, but not in WT cells. In vitro assays showed CXCR2 activation by GROα and MIP2α, two murine endogenous ligands and functional homologs of CXCL8, reduces cellular viability and triggers apoptosis in a dose dependent manner, while treatment with reparixin, a non-competitive allosteric CXCR2 inhibitor, effectively counteracts GROα and MIP2α toxicity, significantly inhibiting the chemokine-induced cell death. Altogether, data further support a role of CXCR2 axis in ALS etiopathogenesis and confirm its pharmacological modulation as a candidate therapeutic strategy.

Allosteric inhibition of CXCR1/2 by an orally bioavailable chemokine receptor inhibitor attenuates allergic lung inflammation in mice

CXCR1/2 chemokine receptors are expressed on the surface of inflammatory cells. We previously reported that parenterally-administered reparixin, a noncompetitive allosteric inhibitor of CXCR1 and CXCR2, inhibits allergic airway inflammation. Ladarixin is a second generation orally bioavailable dual CXCR1/2 inhibitor that binds an allosteric pocket of the trans-membrane region of both receptors with a 100-fold higher affinity than first generation CXCR1/2 inhibitors. Here we investigated whether oral ladarixin also inhibits allergic airway inflammation.

The effect of reparixin on survival in patients at high riskfor in-hospital mortality: a meta-analysis of randomized trials.

IntroductionA great number of anti-inflammatory drugs have been suggested in the treatment of SARS-CoV-2 infection. Reparixin, a non-competitive allosteric inhibitor of the CXCL8 (IL-8) receptors C-X-C chemokine receptor type 1 (CXCR1) and C-X-C chemokine receptor type 2 (CXCR2), has already been tried out as a treatment in different critical settings. Due to the contrasting existing literature, we decided to perform the present meta-analysis of randomized controlled trials (RCTs) to investigate the effect of the use of reparixin on survival in patients at high risk for in-hospital mortality.MethodsWe created a search strategy to include any human RCTs performed with reparixin utilization in patients at high risk for in-hospital mortality, excluding oncological patients. Two trained, independent authors searched PubMed, EMBASE, and the Cochrane Central Register of Controlled Trials (CENTRAL) for appropriate studies. Furthermore, references of review articles and included RCTs were screened to identify more studies. No language restrictions were enforced. To assess the risk of bias of included trials, the Revised Cochrane risk-of-bias tool for randomized trials (RoB 2) was used.ResultsOverall, six studies were included and involved 406 patients (220 received reparixin and 186 received the comparator). The all-cause mortality in the reparixin group was significantly lower than that in the control group [5/220 (2.3%) in the reparixin group vs. 12/186 (6.5%) in the control group, odds ratio = 0.33 (95% confidence interval 0.12 to 0.96), p-value for effect 0.04, p for heterogeneity 0.20, I 2 = 36%]. In addition, no difference in the rate of pneumonia, sepsis, or non-serious infections was shown between the two groups.ConclusionOur meta-analysis of randomized trials suggests that short-term inhibition of CXCL8 activity improved survival in patients at high risk for in-hospital mortality without increasing the risk of infection.Meta-analysis registrationPROSPERO, identifier CRD42021254467.

CXCL1-CXCR1/2 signaling is induced in human temporal lobe epilepsy and contributes to seizures in a murine model of acquired epilepsy

CXCL1, a functional murine orthologue of the human chemokine CXCL8 (IL-8), and its CXCR1 and CXCR2 receptors were investigated in a murine model of acquired epilepsy developing following status epilepticus (SE) induced by intra-amygdala kainate. CXCL8 and its receptors were also studied in human temporal lobe epilepsy (TLE). The functional involvement of the chemokine in seizure generation and neuronal cell loss was assessed in mice using reparixin (formerly referred to as repertaxin), a non-competitive allosteric inhibitor of CXCR1/2 receptors.We found a significant increase in hippocampal CXCL1 level within 24 h of SE onset that lasted for at least 1 week. No changes were measured in blood. In analogy with human TLE, immunohistochemistry in epileptic mice showed that CXCL1 and its two receptors were increased in hippocampal neuronal cells. Additional expression of these molecules was found in glia in human TLE.Mice were treated with reparixin or vehicle during SE and for additional 6 days thereafter, using subcutaneous osmotic minipumps. Drug-treated mice showed a faster SE decay, a reduced incidence of acute symptomatic seizures during 48 h post-SE, and a delayed time to spontaneous seizures onset compared to vehicle controls. Upon reparixin discontinuation, mice developed spontaneous seizures similar to vehicle mice, as shown by EEG monitoring at 14 days and 2.5 months post-SE. In the same epileptic mice, reparixin reduced neuronal cell loss in the hippocampus vs vehicle-injected mice, as assessed by Nissl staining at completion of EEG monitoring.Reparixin administration for 2 weeks in mice with established chronic seizures, reduced by 2-fold on average seizure number vs pre-treatment baseline, and this effect was reversible upon drug discontinuation. No significant changes in seizure number were measured in vehicle-injected epileptic mice that were EEG monitored in parallel.Data show that CXCL1-IL-8 signaling is activated in experimental and human epilepsy and contributes to acute and chronic seizures in mice, therefore representing a potential new target to attain anti-ictogenic effects.

Abstract 21: LiP-MS, a machine learning-based chemoproteomic approach to identify drug targets in complex proteomes

Abstract Background Target identification is a critical step in elucidating the mechanism of action (MoA) for bioactive compounds. For target-based and phenotypic drug discovery pipelines, extensive potential target knowledge for a lead compound provides essential insights that enable potency and selectivity optimization. The tedious process of target deconvolution for a compound often necessitates a plethora of biochemical and biophysical techniques. To expand the toolbox of unbiased target ID approaches, we developed a novel workflow combining limited proteolysis with quantitative mass spectrometry (LiP-MS) that exploits the drug binding phenomena of protein structural alterations and steric hindrance. Advantageously, LiP-MS's unique peptide-centric focus exploits signature peptide detection to discern ligand binding. Additionally, the LiP-MS workflow enables binding affinity estimation (EC50) and binding site prediction. Here we demonstrate the performance of LiP-MS using two well-characterized kinase inhibitors (KIs), an AstraZeneca CDK9 inhibitor (AZ) and Selumetinib (SE). Methods Mechanically sheared HeLa or U2OS cell lysate was incubated with compound at multiple concentrations. Next, a limited proteolytic digest was performed using proteinase K. After quenching this digestion, lysate was trypsin digested to peptides for mass spectrometry analysis. The resulting peptides were analyzed quantitatively using data-independent acquisition (DIA)-MS. Results Herein, we use LiP-MS to unbiasedly identify unique peptides generated by the binding of two distinct classes of kinase inhibitors in human cell lysate. For the ATP-competitive inhibitor AZ, LiP-Quant shows a strong enrichment for CDKs in the target space defined by LiP score, including CDK1, 2, 4, 6, 9 and 11A. In addition, our data indicates that this KI targets members of the CDKs with different selectivity, with CDK9 displaying the highest compound affinity (nM range). For Selumetinib, a non-competitive allosteric inhibitor, LiP-MS clearly identified the direct targets MEK1/2 as the main hits by LiP score. Both cases represent a highly specific enrichment given that we quantified > 120,000 peptides in each of the experiments. These findings confirm our approach's ability to identify genuine drug targets regardless of drug MoA in a complex biological matrix. Importantly, in both KI target ID studies, identified LiP peptides could be successfully deployed to map compound binding site, demonstrating the potential of LiP-MS to pinpoint regions involved in drug-protein interactions. Conclusions Collectively, this data demonstrates that LiP-MS can be used to effectively identify protein drug targets and characterize the binding properties in complex proteomes independent of the compound's MoA and without compound modification or labeling. These capabilities make LiP-MS a powerful addition to the target ID toolbox. Citation Format: Nigel Beaton, Yuehan Feng, Roland Bruderer, Adam Hendricks, Ghaith Hamza, Eric Miele, Rick Davies, Kristina Beeler, Ilaria Piazza, Paola Picotti, Paola Castaldi, Lukas Reiter. LiP-MS, a machine learning-based chemoproteomic approach to identify drug targets in complex proteomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 21.

Identification of novel Epac2 antagonists through in silico and in vitro analyses

cAMP-dependent guanine nucleotide exchange factor (Epac) is a key regulator in signal transduction and represents an excellent drug target to be investigated against various diseases. To date, very few modulators selective for Epac are available; however, there is still an unmet need of isoform-selective inhibitors. In the present study, ligand-based pharmacophores were designed to investigating structurally diverse molecules as Epac2 inhibitors. Pharmacophore models were developed using reported allosteric site inhibitors. The developed models were used to screen 95 thousand compounds from the National Cancer Institute (NCI), Maybride, and our in-house ICCBS Database. The binding mode and efficiency of the screened hits was investigated using molecular docking simulation on the allosteric site of Epac2 apo-protein (PDB ID: 2BYV) followed by ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) profiling Furthermore, obtained in silico screened hits were subjected to in vitro assay for insulin secretion. We identified, three lead molecules RDR02145, AAK-399, and AAD-026 reducing, insulin secretion. Remarkably, a higher inhibitory effect on insulin secretion was observed in AAK-399, and AAD-026 as compared to that of standard Epac2 non-competitive allosteric site inhibitor, MAY0132. Furthermore, Dynamic simulation studies of lead compounds proved the structural stability of the Epac2 auto-inhibited state. These findings underline the potential of these compounds as valuable pharmacological tools for designing future selective probes to inhibit the Epac-mediated signaling pathway.

Recent Advances in c-Jun N-Terminal Kinase (JNK) Inhibitors.

c-Jun N-Terminal Kinases (JNKs), members of the Mitogen-Activated Protein Kinase (MAPK) signaling pathway, play a key role in the pathogenesis of many diseases including cancer, inflammation, Parkinson's disease, Alzheimer's disease, cardiovascular disease, obesity, and diabetes. Therefore, JNKs represent new and excellent target by therapeutic agents. Many JNK inhibitors based on different molecular scaffolds have been discovered in the past decade. However, only a few of them have advanced to clinical trials. The major obstacle for the development of JNK inhibitors as therapeutic agents is the JNKisoform selectivity. In this review, we describe the recent development of JNK inhibitors, including ATP competitive and ATP non-competitive (allosteric) inhibitors, bidentatebinding inhibitors and dual inhibitors, the challenges, and the future direction of JNK inhibitors as potential therapeutic agents.

Drugging K-RasG12C through covalent inhibitors: Mission possible?

Ras, whose mutants are present in approximately 30% of human tumours, is one of the most important oncogenes. Drugging Ras is thus regarded as the quest for the Holy Grail in cancer therapeutics development. Despite more than three decades of efforts, drug discovery targeting Ras constantly fails, rendering Ras undruggable, due to its smooth surface and picomolar affinity towards guanosine substrates. The most frequently mutated isoform of Ras is K-Ras, accounting for >85% of Ras-driven cancers, and one majority of them is the G12C mutation. Recent advances in structural biology shed light on drugging Ras, and one of the cutting-edge breakthroughs is the design of covalent G12C-specific inhibitors targeting the mutated cysteine. This type of inhibitor can be classified into substrate-competitive orthosteric inhibitors and non-competitive allosteric inhibitors. They display improved selectivity and enhanced potency due to their G12-specific and irreversible covalent binding nature. Thus, they represent a new hope for revolutionizing the conventional characterization of Ras as "undruggable" and pave a promising avenue for further drug discovery. Here, we provide comprehensive structural and medicinal chemical insights into K-Ras covalent inhibitors specific for the G12C mutant. We first present an in-depth analysis of the conformations of the inhibitor binding pockets. Then, all the latest covalent ligands selectively inhibiting K-RasG12C are reviewed. Finally, we examine the current challenges faced by this new class of anti-Ras inhibitors.

Cdk 4/6 inhibitors revolutionized breast cancer therapy

The cyclin-CDK inhibitors of the Cip/Kip family p21Cip1, p27Kip1, and p57Kip2 have emerged as multifaceted proteins with functions beyond cell cycle regulation. They are used to treat cancers by preventing over proliferation of cancer cells. In metazoans, two CKI gene families have been defined based on their evolutionary origins, structure, and CDK specificities. A vast body of literature has described the importance of p21, p27, and p57 in restraining proliferation during development, differentiation, and response to cellular stresses, although each has specific biological functions that distinguish it from the other family members. Thus, different anti-proliferative signals tend to cause elevated expression of only a subset of the Cip/Kip proteins. Following a recent and detailed review on the subject. We concentrate our attention on an updated list of compounds under clinical evaluation (phase I/II/III) and discuss their mode of action as ATP-competitive inhibitors Also, tentative progress for forthcoming potential ATP non-competitive inhibitors and allosteric inhibitors will be discussed.

Anticancer applications of allosteric inhibitors of proteasome.

e23066 Background: Proteasome as a hub protease of the ubiquitin proteasome pathway is an established anticancer drug target. Several drugs that inhibit proteasome are currently used to successfully treat aggressive blood cancers. These drugs are based on their competition with protein substrates of proteasome. However, efficacy of these drugs toward solid cancers is inadequate. Besides, the side effects and developing drug resistance are increasingly hampering the therapy. Therefore, there is an unmet challenge to develop new types of proteasome targeting compounds that are efficient against solid cancers and utilize other mechanisms to stop proteasome. Here we present a compound with a novel molecular mechanism, potentially bypassing limitations of the available drugs. Methods: We rationally designed and synthesized a series of small molecule “B” compounds, derivatives of a binding domain of seco-rapamycin that noncompetitively interfere with peptidase activities of proteasome. We tested effects of the compounds in vitro on purified proteasome, in cellulo with selected cancer cell lines and in a xenograft mouse model of prostate cancer. Results: We found that compound B1 binds to the catalytic core of proteasome far from the catalytic sites, destabilizes assembly of the 26S proteasome responsible for digest of polyUb substrates and allosterically inhibits its proteolytic activities. Molecularly, B1 impedes the gating mechanism responsible for substrate uptake as found with AFM. Tryptophan fluorescence indicates that B1 changes proteasome fold and the binding mode of competitive inhibitors. B1 substantially decreases viability of selected cancer cell lines and shifts their mechanical phenotype toward noncancerous status. B1 synergizes with bortezomib decreasing the IC50 5-10 fold. In a xenograft hormone resistant prostate cancer model, B1 treatment leads to shrinkage of the tumor size, decreases enumeration of aggressive, EpCAM+ CTCs and shifts the macrophage profile toward predator M1 type. Conclusions: B1 compounds constitute a new class of noncompetitive allosteric inhibitors of proteasome that could be useful to develop to treat aggressive prostate cancers alone or in synergy with competitive inhibitors.

Role of the Ion Channel Extracellular Collar in AMPA Receptor Gating

AMPA subtype ionotropic glutamate receptors mediate fast excitatory neurotransmission and are implicated in numerous neurological diseases. Ionic currents through AMPA receptor channels can be allosterically regulated via different sites on the receptor protein. We used site-directed mutagenesis and patch-clamp recordings to probe the ion channel extracellular collar, the binding region for noncompetitive allosteric inhibitors. We found position and substitution-dependent effects for introduced mutations at this region on AMPA receptor gating. The results of mutagenesis suggested that the transmembrane domains M1, M3 and M4, which contribute to the ion channel extracellular collar, undergo significant relative displacement during gating. We used molecular dynamics simulations to predict an AMPA receptor open state structure and rationalize the results of mutagenesis. We conclude that the ion channel extracellular collar plays a distinct role in gating and represents a hub for powerful allosteric modulation of AMPA receptor function that can be used for developing novel therapeutics.

Allosteric Communication Disrupted by a Small Molecule Binding to the Imidazole Glycerol Phosphate Synthase Protein-Protein Interface.

Allosteric enzymes regulate a wide range of catalytic transformations, including biosynthetic mechanisms of important human pathogens, upon binding of substrate molecules to an orthosteric (or active) site and effector ligands at distant (allosteric) sites. We find that enzymatic activity can be impaired by small molecules that bind along the allosteric pathway connecting the orthosteric and allosteric sites, without competing with endogenous ligands. Noncompetitive allosteric inhibitors disrupted allostery in the imidazole glycerol phosphate synthase (IGPS) enzyme from Thermotoga maritima as evidenced by nuclear magnetic resonance, microsecond time-scale molecular dynamics simulations, isothermal titration calorimetry, and kinetic assays. The findings are particularly relevant for the development of allosteric antibiotics, herbicides, and antifungal compounds because IGPS is absent in mammals but provides an entry point to fundamental biosynthetic pathways in plants, fungi, and bacteria.

<i>In Silico</i> Evaluation of Anti-Malarial Agents from <i>Hoslundia opposita</i> as Inhibitors of <i>Plasmodium falciparum</i> Lactate Dehydrogenase (P<i>f</i>LDH) Enzyme

Malaria has continued to be a health and economic problem in Africa and the world at large. Many anti-malarial drugs have been rendered ineffective due to the emergence of resistant strains of Plamodium falciparum. A key malaria parasite enzyme in glycolytic pathway, P. falciparum lactate dehydrogenase (PfLDH) is specially targeted for anti-malarial drugs development. Thus, the aim of this investigation was to determine the in silico inhibition effects of antimalarial compounds from Hoslundia opposita Vahl. namely hoslundin, hoslundal and hoslunddiol on PfLDH enzyme. The compounds were docked to the three-dimensional structure of PfLDH as enzyme using AutoDock Vina in PyRx virtual screening software. Binding affinity and position of the inhibitors were evaluated using PyMol software. The PfLDH enzyme showed two binding sites: the cofactors binding site (Site A) and secondary binding site (Site B). In the absence of the cofactor all ligands showed higher affinity than NADH, and were bound to the cofactors binding site (Site A). When docked in the presence of the cofactor, site B was the preferred binding site. Binding to cofactor site with higher binding energy than NADH suggests that these ligands could act as preferential competitive inhibitors of PfLDH. However, the binding to site B also suggests that they may be non-competitive allosteric inhibitors. Amino acid residues Gly99, Asn140, Phe100 and Thr97 were indicated to form hydrogen bonds with Hoslundin. Hoslunddiol showed hydrogen bonding with Thr97 and Met30, while Hoslundal formed hydrogen bond with Thr101 and Asn140.

DF2755A, a novel non-competitive allosteric inhibitor of CXCR1/2, reduces inflammatory and post-operative pain

The activation of CXCR1/2 has been implicated in the genesis of inflammatory and postoperative pain. Here, we investigated a novel orally acting allosteric inhibitor of CXCR1/2 (DF2755A) and evaluated its antinociceptive effect in several models of inflammatory and post-operatory pain. DF2755A was tested in vitro for efficacy in the chemotaxis assay, selectivity and toxicity. In vivo, C57Bl/6 mice were treated orally with DF2755A and the following experiments were performed: pharmacokinetic profile; inflammatory hyperalgesia models using electronic pressure meter test; neutrophil migration assay assessed by myeloperoxidase assay. DF2755A selectively inhibited neutrophil chemotaxis induced by CXCR1/2 ligands without effect on CXCL8 binding to neutrophils. A single mutation of the allosteric site at CXCR1 abrogated the inhibitory effect of DF2755A on CXCL8-induced chemotaxis. DF2755A given orally was well absorbed (88.2%), and it was able to reduce, in a dose (3–30mg/kg)-dependent manner, inflammatory hyperalgesia induced by carrageenan, LPS and CXCL1/KC as well as neutrophil recruitment and IL-1β production. In addition, DF2755A was able to reduce post-incisional nociception. Therapeutic treatment with DF2755A reduced CFA-induced inflammatory hyperalgesia even when injected intrathecally. The present results indicate that DF2755A is a novel selective allosteric inhibitor of CXCR1/2 with a favorable oral pharmacokinetic profile. Furthermore, the results might suggest that DF2755A might be a candidate of a novel therapeutic option to control inflammatory and post-operative pain.

Probing the Role of Conformational Entropy in Protein-Inhibitor Binding

In silico drug discovery has long held the promise to revolutionize the way drugs are designed, but has largely failed to deliver. Historically inhibitors were designed based mainly on static structures that emphasized interaction energetics but ignored potential contributions from conformational entropy. This issue has been difficult to address experimentally. Recently, we have developed an NMR-based approach that employs measures of motion as a proxy for the conformational entropy. Using an empirical calibration, we have shown that the resulting “entropy meter” is both robust and general. We hypothesize that conformational entropy represents a critical “missing piece” in rational drug design. Using the “entropy meter,” we examine the effect of conformational entropy on protein-inhibitor interactions using the drug target and mitogen-activated protein kinase (MAPK) p38α as a model system. Given the relatively large size of this protein, all studies were performed in a perdeuterated background. Using standard methods, we have obtained nearly complete ILV methyl assignments of this protein. 15N and 13C NMR spin relaxation experiments were used to determine the sub-ns dynamics of the protein both in the apo state and bound to either a competitive and a non-competitive (allosteric) inhibitor. In both cases, we observe the a large unfavorable entropic effect of binding. We find that the conformational entropy change upon binding represents a significant contribution to the overall binding free energy. These results promote further studies to understand how classes of inhibitors may modulate the dynamics and corresponding internal entropy of protein targets, and how this information can be used to design better lead compounds.This work was supported by NIH Grant GM100910

CXCR1/2 inhibition blocks and reverses type 1 diabetes in mice.

Chemokines and their receptors have been associated with or implicated in the pathogenesis of type 1 diabetes (T1D), but the identification of a single specific chemokine/receptor pathway that may constitute a suitable target for the development of therapeutic interventions is still lacking. Here, we used multiple low-dose (MLD) streptozotocin (STZ) injections and the NOD mouse model to investigate the potency of CXCR1/2 inhibition to prevent inflammation- and autoimmunity-mediated damage of pancreatic islets. Reparixin and ladarixin, noncompetitive allosteric inhibitors, were used to pharmacologically blockade CXCR1/2. Transient blockade of said receptors was effective in preventing inflammation-mediated damage in MLD-STZ and in preventing and reversing diabetes in NOD mice. Blockade of CXCR1/2 was associated with inhibition of insulitis and modification of leukocytes distribution in blood, spleen, bone marrow, and lymph nodes. Among leukocytes, CXCR2(+) myeloid cells were the most decreased subpopulations. Together these results identify CXCR1/2 chemokine receptors as "master regulators" of diabetes pathogenesis. The demonstration that this strategy may be successful in preserving residual β-cells holds the potential to make a significant change in the approach to management of human T1D.

Cyclin-dependent kinase inhibitors as marketed anticancer drugs: where are we now? A short survey.

In the early 2000s, the anticancer drug imatinib (Glivec®) appeared on the market, exhibiting a new mode of action by selective kinase inhibition. Consequently, kinases became a validated therapeutic target, paving the way for further developments. Although these kinases have been thoroughly studied, none of the compounds commercialized since then target cyclin-dependent kinases (CDKs). Following a recent and detailed review on the subject by Galons et al., we concentrate our attention on an updated list of compounds under clinical evaluation (phase I/II/III) and discuss their mode of action as ATP-competitive inhibitors. CDK inhibition profiles and clinical development stages are reported for the 14 compounds under clinical evaluation. Also, tentative progress for forthcoming potential ATP non-competitive inhibitors and allosteric inhibitors are briefly described, along with their limitations.

Characterization of a Cdc42 Protein Inhibitor and Its Use as a Molecular Probe

Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.

Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 non‐competitive allosteric inhibitor

DF 2156A is a new dual inhibitor of IL-8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential. The binding mode, molecular mechanism of action and selectivity were investigated using chemotaxis of L1.2 transfectants and human leucocytes, in addition to radioligand and [(35) S]-GTPγS binding approaches. The therapeutic potential of DF 2156A was evaluated in acute (liver ischaemia and reperfusion) and chronic (sponge-induced angiogenesis) experimental models of inflammation. A network of polar interactions stabilized by a direct ionic bond between DF 2156A and Lys(99) on CXCR1 and the non-conserved residue Asp(293) on CXCR2 are the key determinants of DF 2156A binding. DF 2156A acted as a non-competitive allosteric inhibitor blocking the signal transduction leading to chemotaxis without altering the binding affinity of natural ligands. DF 2156A effectively and selectively inhibited CXCR1/CXCR2-mediated chemotaxis of L1.2 transfectants and leucocytes. In a murine model of sponge-induced angiogenesis, DF 2156A reduced leucocyte influx, TNF-α production and neovessel formation. In vitro, DF 2156A prevented proliferation, migration and capillary-like organization of HUVECs in response to human IL-8. In a rat model of liver ischaemia and reperfusion (I/R) injury, DF 2156A decreased PMN and monocyte-macrophage infiltration and associated hepatocellular injury. DF 2156A is a non-competitive allosteric inhibitor of both IL-8 receptors CXCR1 and CXCR2. It prevented experimental angiogenesis and hepatic I/R injury in vivo and, therefore, has therapeutic potential for acute and chronic inflammatory diseases.

Target protein interactions of indole‐3‐carbinol and the highly potent derivative 1‐benzyl‐I3C with the C‐terminal domain of human elastase uncouples cell cycle arrest from apoptotic signaling

Elastase is the only currently identified target protein for indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin in cruciferous vegetables such as broccoli, cabbage, and Brussels sprouts that induces a cell cycle arrest and apoptosis of human breast cancer cells. In vitro elastase enzymatic assays demonstrated that I3C and at lower concentrations its more potent derivative 1-benzyl-indole-3-carbinol (1-benzyl-I3C) act as non-competitive allosteric inhibitors of elastase activity. Consistent with these results, in silico computational simulations have revealed the first predicted interactions of I3C and 1-benzyl-I3C with the crystal structure of human neutrophil elastase, and identified a potential binding cluster on an external surface of the protease outside of the catalytic site that implicates elastase as a target protein for both indolecarbinol compounds. The Δ205 carboxyterminal truncation of elastase, which disrupts the predicted indolecarbinol binding site, is enzymatically active and generates a novel I3C resistant enzyme. Expression of the wild type and Δ205 elastase in MDA-MB-231 human breast cancer cells demonstrated that the carboxyterminal domain of elastase is required for the I3C and 1-benzyl-I3C inhibition of enzymatic activity, accumulation of the unprocessed form of the CD40 elastase substrate (a tumor necrosis factor receptor family member), disruption of NFκB nuclear localization and transcriptional activity, and induction of a G1 cell cycle arrest. Surprisingly, expression of the Δ205 elastase molecule failed to reverse indolecarbinol stimulated apoptosis, establishing an elastase-dependent bifurcation point in anti-proliferative signaling that uncouples the cell cycle and apoptotic responses in human breast cancer cells.