In Silico Development of Combinatorial Therapeutic Approaches Targeting Key Signaling Pathways in Metabolic Syndrome.

PURPOSE: Dysregulations of key signaling pathways in metabolic syndrome are multifactorial, eventually leading to cardiovascular events. Hyperglycemia in conjunction with dyslipidemia induces insulin resistance and provokes release of proinflammatory cytokines resulting in chronic inflammation, accelerated lipid peroxidation with further development of atherosclerotic alterations and diabetes. We have proposed a novel combinatorial approach using FDA approved compounds targeting IL-17a and DPP4 to ameliorate a significant portion of the clustered clinical risks in patients with metabolic syndrome. In our current research we have modeled the outcomes of metabolic syndrome treatment using two distinct drug classes. METHODS: Targets were chosen based on the clustered clinical risks in metabolic syndrome: dyslipidemia, insulin resistance, impaired glucose control, and chronic inflammation. Drug development platform, BIOiSIM™, was used to narrow down two different drug classes with distinct modes of action and modalities. Pharmacokinetic and pharmacodynamic profiles of the most promising drugs were modeling showing predicted outcomes of combinatorial therapeutic interventions. RESULTS: Preliminary studies demonstrated that the most promising drugs belong to DPP-4 inhibitors and IL-17A inhibitors. Evogliptin was chosen to be a candidate for regulating glucose control with long term collateral benefit of weight loss and improved lipid profiles. Secukinumab, an IL-17A sequestering agent used in treating psoriasis, was selected as a repurposed candidate to address the sequential inflammatory disorders that follow the first metabolic insult. CONCLUSIONS: Our analysis suggests this novel combinatorial therapeutic approach inducing DPP4 and Il-17a suppression has a high likelihood of ameliorating a significant portion of the clustered clinical risk in metabolic syndrome.

Synthetic fluorescent MYC probe: Inhibitor binding site elucidation and development of a high-throughput screening assay.

We report the discovery of a fluorescent small molecule probe. This probe exhibits an emission increase in the presence of the oncoprotein MYC that can be attenuated by a competing inhibitor. Hydrogen-deuterium exchange mass spectrometry analysis, rationalized by induced-fit docking, suggests it binds to the "coiled-coil" region of the leucine zipper domain. Point mutations of this site produced functional MYC constructs resistant to inhibition in an oncogenic transformation assay by compounds that displace the probe. Utilizing this probe, we have developed a high-throughput assay to identify MYC inhibitor scaffolds. Screening of a diversity library (N = 1408, 384-well) and a library of pharmacologically active compounds (N = 1280, 1536-well) yielded molecules with greater drug-like properties than the probe. One lead is a potent inhibitor of oncogenic transformation and is specific for MYC relative to resistant mutants and transformation-inducing oncogenes. This method is simple, inexpensive, and does not require protein modification, DNA binding, or the dimer partner MAX. This assay presents an opportunity for MYC inhibition researchers to discover unique scaffolds.

Monoclonal Antibodies for Combating Synthetic Opioid Intoxication.

Opioid abuse in the United States has been declared a national crisis and is exacerbated by an inexpensive, readily available, and illicit supply of synthetic opioids. Specifically, fentanyl and related analogues such as carfentanil pose a significant danger to opioid users due to their high potency and rapid acting depression of respiration. In recent years these synthetic opioids have become the number one cause of drug-related deaths. In our research efforts to combat the public health threat posed by synthetic opioids, we have developed monoclonal antibodies (mAbs) against the fentanyl class of drugs. The mAbs were generated in hybridomas derived from mice vaccinated with a fentanyl conjugate vaccine. Guided by a surface plasmon resonance (SPR) binding assay, we selected six hybridomas that produced mAbs with 10-11 M binding affinity for fentanyl, yet broad cross-reactivity with related fentanyl analogues. In mouse antinociception models, our lead mAb (6A4) could blunt the effects of both fentanyl and carfentanil in a dose-responsive manner. Additionally, mice pretreated with 6A4 displayed enhanced survival when subjected to fentanyl above LD50 doses. Pharmacokinetic analysis revealed that the antibody sequesters large amounts of these drugs in the blood, thus reducing drug biodistribution to the brain and other tissue. Lastly, the 6A4 mAb could effectively reverse fentanyl/carfentanil-induced antinociception comparable to the opioid antagonist naloxone, the standard of care drug for treating opioid overdose. While naloxone is known for its short half-life, we found the half-life of 6A4 to be approximately 6 days in mice, thus monoclonal antibodies could theoretically be useful in preventing renarcotization events in which opioid intoxication recurs following quick metabolism of naloxone. Our results as a whole demonstrate that monoclonal antibodies could be a desirable treatment modality for synthetic opioid overdose and possibly opioid use disorder.

Synthetic molecules for disruption of the MYC protein-protein interface.

MYC is a key transcriptional regulator involved in cellular proliferation and has established roles in transcriptional elongation and initiation, microRNA regulation, apoptosis, and pluripotency. Despite this prevalence, functional chemical probes of MYC function at the protein level have been limited. Previously, we discovered 5a, that binds to MYC with potency and specificity, downregulates the transcriptional activities of MYC and shows efficacy in vivo. However, this scaffold posed intrinsic pharmacokinetic liabilities, namely, poor solubility that precluded biophysical interrogation. Here, we developed a screening platform based on field-effect transistor analysis (Bio-FET), surface plasmon resonance (SPR), and a microtumor formation assay to analyze a series of new compounds aimed at improving these properties. This blind SAR campaign has produced a new lead compound of significantly increased in vivo stability and solubility for a 40-fold increase in exposure. This probe represents a significant advancement that will not only enable biophysical characterization of this interaction and further SAR, but also contribute to advances in understanding of MYC biology.

Design, synthesis and optimization of 7-substituted-pyrazolo[4,3-b]pyridine ALK5 (activin receptor-like kinase 5) inhibitors.

A series of potent ALK5 inhibitors were designed using a SBDD approach and subsequently optimized to improve drug likeness. Starting with a 4-substituted quinoline screening hit, SAR was conducted using a ALK5 binding model to understand the binding site and optimize activity. The resulting inhibitors displayed excellent potency but were limited by high in vitro clearance in rat and human microsomes. Using a scaffold morphing strategy, these analogs were transformed into a related pyrazolo[4,3-b]pyridine series with improved ADME properties.

Design, synthesis and optimization of novel Alk5 (activin-like kinase 5) inhibitors.

Using SBDD, a series of 4-amino-7-azaindoles were discovered as a novel class of Alk5 inhibitors that are potent in both Alk5 enzymatic and cellular assays. Subsequently a ring cyclization strategy was utilized to improve ADME properties leading to the discovery of a series of 1H-imidazo[4,5-c]pyridin-2(3H)-one drug like Alk5 inhibitors.

Fragment-based drug discovery of potent and selective MKK3/6 inhibitors.

The MAPK signaling cascade, comprised of several linear and intersecting pathways, propagates signaling into the nucleus resulting in cytokine and chemokine release. The Map Kinase Kinase isoforms 3 and 6 (MKK3 and MKK6) are responsible for the phosphorylation and activation of p38, and are hypothesized to play a key role in regulating this pathway without the redundancy seen in downstream effectors. Using FBDD, we have discovered efficient and selective inhibitors of MKK3 and MKK6 that can serve as tool molecules to help further understand the role of these kinases in MAPK signaling, and the potential impact of inhibiting kinases upstream of p38.

Onchocerca volvulus-neurotransmitter tyramine is a biomarker for river blindness.

Onchocerciasis, also known as "river blindness", is a neglected tropical disease infecting millions of people mainly in Africa and the Middle East but also in South America and Central America. Disease infectivity initiates from the filarial parasitic nematode Onchocerca volvulus, which is transmitted by the blackfly vector Simulium sp. carrying infectious third-stage larvae. Ivermectin has controlled transmission of microfilariae, with an African Program elimination target date of 2025. However, there is currently no point-of-care diagnostic that can distinguish the burden of infection--including active and/or past infection--and enable the elimination program to be effectively monitored. Here, we describe how liquid chromatography-MS-based urine metabolome analysis can be exploited for the identification of a unique biomarker, N-acetyltyramine-O,ß-glucuronide (NATOG), a neurotransmitter-derived secretion metabolite from O. volvulus. The regulation of this tyramine neurotransmitter was found to be linked to patient disease infection, including the controversial antibiotic doxycycline treatment that has been shown to both sterilize and kill adult female worms. Further clues to its regulation have been elucidated through biosynthetic pathway determination within the nematode and its human host. Our results demonstrate that NATOG tracks O. volvulus metabolism in both worms and humans, and thus can be considered a host-specific biomarker for onchocerciasis progression. Liquid chromatography-MS-based urine metabolome analysis discovery of NATOG not only has broad implications for a noninvasive host-specific onchocerciasis diagnostic but provides a basis for the metabolome mining of other neglected tropical diseases for the discovery of distinct biomarkers and monitoring of disease progression.

Rapid Determination of the Specificity Constant of Irreversible Inhibitors (kinact/KI) by Means of an Endpoint Competition Assay.

Owing to their covalent target occupancy, irreversible inhibitors require low exposures and offer long duration, and their use thus represents a powerful strategy for achieving pharmacological efficacy. Importantly, the potency metric of irreversible inhibitors is kinact/KI not IC50. A simple approach to measuring kinact/KI was developed that makes use of an irreversible probe for competitive assays run to completion against test compounds. In this system, the kinact/KI value of the test compound is equal to (kinact/KI)probe ×[probe]/IC50. The advantages of this method include simplicity, high throughput, and application to all target classes, and it only requires an in-depth kinetic evaluation of the probe.

Probing BoNT/A protease exosites: implications for inhibitor design and light chain longevity.

Botulinum neurotoxin serotype A (BoNT/A) is one of the most lethal toxins known. Its extreme toxicity is due to its light chain (LC), a zinc protease that cleaves SNAP-25, a synaptosome-associated protein, leading to the inhibition of neuronal activity. Studies on BoNT/A LC have revealed that two regions, termed exosites, can play an important role in BoNT catalytic activity. A clear understanding of how these exosites influence neurotoxin catalytic activity would provide a critical framework for deciphering the mechanism of SNAP-25 cleavage and the design of inhibitors. Herein, based on the crystallographic structure of BoNT/A LC complexed with its substrate, we designed an α-exosite binding probe. Experiments with this unique probe demonstrated that α-exosite binding enhanced both catalytic activity and stability of the LC. These data help delineate why α-exosite binding is needed for SNAP-25 cleavage and also provide new insights into the extended lifetime observed for BoNT/A LC in vivo.

ATP allosterically activates the human 5-lipoxygenase molecular mechanism of arachidonic acid and 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid.

5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid. This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE (dehydration of the hydroperoxide) has a rate of substrate capture (Vmax/Km) significantly lower than that of AA hydroperoxidation (oxidation of AA to form the hydroperoxide); however, hyperbolic kinetic parameters for ATP activation indicate a similar activation for AA and 5(S)-HpETE. Solvent isotope effect results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly because of a lowering of the dependence of the rate-limiting step on hydrogen atom abstraction and an increase in the dependency on hydrogen bond rearrangement. Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation.

A simple and widely applicable hit validation strategy for protein-protein interaction inhibitors based on a quantitative ligand displacement assay.

Identification of inhibitors for protein-protein interactions (PPIs) from high-throughput screening (HTS) is challenging due to the weak affinity of primary hits. We present a hit validation strategy of PPI inhibitors using quantitative ligand displacement assay. From an HTS for Bcl-xL/Mcl-1 inhibitors, we obtained a hit candidate, I1, which potentially forms a reactive Michael acceptor, I2, inhibiting Bcl-xL/Mcl-1 through covalent modification. We confirmed rapid reversible and competitive binding of I1 with a probe peptide, suggesting non-covalent binding. The advantages of our approach over biophysical assays include; simplicity, higher throughput, low protein consumption and universal application to PPIs including insoluble membrane proteins.

Benzoquinones as inhibitors of botulinum neurotoxin serotype A.

Although botulinum neurotoxin serotype A (BoNT/A) is known for its use in cosmetics, it causes a potentially fatal illness, botulism, and can be used as a bioterror weapon. Many compounds have been developed that inhibit the BoNTA zinc-metalloprotease light chain (LC), however, none of these inhibitors have advanced to clinical trials. In this study, a fragment-based approach was implemented to develop novel covalent inhibitors of BoNT/A LC. First, electrophilic fragments were screened against BoNT/A LC, and benzoquinone (BQ) derivatives were found to be active. In kinetic studies, BQ compounds acted as irreversible inhibitors that presumably covalently modify cysteine 165 of BoNT/A LC. Although most BQ derivatives were highly reactive toward glutathione in vitro, a few compounds such as natural product naphthazarin displayed low thiol reactivity and good BoNT/A inhibition. In order to increase the potency of the BQ fragment, computational docking studies were employed to elucidate a scaffold that could bind to sites adjacent to Cys165 while positioning a BQ fragment at Cys165 for covalent modification; 2-amino-N-arylacetamides met these criteria and when linked to BQ displayed at least a 20-fold increase in activity to low µM IC50 values. Unlike BQ alone, the linked-BQ compounds demonstrated only weak irreversible inhibition and therefore acted mainly as non-covalent inhibitors. Further kinetic studies revealed a mutual exclusivity of BQ covalent inactivation and competitive inhibitor binding to sites adjacent to Cys165, refuting the viability of the current strategy for developing more potent irreversible BoNT/A inhibitors. The highlights of this study include the discovery of BQ compounds as irreversible BoNT/A inhibitors and the rational design of low µM IC50 competitive inhibitors that depend on the BQ moiety for activity.

Mechanistic insights into the LsrK kinase required for autoinducer-2 quorum sensing activation.

In enteric bacteria, the kinase LsrK catalyzes the phosphorylation of the C5-hydroxyl group in the linear form of 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). This phosphorylation is required for AI-2 sequestration in the cytoplasm and subsequent derepression of AI-2-related genes necessary for quorum development. While LsrK is a critical enzyme within the DPD quorum sensing relay system, kinetic details of this kinase have yet to be reported. A continuous UV-vis spectrophotometric assay was developed that allowed steady-state kinetic analysis of LsrK to be undertaken with the substrates ATP and DPD. The data was most consistent with a rapid equilibrium ordered mechanism with ATP binding first: kcat (7.4 ± 0.6 s(-1)), Km,ATP (150 ± 30 µM) and Km(app),DPD (1.0 ± 0.2 mM). The assay also allowed a DPD substrate profile to be conducted, which provided an unexpected biochemical disconnect between the previous agonist/antagonist cell-based reporter assay and the LsrK assay presented herein. Together these findings raise the importance of LsrK and lay the foundation not only for further understanding of this enzyme and its critical biological role but also for the rational design of regulatory molecules targeting AI-2 quorum sensing in pathogenic bacteria.

Probing the effects of hapten stability on cocaine vaccine immunogenicity.

Judicious hapten design has been shown to be of importance when trying to generate a viable vaccine against a drug of abuse. Hapten design has typically been predicated upon faithfully emulating the unique chemical architecture that each drug presents. However, the need for drug-hapten congruency may also compromise vaccine immunogenicity if the drug-hapten conjugate possesses chemical epitope instability. There has been no systematic study on the impact of hapten stability as it relates to vaccine immunogenicity. As a starting point, we have probed the stability of a series of cocaine haptens through varying several of its structural elements, including functionality at the C2-position, the nature of the linker, and its site of attachment. Accordingly, a hydrolytic stability profile of four cocaine haptens (GNNA, GNNS, GNE, and GNC) was produced, and these results were compared through each hapten's immunological properties, which were generated via active vaccination. From this group of four, three of the haptens, GNE, GNNA, and GNC, were further examined in an animal behavioral model, and findings here were again measured in relationship to hapten stability. We demonstrate a corresponding relationship between the half-life of the hapten and its immunogenicity, wherein haptens presenting a fully representative cocaine framework elicited higher concentrations of cocaine-specific IgG in sera and also conferred better protection against cocaine-induced locomotor activity. Our results indicate that hapten half-life plays an important role in vaccine immunogenicity and this in turn can impact animal behavioral effects when challenged with a drug of abuse.

Targeted Reversible Covalent Modification of a Noncatalytic Lysine of the Krev Interaction Trapped 1 Protein Enables Site-Directed Screening for Protein-Protein Interaction Inhibitors.

The covalent reversible modification of proteins is a validated strategy for the development of probes and candidate therapeutics. However, the covalent reversible targeting of noncatalytic lysines is particularly challenging. Herein, we characterize the 2-hydroxy-1-naphthaldehyde (HNA) fragment as a targeted covalent reversible ligand of a noncatalytic lysine (Lys720) of the Krev interaction trapped 1 (KRIT1) protein. We show that the interaction of HNA with KRIT1 is highly specific, results in prolonged residence time of >8 h, and inhibits the Heart of glass 1 (HEG1)-KRIT1 protein-protein interaction (PPI). Screening of HNA derivatives identified analogs exhibiting similar binding modes as the parent fragment but faster target engagement and stronger inhibition activity. These results demonstrate that HNA is an efficient site-directing fragment with promise in developing HEG1-KRIT1 PPI inhibitors. Further, the aldimine chemistry, when coupled with templating effects that promote proximity, can produce a long-lasting reversible covalent modification of noncatalytic lysines.

A randomized, double-blind, placebo controlled, phase 1 study of the safety, tolerability, pharmacokinetics, and pharmacodynamics of LB-102, a selective dopamine D2/3/5-HT7 inhibitor.

LB-102 is an N-methylated analogue of amisulpride under development to treat schizophrenia. LB-102 was evaluated in a Phase 1, double-blind, placebo-controlled, clinical study to evaluate safety and pharmacokinetics. This was a first-in-human study examining single and multiple doses of LB-102 administered orally in 64 healthy volunteers. Dosing in the single ascending dose (SAD) portion of the study was initially planned to be 50, 100, 200, and 400 mg, with doses in the multiple ascending dose (MAD) portion to be determined based on observations in the SAD portion. As a result of two cases of EPS (acute dystonia) at 200 mg in the MAD portion of the study, dosing of that arm was discontinued and doses for the remaining cohort were decreased to 150 mg/day. Dose escalation was guided by safety and plasma concentrations of LB-102 compared to a translational model. LB-102 was generally safe and well-tolerated, and clinical lab values were unremarkable at all doses, save for prolactin which was transiently elevated in the majority of subjects treated with LB-102; there were no clinical observations associated with the increases in prolactin elevation. There was evidence of transient QT interval prolongation at the 200 mg dose, none of which resulted in clinical observation or triggered stopping criteria. There were four instances of EPS (acute dystonia), typically associated with dopamine receptor occupancy in excess of 80%, one at 100 mg QD, one at 75 mg BID, and two at 100 mg BID. A phase 2 clinical study of LB-102 in schizophrenia patients with PANSS as primary endpoint is being planned.

Identification of Slow-Binding Inhibitors of the BoNT/A Protease.

Botulinum neurotoxin A (BoNT/A) is a lethal toxin, which causes botulism, and is categorized as a bioterrorism threat, which causes flaccid paralysis and death. Botulinum A neurotoxicity is governed through its light chain (LC), a zinc metalloprotease. Pharmacological investigations aimed at negating BoNT/A's LC have typically looked to inhibitors that have been shown to inhibit the light chain's activity by reversible zinc chelation within its active site. This report outlines the first examples of nonpeptidic inhibitors of the BoNT/A LC that possess slow-binding kinetics, a needed logic to counteract the longevity of BoNT/A. Cyclopropane, alkyl, and alkenyl derivatives of 2,4-dichlorocinamic hydroxamic acid (DCHA) were shown to possess both one-step and two-step slow-binding kinetics. Structure-kinetic relationships (SKRs) were observed and were rationalized with the aid of docking models that predicted improved interactions with residues within a hydrophobic cleft adjacent to the active site.

Biochemical characterization of TAK-593, a novel VEGFR/PDGFR inhibitor with a two-step slow binding mechanism.

Inhibition of tumor angiogenesis leads to a lack of oxygen and nutrients in the tumor and therefore has become a standards of care for many solid tumor therapies. Dual inhibition of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) protein kinase activities is a popular strategy for targeting tumor angiogenesis. We discovered that TAK-593, a novel imidazo[1,2-b]pyridazine derivative, potently inhibits tyrosine kinases from the VEGFR and PDGFR families. TAK-593 was highly selective for these families, with an IC(50) >1 µM when tested against more than 200 protein and lipid kinases. TAK-593 displayed competitive inhibition versus ATP. In addition, TAK-593 inhibited VEGFR2 and PDGFRß in a time-dependent manner, classifying it as a type II kinase inhibitor. Analysis of enzyme-inhibitor preincubation experiments revealed that the binding of TAK-593 to VEGFR2 and PDGFRß occurs via a two-step slow binding mechanism. Dissociation of TAK-593 from VEGFR2 was extremely slow (t(1/2) >17 h), and the affinity of TAK-593 at equilibrium (K(i)*) was less than 25 pM. Ligand displacement analysis with a fluorescent tracer confirmed the slow dissociation of TAK-593. The dissociation rate constants were in good agreement between the activity and ligand displacement data, and both analyses supported slow dissociation of TAK-593. The long residence time of TAK-593 may achieve an extended pharmacodynamic effect on VEGFR2 and PDGFRß kinases in vivo that differs substantially from its observed pharmacokinetic profile.

Biochemical characterization of a novel type-II VEGFR2 kinase inhibitor: comparison of binding to non-phosphorylated and phosphorylated VEGFR2.

A pyrrolo[3,2-d]pyrimidine-based type-II vascular endothelial growth factor receptor 2 (VEGFR2) kinase inhibitor, compound 20d, displayed time-dependent inhibition of the non-phosphorylated catalytic domain of VEGFR2. In contrast, 20d did not show time-dependent inhibition of the phosphorylated enzyme. Dissociation of 20d from non-phosphorylated VEGFR2 was slow and the half-life of the complex was longer than 4h. In contrast, dissociation of 20d from the phosphorylated enzyme was very fast (half-life <5min). A fluorescent tracer based displacement assay and surface plasmon resonance (SPR) analysis confirmed the slow dissociation of 20d from only non-phosphorylated VEGFR2. Thus, activity based and binding kinetic analyses both supported slow dissociation of 20d from only non-phosphorylated VEGFR2. Additionally SPR analysis revealed that association rates were rapid and nearly identical for these two phosphorylation forms of VEGFR2. From these results, the preferential effect of 20d on non-phosphorylated VEGFR2 is dominated by its slow dissociation from the enzyme and this characteristically long residence time may increase its potency in vivo. The present findings may assist in the design of novel type-II kinase inhibitors.