Evaluation of a hapten conjugate vaccine against the "zombie drug" xylazine.

Xylazine has emerged as a primary adulterant in fentanyl, exacerbating the complexity of the opioid crisis. Yet, there is no approved drug that can reverse xylazine's pathophysiology. As a prelude to monoclonal antibodies being assessed as a viable therapeutic, a vaccine inquiry was conducted evaluating the immune response in reversing xylazine induced behavior effects.

Discovery of PLD4 modulators by high-throughput screening and kinetic analysis.

Phospholipase D3 (PLD3) and D4 (PLD4) are endolysosomal exonucleases of ssDNA and ssRNA that regulate innate immunity. Polymorphisms of these enzymes are correlated with numerous human diseases, including Alzheimer's, rheumatoid arthritis, and systemic sclerosis. Pharmacological modulation of these immunoregulatory proteins may yield novel immunotherapies and adjuvants. A previous study reported a high-throughput screen (N = 17,952) that discovered a PLD3-selective activator and inhibitor, as well as a nonselective inhibitor, but failed to identify selective modulators of PLD4. However, modulators selective for PLD4 are therapeutically pertinent, since recent reports have shown that regulating this protein has direct implications in cancer and autoimmune diseases. Furthermore, the high expression of PLD4 in dendritic and myeloid cells, in comparison to the broader expression of PLD3, presents the opportunity for a cell-targeted immunotherapy. Here, we describe screening of an expended diversity library (N = 45,760) with an improved platform and report the discovery of one inhibitor and three activators selective for PLD4. Furthermore, kinetic modeling and structural analysis suggest mechanistic differences in the modulation of these hits. These findings further establish the utility of this screening platform and provide a set of chemical scaffolds to guide future small-molecule development for this novel immunoregulator target.

An Engineered Human-Antibody Fragment with Fentanyl Pan-Specificity That Reverses Carfentanil-Induced Respiratory Depression.

The opioid overdose crisis primarily driven by potent synthetic opioids resulted in more than 500,000 deaths in the US over the last 20 years. Though naloxone, a short-acting medication, remains the primary treatment option for temporarily reversing opioid overdose effects, alternative countermeasures are needed. Monoclonal antibodies present a versatile therapeutic opportunity that can be tailored to synthetic opioids and help prevent post-treatment renarcotization. The ultrapotent analog carfentanil is especially concerning due to its unique pharmacological properties. With this in mind, we generated a fully human antibody through a drug-specific B cell sorting strategy with a combination of carfentanil and fentanyl probes. The resulting pan-specific antibody was further optimized through scFv phage display, producing C10-S66K. This monoclonal antibody displays high affinity to carfentanil, fentanyl, and other analogs and reversed carfentanil-induced respiratory depression. Additionally, X-ray crystal structures with carfentanil and fentanyl bound provided structural insight into key drug:antibody interactions.

Development of a vaccine against the synthetic opioid U-47700.

Opioid use disorders and overdose have become a major public health concern in recent years. U-47700, a New psychoactive substances (NPS) opioid, also known as "pinky" or "pink" has been identified as a new threat in the drug supply because of its potency and abuse potential. Conjugate vaccines that can produce antibodies against target drug molecules have emerged as a promising tool to treat substance use disorders. Herein, we report the design, synthesis, and in vivo characterization of a U-47700 vaccine. The vaccine demonstrated favorable results with rodents producing elevated levels of antibody titer and sub-micromolar affinity to U-47700. In addition, antibodies generated by the vaccine effectively mitigated drug-induced effects by preventing the drug from penetrating the blood-brain barrier, which was verified by antinociception and drug biodistribution studies. The development of a vaccine against U-47700 and other NPS opioids contributes to the continued advancement of non-conventional pharmacological treatments to address the global opioid epidemic.

An Engineered Human-Antibody Fragment with Fentanyl Pan-Specificity that Reverses Carfentanil-Induced Respiratory Depression.

The opioid overdose crisis primarily driven by potent synthetic opioids resulted in more than 500,000 deaths in the US over the last 20 years. Though naloxone, a short acting medication, remains the primary treatment option for temporarily reversing opioid overdose effects, alternative countermeasures are needed. Monoclonal antibodies present a versatile therapeutic opportunity that can be tailored for synthetic opioids and that can help prevent post-treatment renarcotization. The ultrapotent analog carfentanil, is especially concerning due to its unique pharmacological properties. With this in mind, we generated a fully human antibody through a drug-specific B cell sorting strategy with a combination of carfentanil and fentanyl probes. The resulting pan-specific antibody was further optimized through scFv phage display. This antibody, C10-S66K, displays high affinity to carfentanil, fentanyl, and other analogs, and reversed carfentanil-induced respiratory depression. Additionally, x-ray crystal structures with carfentanil and fentanyl bound provided structural insight into key drug:antibody interactions.

Pharmacological GHSR (ghrelin receptor) blockade reduces alcohol binge-like drinking in male and female mice.

Ghrelin is a peptide that is produced by endocrine cells that are primarily localized in the stomach. Ghrelin receptors (GHSR) are expressed in the brain and periphery. Preclinical and clinical studies support a role for ghrelin in alcohol drinking and seeking. The GHSR has been suggested to be a potential pharmacotherapeutic target for alcohol use disorder (AUD). However, the role of the ghrelin system and its potential modulation by biological sex on binge-like drinking has not been comprehensively investigated. The present study tested six GHSR antagonists in an alcohol binge-like drinking procedure in male and female mice. Systemic administration of the GHSR antagonists JMV2959, PF-5190457, PF-6870961, and HM-04 reduced alcohol intake in both male and female mice. YIL-781 decreased intake in males, and LEAP2 (likely peripherally restricted) did not reduce intake in mice of either sex. We also administered LEAP2 and JMV2959 intracerebroventricularly to investigate whether the effects of GHSR blockade on alcohol intake are mediated by central receptors. The central administration of LEAP2 and JMV2959 decreased alcohol intake, particularly in high-drinking animals. Finally, in a preliminary experiment, an anti-ghrelin vaccine was examined for its potential effect on binge-like drinking and had no effect. In all experiments, there was a lack of meaningful sex differences. These findings suggest that central GHSR mediates binge-like alcohol intake. These data reveal novel pharmacological compounds with translational potential in the treatment of AUD and provide further evidence of the GHSR as a potential treatment target for AUD.

Catalytic Antibody Blunts Carfentanil-Induced Respiratory Depression.

Carfentanil, the most potent of the fentanyl analogues, is at the forefront of synthetic opioid-related deaths, second to fentanyl. Moreover, the administration of the opioid receptor antagonist naloxone has proven inadequate for an increasing number of opioid-related conditions, often requiring higher/additional doses to be effective, as such interest in alternative strategies to combat more potent synthetic opioids has intensified. Increasing drug metabolism would be one strategy to detoxify carfentanil; however, carfentanil's major metabolic pathways involve N-dealkylation or monohydroxylation, which do not lend themselves readily to exogenous enzyme addition. Herein, we report, to our knowledge, the first demonstration that carfentanil's methyl ester when hydrolyzed to its acid was found to be 40,000 times less potent than carfentanil in activating the µ-opioid receptor. Physiological consequences of carfentanil and its acid were also examined through plethysmography, and carfentanil's acid was found to be incapable of inducing respiratory depression. Based upon this information, a hapten was chemically synthesized and immunized, allowing the generation of antibodies that were screened for carfentanil ester hydrolysis. From the screening campaign, three antibodies were found to accelerate the hydrolysis of carfentanil's methyl ester. From this series of catalytic antibodies, the most active underwent extensive kinetic analysis, allowing us to postulate its mechanism of hydrolysis against this synthetic opioid. In the context of potential clinical applications, the antibody, when passively administered, was able to reduce respiratory depression induced by carfentanil. The data presented supports further development of antibody catalysis as a biologic strategy to complement carfentanil overdose reversal.

Demystifying the Druggability of the MYC Family of Oncogenes.

The MYC family of oncogenes (MYC, MYCN, and MYCL) encodes a basic helix-loop-helix leucine zipper (bHLHLZ) transcriptional regulator that is responsible for moving the cell through the restriction point. Through the HLHZIP domain, MYC heterodimerizes with the bHLHLZ protein MAX, which enables this MYC-MAX complex to bind to E-box regulatory DNA elements thereby controlling transcription of a large group of genes and their proteins. Translationally, MYC is one of the foremost oncogenic targets, and deregulation of expression of the MYC family gene/proteins occurs in over half of all human tumors and is recognized as a hallmark of cancer initiation and maintenance. Additionally, unexpected roles for this oncoprotein have been found in cancers that nominally have a non-MYC etiology. Although MYC is rarely mutated, its gain of function in cancer results from overexpression or from amplification. Moreover, MYC is a pleiotropic transcription factor possessing broad pathogenic prominence making it a coveted cancer target. A widely held notion within the biomedical research community is that the reliable modulation of MYC represents a tremendous therapeutic opportunity given its role in directly potentiating oncogenesis. However, the MYC-MAX heterodimer interaction contains a large surface area with a lack of well-defined binding sites creating the perception that targeting of MYC-MAX is forbidding. Here, we discuss the biochemistry behind MYC and MYC-MAX as it relates to cancer progression associated with these transcription factors. We also discuss the notion that MYC should no longer be regarded as undruggable, providing examples that a therapeutic window is achievable despite global MYC inhibition.

Development of an Effective Monoclonal Antibody against Heroin and Its Metabolites Reveals Therapies Have Mistargeted 6-Monoacetylmorphine and Morphine over Heroin.

The opioid epidemic is a global public health crisis that has failed to abate with current pharmaceutical treatments. Moreover, these FDA-approved drugs possess numerous problems such as adverse side effects, short half-lives, abuse potential, and recidivism after discontinued use. An alternative treatment model for opioid use disorders is immunopharmacotherapy, where antibodies are produced to inhibit illicit substances by sequestering the drug in the periphery. Immunopharmacotherapeutics against heroin have engaged both active and passive vaccines targeting heroin's metabolites, 6-monoacetylmorphine (6-AM) and morphine, since decades of research have stated that heroin's psychoactive and lethal effects are mainly attributed to these compounds. However, concerted efforts to develop effective immunopharmacotherapies against heroin abuse have faced little clinical advancement, suggesting a need for reassessing drug target selection. To address this issue, four unique monoclonal antibodies were procured with distinct affinity to either heroin, 6-AM, or morphine. Examination of these antibodies through in vitro and in vivo tests revealed monoclonal antibody 11D12 as the optimal therapeutic and provided crucial insights into the key chemical species to target for blunting heroin's psychoactive and lethal effects. These findings offer clarification into the problematic attempts of therapeutics targeting heroin's metabolites and provide a path forward for future heroin immunopharmacotherapy development.

Polyphosphazene: A New Adjuvant Platform for Cocaine Vaccine Development.

Cocaine is a highly addictive drug that has seen a steady uptrend causing severe health problems worldwide. Currently, there are no approved therapeutics for treating cocaine use disorder; hence, there is an urgent need to identify new medications. Immunopharmacotherapeutics is a promising approach utilizing endogenous antibodies generated through active vaccination, and if properly programmed, can blunt a drug's psychoactive and addictive effects. However, drug vaccine efficacy has largely been limited by the modest levels of antibodies induced. Herein, we explored an adjuvant system consisting of a polyphosphazene macromolecule, specifically poly[di(carboxylatoethylphenoxy)-phosphazene] (PCEP), a biocompatible synthetic polymer that was solicited for improved cocaine conjugate vaccine delivery performance. Our results demonstrated PCEP's superior assembling efficiency with a cocaine hapten as well as with the combined adjuvant CpG oligodeoxynucleotide (ODN). Importantly, this combination led to a higher titer response, balanced immunity, successful sequestering of cocaine in the blood, and a reduction in the drug in the brain. Moreover, a PCEP-cocaine conjugate vaccine was also found to function well via intranasal administration, where its efficacy was demonstrated through the antibody titer, affinity, mucosal IgA production, and a reduction in cocaine's locomotor activity. Overall, a comprehensive evaluation of PCEP integrated within a cocaine vaccine established an advance in the use of synthetic adjuvants in the drugs of abuse vaccine field.

Investigation of Salicylanilides as Botulinum Toxin Antagonists.

Botulinum neurotoxin serotype A (BoNT/A) is recognized by the Centers for Disease Control and Prevention (CDC) as the most potent toxin and as a Tier 1 biowarfare agent. The severity and longevity of botulism stemming from BoNT/A is of significant therapeutic concern, and early administration of antitoxin-antibody therapy is the only approved pharmaceutical treatment for botulism. Small molecule therapeutic strategies have targeted both the heavy chain (HC) and the light chain (LC) catalytic active site and α-/ß-exosites. The LC translocation mechanism has also been studied, but an effective, nontoxic inhibitor remains underexplored. In this work, we screened a library of salicylanilides as potential translocation inhibitors. Potential leads following a primary screen were further scrutinized to identify sal30, which has a cellular minimal concentration of a drug that is required for 50% inhibition (IC50) value of 141 nM. The inquiry of salicylanilide sal30's mechanism of action was explored through a self-quenched fluorogenic substrate conjugated to bovine serum albumin (DQ-BSA) fluorescence, confocal microscopy, and vacuolar H+-ATPase (V-ATPase) inhibition assays. The summation of these findings imply that endolysosomal proton translocation through the protonophore mechanism of sal30 causes endosome pH to increase, which in turn prevents LC translocation into cytosol, a process that requires an acidic pH. Thus, the inhibition of BoNT/A activity by salicylanilides likely occurs through disruption of pH-dependent endosomal LC translocation. We further probed BoNT inhibition by sal30 using additivity analysis studies with bafilomycin A1, a known BoNT/A LC translocation inhibitor, which indicated the absence of synergy between the two ionophores.

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.

A Vaccine against Benzimidazole-Derived New Psychoactive Substances That Are More Potent Than Fentanyl.

New psychoactive substance (NPS) opioids have proliferated within the international drug market. While synthetic opioids are traditionally composed of fentanyl analogues, benzimidazole-derived isotonitazene and its derivatives are the current NPS opioids of concern. Hence, in this study, we implement immunopharmacotherapy wherein antibodies are produced with high titers and nanomolar affinity to multiple benzimidazole-derived NPS opioids (BNO). Notably, these antibodies blunt psychoactive and physiological repercussions from BNO exposure, which was observed through antinociception, whole-body plethysmography, and blood-brain biodistribution studies. Moreover, we detail previously unreported pharmacokinetics of these drugs, which explains the struggle of traditional pharmaceutical opioid antagonists against BNO substances. These findings provide further insight into the in vivo effects of BNO drugs and the development of effective broad-spectrum therapeutics against NPS opioids.

Development of effective therapeutics for polysubstance use disorders.

Traditional pharmacotherapies for substance use disorders have focused on mono-substance abuse. However, recent epidemiological studies have found polysubstance use disorders (PUD) are becoming more prevalent and the abuse of adulterated drugs has led to increasing unintentional overdose deaths. Unfortunately, there are no approved pharmacological agents for PUD. Hence, a therapeutic model of interest to address this growing epidemic is immunopharmacotherapy, where individuals are inoculated with conjugate vaccines formulated with haptens that mimic the drug of abuse. These conjugate vaccines have demonstrated significant therapeutic potential against mono-substance abuse, thus recent studies have applied this model to address PUD. This review presents immunopharmacotherapeutic advancements against polysubstance abuse and discusses necessary developments for conjugate vaccines in order to effectively treat this unaddressed epidemic.

Isoxazole-9 reduces enhanced fear responses and retrieval in ethanol-dependent male rats.

Plasticity in the dentate gyrus (DG) is strongly influenced by ethanol, and ethanol experience alters long-term memory consolidation dependent on the DG. However, it is unclear if DG plasticity plays a role in dysregulation of long-term memory consolidation during abstinence from chronic ethanol experience. Outbred male Wistar rats experienced 7 weeks of chronic intermittent ethanol vapor exposure (CIE). Seventy-two hours after CIE cessation, CIE and age-matched ethanol-naïve Air controls experienced auditory trace fear conditioning (TFC). Rats were tested for cue-mediated retrieval in the fear context either twenty-four hours (24 hr), ten days (10 days), or twenty-one days (21 days) later. CIE rats showed enhanced freezing behavior during TFC acquisition compared to Air rats. Air rats showed significant fear retrieval, and this behavior did not differ at the three time points. In CIE rats, fear retrieval increased over time during abstinence, indicating an incubation in fear responses. Enhanced retrieval at 21 days was associated with reduced structural and functional plasticity of ventral granule cell neurons (GCNs) and reduced expression of synaptic proteins important for neuronal plasticity. Systemic treatment with the drug Isoxazole-9 (Isx-9; small molecule that stimulates DG plasticity) during the last week and a half of CIE blocked altered acquisition and retrieval of fear memories in CIE rats during abstinence. Concurrently, Isx-9 modulated the structural and functional plasticity of ventral GCNs and the expression of synaptic proteins in the ventral DG. These findings identify that abstinence-induced disruption of fear memory consolidation occurs via altered plasticity within the ventral DG, and that Isx-9 prevented these effects.

Broadly Neutralizing Synthetic Cannabinoid Vaccines.

Synthetic cannabinoids (SCs) constitute a significant portion of psychoactive substances forming a major public health risk. Due to the wide variety of SCs, broadly neutralizing antibodies generated by active immunization present an intriguing pathway to combat cannabinoid use disorder. Here, we probed hapten design for antibody affinity and cross reactivity against two classes of SCs. Of the 10 haptens screened, 3 vaccine groups revealed submicromolar IC50, each targeting 5-6 compounds in our panel of 22 drugs. Moreover, SCs were successfully sequestered when administered by vaping or intraperitoneal injection, which was confirmed within animal models by observing locomotion, body temperature, and pharmacokinetics. We also discovered synergistic effects to simultaneously blunt two drug classes through an admixture vaccine approach. Collectively, our study provides a comprehensive foundation for the development of vaccines against SCs.

Immunopharmacotherapeutic advancements in addressing methamphetamine abuse.

Methamphetamine (METH) is an illicit psychostimulant that is known to account for substance abuse disorders globally, second only to opioids, yet has no approved pharmacotherapies. Traditional therapies employ small molecule agonists or antagonists for substance use disorders or overdose reversal by targeting drug-specific receptors in the brain. However, the comprehensive mechanism of METH on multiple sites within the central nervous system (CNS) implies its receptors lack the high affinity and specificity required for an "ideal" drug target. The alternative to pharmacotherapies is to sequester abused drugs in the periphery, effectively eliminating the effects from CNS receptor occupation through pharmacokinetic antagonism. This review presents updates on immunopharmacotherapeutic advancements in addressing methamphetamine abuse by focusing on the cultivation of research optimization strategies regarding hapten chemistry, carrier proteins, and adjuvants implemented in active immunization. Furthermore, we discuss necessary developments for each component of active immunopharmacotherapies and the future of active vaccines in treating METH use disorder.

Salicylanilides Reduce SARS-CoV-2 Replication and Suppress Induction of Inflammatory Cytokines in a Rodent Model.

SARS-CoV-2 virus has recently given rise to the current COVID-19 pandemic where infected individuals can range from being asymptomatic, yet highly contagious, to dying from acute respiratory distress syndrome. Although the world has mobilized to create antiviral vaccines and therapeutics to combat the scourge, their long-term efficacy remains in question especially with the emergence of new variants. In this work, we exploit a class of compounds that has previously shown success against various viruses. A salicylanilide library was first screened in a SARS-CoV-2 activity assay in Vero cells. The most efficacious derivative was further evaluated in a prophylactic mouse model of SARS-CoV-2 infection unveiling a salicylanilide that can reduce viral loads, modulate key cytokines, and mitigate severe weight loss involved in COVID-19 infections. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and a previously established favorable pharmacokinetic profile for the lead salicylanilide renders salicylanilides in general as promising therapeutics for COVID-19.

Use of Crystallography and Molecular Modeling for the Inhibition of the Botulinum Neurotoxin A Protease.

Botulinum neurotoxins (BoNTs) are extremely toxic and have been deemed a Tier 1 potential bioterrorism agent. The most potent and persistent of the BoNTs is the "A" serotype, with strategies to counter its etiology focused on designing small-molecule inhibitors of its light chain (LC), a zinc-dependent metalloprotease. The successful structure-based drug design of inhibitors has been confounded as the LC is highly flexible with significant morphological changes occurring upon inhibitor binding. To achieve greater success, previous and new cocrystal structures were evaluated from the standpoint of inhibitor enantioselectivity and their effect on active-site morphology. Based upon these structural insights, we designed inhibitors that were predicted to take advantage of π-π stacking interactions present in a cryptic hydrophobic subpocket. Structure-activity relationships were defined, and X-ray crystal structures and docking models were examined to rationalize the observed potency differences between inhibitors.

Irreversible inhibition of BoNT/A protease: proximity-driven reactivity contingent upon a bifunctional approach.

Botulinum neurotoxin A (BoNT/A) is categorized as a Tier 1 bioterrorism agent and persists within muscle neurons for months, causing paralysis. A readily available treatment that abrogates BoNT/A's toxicity and longevity is a necessity in the event of a widespread BoNT/A attack and for clinical treatment of botulism, yet remains an unmet need. Herein, we describe a comprehensive warhead screening campaign of bifunctional hydroxamate-based inhibitors for the irreversible inhibition of the BoNT/A light chain (LC). Using the 2,4-dichlorocinnamic hydroxamic acid (DCHA) metal-binding pharmacophore modified with a pendent warhead, a total of 37 compounds, possessing 13 distinct warhead types, were synthesized and evaluated for time-dependent inhibition against the BoNT/A LC. Iodoacetamides, maleimides, and an epoxide were found to exhibit time-dependent inhibition and their k GSH measured as a description of reactivity. The epoxide exhibited superior time-dependent inhibition over the iodoacetamides, despite reacting with glutathione (GSH) 51-fold slower. The proximity-driven covalent bond achieved with the epoxide inhibitor was contingent upon the vital hydroxamate-Zn2+ anchor in placing the warhead in an optimal position for reaction with Cys165. Monofunctional control compounds exemplified the necessity of the bifunctional approach, and Cys165 modification was confirmed through high-resolution mass spectrometry (HRMS) and ablation of time-dependent inhibitory activity against a C165A variant. Compounds were also evaluated against BoNT/A-intoxicated motor neuron cells, and their cell toxicity, serum stability, and selectivity against matrix metalloproteinases (MMPs) were characterized. The bifunctional approach allows the use of less intrinsically reactive electrophiles to intercept Cys165, thus expanding the toolbox of potential warheads for selective irreversible BoNT/A LC inhibition. We envision that this dual-targeted strategy is amenable to other metalloproteases that also possess non-catalytic cysteines proximal to the active-site metal center.