Mark S. Hixon

Loss of the functional Adenomatous Polyposis Coli (APC-LOF) tumor suppressor gene represents the disease-initiating event in most colorectal cancer (CRC) cases. A newly identified dependency between PRMT5 and APC-LOF suggests that inhibiting PRMT5 may help intercept CRC. To circumvent hematological toxicities associated with orally bioavailable first-generation PRMT5 inhibitors, we aimed to limit systemic exposure after oral administration. We describe our efforts, challenges, and compound evaluation workflow resulting in gut-restricted PRMT5 inhibitors. A two-pronged approach was envisioned, consisting of (1) minimizing passive absorption, and (2) maximizing systemic clearance by incorporation of a metabolic “soft spot”. This resulted in 9 and 18, displaying low absorption in preclinical species and high first-pass extraction mediated by aldehyde oxidase. 9 and 18 demonstrated in vivo colon pharmacodynamics without signs of systemic on-target toxicity, confirming gut-restriction. Administering 9 to dextran sodium sulfate (DSS)-treated polyp-bearing ApcMin/+ mice significantly reduced polyp number, indicating local treatment efficacy.

As many prophylactics targeting SARS-CoV-2 are aimed at the spike protein receptor-binding domain (RBD), we examined the risk of immune evasion from previously published RBD-targeting neutralizing antibodies (nAbs). Epitopes for RBD-targeting nAbs overlap one another substantially and can give rise to escape mutants with ACE2 affinities comparable to wild type that still infect cells in vitro . We used evolutionary modeling to predict the frequency of immune escape before and after the widespread presence of nAbs due to vaccines, passive immunization or natural immunity. Our modeling suggests that SARS-CoV-2 mutants with one or two mildly deleterious mutations are expected to exist in high numbers due to neutral genetic variation, and consequently resistance to single or double antibody combinations can develop quickly under positive selection. One Sentence Summary: SARS-CoV-2 will evolve quickly to evade widely deployed spike RBD-targeting monoclonal antibodies, requiring combinations that rely on at least three antibodies targeting distinct epitopes to suppress viral immune evasion.

Clostridioides difficile infection (CDI) causes serious and sometimes fatal symptoms like diarrhea and pseudomembraneous colitis. Although antibiotics for CDI exist, they are either expensive or cause recurrence of the infection due to their altering the colonic microbiota which is necessary to suppress the infection. Here, we leverage a class of known membrane-targeting compounds that we previously showed to have broad inhibitory activity across multiple C. difficile strains while preserving the microbiome to develop an efficacious agent. A new series of salicylanilides was synthesized and the most potent analog was selected through an in vitro inhibitory assay to evaluate its pharmacokinetic parameters and potency in a CDI mouse model. The results revealed reduced recurrence of CDI and disturbance of the microbiota in mice compared to standard-of-care vancomycin, thus paving the way for novel therapy that can potentially target the cell membrane of C. difficile to minimize relapse in the recovering patient.

Benzamide antipsychotics such as amisulpride are dosed as racemates though efficacy is assumed to be mediated through S enantiomer binding to D₂ receptors. At prescribed doses, the benzamides likely display polypharmacy since brain exposure should be sufficient to engage the 5-HT₇ receptors, as well. Curiously, the studies herein reveal that racemic dosing is required to engage both targets since the D₂ receptor has an almost 40-fold selectivity for the S enantiomer, while the 5-HT₇ receptor has greater than 50-fold preference for the R enantiomer.

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.

The parasitic disease onchocerciasis is the second leading cause of preventable blindness, afflicting more than 18 million people worldwide. Despite an available treatment, ivermectin, and control efforts by the World Health Organization, onchocerciasis remains a burden in many regions. With an estimated 120 million people living in areas at risk of infection, efforts are now shifting from prevention to surveillance and elimination. The lack of a robust, point-of-care diagnostic for an active Onchocerca infection has been a limiting factor in these efforts. Previously, we reported the discovery of the biomarker N-acetyl-tyramine-O-glucuronide (NATOG) in human urine samples and its ability to track treatment progression between medicated patients relative to placebo; we also established its capability to monitor disease burden in a jird model. NATOG is a human-produced metabolite of tyramine, which itself is produced as a nematode neurotransmitter. The ability of NATOG to distinguish between active and past infection overcomes the limitations of antibody biomarkers and PCR methodologies. Lateral flow immunoassay (LFIA) diagnostics offer the versatility and simplicity to be employed in the field and are inexpensive enough to be utilized in large-scale screening efforts. Herein, we report the development and assessment of a NATOG-based urine LFIA for onchocerciasis, which accurately identified 85% of analyzed patient samples (N = 27).

Receptor tyrosine kinase therapies have proven to be efficacious in specific cancer patient populations; however, a significant limitation of tyrosine kinase inhibitor (TKI) treatment is the emergence of resistance mechanisms leading to a transient, partial, or complete lack of response. Combination therapies using agents with synergistic activity have potential to improve response and reduce acquired resistance. Chemoreagent or TKI treatment can lead to increased expression of hepatocyte growth factor (HGF) and/or MET, and this effect correlates with increased metastasis and poor prognosis. Despite MET's role in resistance and cancer biology, MET TKI monotherapy has yielded disappointing clinical responses. In this study, we describe the biological activity of a selective, oral MET TKI with slow off-rate and its synergistic antitumor effects when combined with an anti-HGF antibody. We evaluated the combined action of simultaneously neutralizing HGF ligand and inhibiting MET kinase activity in two cancer xenograft models that exhibit autocrine HGF/MET activation. The combination therapy results in additive antitumor activity in KP4 pancreatic tumors and synergistic activity in U-87MG glioblastoma tumors. Pharmacodynamic characterization of biomarkers that correlate with combination synergy reveal that monotherapies induce an increase in the total MET protein, whereas combination therapy significantly reduces total MET protein levels and phosphorylation of 4E-BP1. These results hold promise that dual targeting of HGF and MET by combining extracellular ligand inhibitors with intracellular MET TKIs could be an effective intervention strategy for cancer patients who have acquired resistance that is dependent on total MET protein. Mol Cancer Ther; 16(7); 1269–78. ©2017 AACR.

Serine hydrolases are susceptible to potent reversible inhibition by boronic acids. Large collections of chemically diverse boronic acid fragments are commercially available because of their utility in coupling chemistry. We repurposed the approximately 650 boronic acid reagents in our collection as a directed fragment library targeting serine hydrolases and related enzymes. Highly efficient hits (LE > 0.6) often result. The utility of the approach is illustrated with the results against autotaxin, a phospholipase implicated in cardiovascular disease.