Margaret E. Olson

Cytidine analogues have conferred highly efficacious antimetabolites with broad utility as antiviral and anticancer agents. However, in many cases, human cytidine deaminase (CDA) converts the cytidine-based inhibitor into an inactive uridine metabolite with diminished potency. Inhibitors of CDA are useful agents to boost the efficacy of cytosine- and cytidine-containing drugs by inhibiting their rapid degradation. Toward the goal of developing CDA inhibitors, and our overarching interest in cytosine deaminase enzymes in general, we developed a real-time fluorescence-based deamination activity assay for CDA using isomorphic nucleoside analogues. Base-modified pyrimidine nucleosides that exhibit differential fluorescence properties as either the cytosine or uracil nucleobase were developed. We found that 5-benzo-2-furyl-2'-deoxycytidine is the best fluorescence reporter when implemented in a CDA enzyme activity assay, which permits detailed measurements of the kinetics of CDA activity in the presence or absence of inhibitors. Utilizing this assay, we then screened our in-house collection of 1054 fragments and found 23 hits that were further studied. Two fragment-sized CDA inhibitors with low micromolar potency (200-300 μM) and good ligand efficiency (>0.3) were identified, thereby conferring promising starting points for future inhibitor development.

On the occasion of the 2023 International Women's Day on March 8, 2023, we want to celebrate and highlight the contributions of many women volunteers in the American Chemical Society Division of Medicinal Chemistry (ACS MEDI).

On the occasion of the 2023 International Women's Day on March 8, 2023, we want to celebrate and highlight the contributions of many women volunteers in the American Chemical Society Division of Medicinal Chemistry (ACS MEDI).

The Young Medicinal Chemists Committee (YMCC) is a part of the larger ACS Division of Medicinal Chemistry(MEDI) and was formed to ensure that MEDI meets the needs ofallmedicinal chemists, including students and early careerscientists. There is a clear need to offer additional, specific opportunities to this group of medicinal chemists within the MEDIdivision. Primary functions of YMCC include facilitating networking and mentorship opportunities, collaborating with internationalmedicinal chemistry societies, and offering social programming for all MEDI members at ACS National Meetings. We are excited tocontinue to engage students and early career chemists through new initiatives and programming in the future. In this Editorial wehighlight current initiatives relevant to early career medicinal chemists and solicit input from the larger medicinal chemistry community.

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.

We describe a comprehensive screening campaign of warheads, linked to a hydroxamate chelating anchor, for the modification of Cys165 within the BoNT/A protease. Engaging thorough enzyme kinetics, we detail a remarkable proximity-driven covalent bond with an epoxide warhead, a weak electrophile; yet, one that possessed superior irreversible inhibition, and pharmacological properties, when compared to intrinsically higher reactive warheads. This directed, selective covalent bond was contingent upon the crucial hydroxamate-Zn2+ chelating interaction as exemplified by examining non-chelating compounds. We discuss previous approaches using non-target selective cysteine-reactive warheads to modify the BoNT/A protease of which none present any therapeutic potential – our bifunctional strategy allows the use of intrinsically less reactive warheads to intercept the cysteine, which will allow for less off-target modifications of such inhibitors. Moreover, we also broach that this bifunctional approach is not a one-off strategy that we believe can be broadly translated to other metalloproteases that possess non-catalytic, yet, nucleophilic residues within the enzymes catalytic sphere.

Hepatitis C virus (HCV) infections represent a global health challenge; however, developing a vaccine for treatment of HCV infection has remained difficult as heterogeneous HCV contains distinct genotypes, and each genotype contains various subtypes and different envelope glycoproteins. Currently, there is no effective preventive vaccine for achieving global control over HCV. In our efforts to improve upon current HCV vaccines we designed a synthetically accessible adjuvant platform, wherein we synthesized 11 novel lipidated tucaresol analogues to assess their immunological potential. Using a tucaresol-based adjuvant approach, truncated lipid-variants together with an engineered E1E2 antigen construct, namely E2ΔTM3, elicited antibody (Ab) responses that were significantly higher than tucaresol. In sum, antibody end-point titer values largely corroborated HCV neutralization data with a simplified lipidated tucaresol variant affording the highest end point titer and % neutralization. This study lays the groundwork for additional permutations in tucaresol adjuvant design, including the examination of other proteins in vaccine development.

Botulinum neurotoxins have remarkable persistence (~weeks to months in cells), outlasting the small molecule inhibitors designed to target them. To address this disconnect, inhibitors bearing two pharmacophores — a zinc binding group and a Cys-reactive warhead — were designed to leverage both affinity and reactivity. A series of 1st generation bifunctional inhibitors was achieved through structure-based inhibitor design. Through X-ray crystallography, engagement of both the catalytic Zn2+ and Cys165 was confirmed. A 2nd generation series improved on affinity by incorporating known reversible inhibitor pharmacophores; the mechanism was confirmed by exhaustive dialysis, mass spectrometry, and in vitro evaluation against the C165S mutant. Finally, a 3rd generation inhibitor was shown to have good cellular activity and low toxicity. In addition to our findings, an alternative method of modeling time-dependent inhibition that simplifies assay set-up and allows comparison of inhibition models is discussed.