Publications

Stereochemistry-Guided Switch from Irreversible to Slow-Binding

Cathepsin L Inhibition

Adrián Fernández-de-la-Pradilla, Jure Loboda, Katarina Karničar, Dusan Turk, Katarzyna Świderek,*

Florenci V. González,* and Vicent Moliner*

ACS Catalysis 2026, 26(10), 9008-9023.

Due to its involvement in a range of disease-related processes, such as cancer metastasis, viral entry, and immune dysregulation, cathepsin L (CatL), a lysosomal cysteine protease, has been proposed as an attractive therapeutic target. In this work, we present the design, synthesis, and mechanistic investigation of two epoxyketone-based dipeptidyl CatL inhibitors (D1 and D2) and two azido-based derivatives (AZA and AMK). From a combination of experimental and computational studies, we have demonstrated the importance of epoxide stereochemistry and azido functionality in determining the mode of inhibition and reactivity. Classical and hybrid QM/MM molecular dynamics (MD) simulations show that only (R)-epoxide (D1) occupies reactive conformations with respect to the catalytic triad (Cys25−His163−Asn187), leading to irreversible covalent inactivation via nucleophilic attack at the epoxide functionality. (S)-Epoxide (D2), on the other hand, occupies non-reactive conformations but can reach a less stable“flipped” conformation, suggesting a very slow SN2-type attack at the carbon adjacent to the azido moiety. The substitution of the epoxide with an azidomethyl ketone (AMK) preserved slow-binding covalent inhibition; however, exploration of the free energy landscape revealed a lower activation barrier, consistent with the retention of activity. Kinetic, mass spectrometry (MS), saturation transfer diﬀerence (STD) NMR, and thermal-shift studies support the computational predictions and show how slight modifications to the stereochemistry and the warhead of

the inhibitor can tune CatL activity from fully irreversible to slow-binding reversible inhibition. These results provide a rational framework for designing covalent inhibitors with tunable residence times and dual reactivity toward CatL that are important in cancer progression and in viral processes.

Macrocycles and stapled-peptides in the fight against SARS-CoV-2: a review

Santo Previti,* Elsa Calcaterra, Florenci V. González, Carla Di Chio, Maria L. Calabrò, Roberta Ettari, Maria Zapallo

European Journal of Medicinal Chemistry 2026, 312, 118828

The development of innovative therapeutic strategies for challenging biological targets has led to a resurgence of interest in macrocyclic and peptide-stapled compounds. The conformationally constrained architectures of these compounds enable high affinity, selectivity, and improved pharmacokinetic profiles compared with linear analogues. These properties position macrocycles and stapled-peptides as promising platforms for the development of next-generation therapeutics, including antiviral agents addressing emerging diseases such as COVID-19. In six years, the scientific community has provided several structure-activity relationship studies in which the anti- SARS-CoV-2 effects of macrocycles and stapled-peptides have been reported. The present review aims to discuss macrocycles and stapled-peptides with inhibitory properties against SARS-CoV-2 infection. A particular focus has been addressed to the design of cyclic entities, effect of ring size, presence of unnatural amino acids, stapling position, stereochemistry, role of linkers, pan-antiviral effects, metabolic stability assessment, and selectivity profile, among others. Comparisons with linear counterparts were discussed, wherever applicable, to elucidate the differences in terms of biological properties towards the target, antiviral effects in cell-based assays, molecular architecture, and proteolytic resistance. Overall, the development of macrocycles and stapled-peptides against SARS-CoV-2 was found to be a productive strategy for the identification of novel and effective antiviral agents. Furthermore, future challenges and perspectives have been discussed.

Design, synthesis and biological evaluation of peptidyl keto vinyl sulfones

as cysteine proteases inhibitors. Effect of the peptide structure on

the activity

Sergio de la Hoz-Rodríguez, Santiago Royo, Christian Kersten, Tanja Schirmeister, Santiago Rodríguez, Gemma Bou-Plaza, Cristian Vicent, Florenci V. González*

Bioorganic Chemistry 2025, 176, 109831

A family of fifteen peptidyl keto vinyl sulfones have been prepared and assayed against the cysteine proteases

human cathepsin L, parasitic rhodesain and Mpro from SARS-CoV-2 virus. The key synthetic step for the preparation

of the keto vinyl sulfone moiety of the inhibitors was achieved through a Nozaki-Hiyama-Kishi (NHK)

reaction between amino aldehydes and (E)-β-iodo(vinyl)sulfones. Inhibitors differ on the peptide sequence and/

or the substituent of the sulfone moiety. Although the inhibitors with a hydrophobic residue at P1 site were

initially prepared to inhibit rhodesain and cathepsin L both belonging to papain family, and the ones with a

glutamine surrogate at P1 site were made for SARS-CoV-2 Mpro, unexpected results were obtained. Interestingly,

the most active inhibitors against Mpro were the ones with Leu at P1 site. Molecular modelling studies suggest the

vinyl sulfone moiety to occupy the S1’ or S1 site of Mpro acting as a glutamine surrogate. Interestingly, hit

compounds are potent dual inhibitors with nanomolar activity against SARS-CoV-2 Mpro (Ki* =75.2–2670 nM)

and subnanomolar activity (Ki* =0.46–678 nM) against human cathepsin L.

Peptidyl keto vinyl amides as improved inhibitors of cathepsin L

Adrián Fernández-de-la-Pradilla, Clara Quílez, Tadej Ursic, Katarina Karnicar,

Gasper Turk, Dusan Turk, Katarzyna Swiderek,* Florenci V. González,* Vicent Moliner*

Bioorganic and Medicinal Chemistry 2025, 137, 118609

Cysteine protease cathepsin L (CatL) is linked to cancer, infection, and neurodegeneration, rendering it a

compelling target for therapy. Expanding upon dipeptidyl enoates (keto vinyl esters, KVE), we introduce

dipeptidyl keto vinyl amides (KVA) as a more stable, tunable Michael-acceptor scaffold. A first set of compounds

(1¡11), that utilized a Z-Leu-Ala backbone, was synthesized based on previous MD simulations and docking

results to prioritize the variants for S′-subsite engagement. Fluorometric assays using recombinant CatL showed

that all behaved as purely reversible inhibitors (non-time-dependent) with low-micromolar to mid-nanomolar Ki

values from 1.47 μM to 116 nM, with KVA outperforming KVE. A second set of more hydrophobic compounds

(42–43) was synthesized on Z-Phe-Leu and provided low nanomolar Ki values. Then, a series of long MD simulations

was done to rationalize the kinetics. Computational results show that Cys25 is close to the warhead,

while hydrogen-bonding to the oxyanion hole (Gln19) and contacts with Asp162 and Gly68, all stabilize binding.

KVA 4 (Leu-Ala, 2,2-difluoroethyl) exhibited the most consistent specific interaction, in agreement with the

lowest experimentally determined Ki values, providing a favorable starting point for further development.

Overall, KVAs emerge as stable, potent CatL inhibitors and a promising platform for selectivity and in cellulo

optimization.

FGA139, a novel cysteine protease inhibitor, exhibits anti-inflammatory and neuroprotective activity and reveals microglial modulation via multi-omics profiling
Ania Canseco-Rodríguez, Florenci V. González, Ana Sánchez-Pérez
Life Sciences 2025, 379, 123880.

Neuroinflammation is a key driver in the progression of numerous brain disorders, with cysteine proteases such as calpains, caspases, and cathepsins playing central roles in inflammatory signaling.

This study investigates FGA139, a novel irreversible inhibitor targeting cysteine proteases. We evaluated the anti-inflammatory properties of FGA139 in lipopolysaccharide (LPS)-activated macrophages (RAW264.7) and microglia (HMC3). In addition, its neuroprotective effects were assessed in differentiated SH-SY5Y neuron-like cells exposed to conditioned media (CM+) derived from the activated immune cells.

FGA139 exhibited a favorable safety profile and robust anti-inflammatory activity, significantly reducing nitric oxide (NO) production in macrophages and TNFα levels in microglia. Conditioned media from both LPS-stimulated immune cells lines (CM+) reduced neurite length in neuronal cells. However, CM+ from HMC3 cells impaired neuronal viability, whereas CM+ from RAW264.7 cells elevated reactive oxygen species (ROS) and NO levels—indicating distinct neurotoxic signatures. Preincubation of neuron-like cells with FGA139 effectively mitigated most of these adverse effects.

Metabolomic analysis of the activated microglia supernatant revealed that FGA139 increased extracellular levels of neuroprotective metabolites, including purines, linoleic acid, and phenyllactic acid. Proteomic data confirmed that FGA139 attenuated M1-like microglial polarization, likely through modulation of pathways associated with zinc transport and vesicle trafficking.

In conclusion, FGA139 demonstrates potent neuroprotective effects and modulates microglial activation. These findings uncover novel mechanisms underlying the beneficial effects of cysteine protease inhibition and support the therapeutic potential of FGA139 in treating neuroinflammatory conditions, positioning it as a promising modulator of microglial function.

Profiling Cysteine Proteases in Neuroinflammatory Cells
Laura Agost-Beltrán, Ania Canseco-Rodríguez, Tanja Schirmeister, Santiago Rodríguez, Ana Sánchez-Pérez, Florenci V. González
ChemMedChem 2024, e202400520.

A new activity-based probe (ABP) for cysteine proteases, FGA139, was designed by attaching a bodipy fluorophore to an irreversible inhibitor's peptide backbone. The probe is synthesized through metathesis and “click” reactions. It profiles activities of cathepsins B, L, and calpain in neurodegenerative cell models, confirming specificity and showing protective effects against oxidative stress and macrophage activation.

Rhodesain inhibitors at the edge of reversibility-irreversibility
Laura Agost-Beltrán, Collin Zimmer, Hans Joachim Räder, Cristian Kernsten, Tanja Schirmeister, Santiago Rodríguez, Florenci V. González
Bioorganic Chemistry 2024, 153, 107830.

A comparative study of Michael acceptor and keto-Michael acceptor inhibitors of the cysteine protease rhodesain has been performed. Five new inhibitors have been prepared bearing the peptide structure of the known cysteine protease inhibitor K11777 and differing on the warhead. For the preparation of the Michael acceptor warhead, a Horner-Wadsworth-Emmons reaction was used. In the synthetic routes of the keto-Michael acceptor warheads, keto-enoate and keto-vinyl sulfone, a metathesis reaction and a radical sulfonylation were the key steps, respectively. Interestingly, keto-Michael acceptors inhibited rhodesain through a dual mode of action, showing reversibility at low inhibitor concentrations and irreversibility at high inhibitor concentrations.

Peptidyl nitroalkene inhibitors of main protease rationalized by computational and crystallographic investigations as antivirals against SARS-CoV-2
Francisco J. Medrano, Sergio de la Hoz-Rodríguez, Sergio Martí, Kemel Arafat, Tanja Schirmeister, Stefan J. Hammerschmidt, Christin Müller, Águeda González-Martínez, Elena Santillana, John Ziebuhr, Antonio Romero, Collin Zimmer, Annabelle Welder, Robert Zimmermann, Alessio Lodola, Katarzyna Swiderek, Vicent Moliner, Florenci V. González
Commun. Chem. 2024, 7, 15.

The coronavirus disease 2019 (COVID-19) pandemic continues to represent a global public health issue. The viral main protease (Mpro) represents one of the most attractive targets for the development of antiviral drugs. Herein we report peptidyl nitroalkenes exhibiting enzyme inhibitory activity against Mpro (Ki: 1–10 μM) good anti-SARS-CoV-2 infection activity in the low micromolar range (EC50: 1–12 μM) without significant toxicity. Additional kinetic studies of compounds FGA145, FGA146 and FGA147 show that all three compounds inhibit cathepsin L, denoting a possible multitarget effect of these compounds in the antiviral activity. Structural analysis shows the binding mode of FGA146 and FGA147 to the active site of the protein. Furthermore, our results illustrate that peptidyl nitroalkenes are effective covalent reversible inhibitors of the Mpro and cathepsin L, and that inhibitors FGA145, FGA146 and FGA147 prevent infection against SARS-CoV-2.

Transcription factors HB21/40/53 trigger inflorescence arrest through abscisic acid accumulation at the end of flowering
Verónica Sánchez-Gerschon, Irene Martínez-Fernández, María R. González-Bermúdez, Sergio de la Hoz-Rodríguez, Florenci V. González, Jorge Lozano-Juste, Cristina Ferrándiz, and Vicente Balanzá
Plant Physiology 2024, 195, 2743.

Flowers, and hence, fruits and seeds, are produced by the activity of the inflorescence meristem after the floral transition. In plants with indeterminate inflorescences, the final number of flowers produced by the inflorescence meristem is determined by the length of the flowering period, which ends with inflorescence arrest. Inflorescence arrest depends on many different factors, such as the presence of seeds, the influence of the environment, or endogenous factors such as phytohormone levels and age, which modulate inflorescence meristem activity. The FRUITFULL-APETALA2 (FUL-AP2) pathway plays a major role in regulating the end of flowering, likely integrating both endogenous cues and those related to seed formation. Among AP2 targets, HOMEOBOX PROTEIN21(HB21) has been identified as a putative mediator of AP2 function in the control of inflorescence arrest. HB21 is a homeodomain leucine zipper transcription factor involved in establishing axillary bud dormancy. Here, we characterized the role of HB21 in the control of the inflorescence arrest at the end of flowering in Arabidopsis (Arabidopsis thaliana). HB21, together with HB40 and HB53, are upregulated in the inflorescence apex at the end of flowering, promoting floral bud arrest. We also show that abscisic acid (ABA) accumulation occurs in the inflorescence apex in an HB-dependent manner. Our work suggests a physiological role of ABA in floral bud arrest at the end of flowering, pointing to ABA as a regulator of inflorescence arrest downstream of the HB21/40/53 genes.

Impact of the Warhead of Dipeptidyl Keto Michael Acceptors on the inhibition Mechanism of Cysteine Protease Cathepsin L
Adrián Fernández-de-la-Pradilla, Santiago Royo, Tanja Schirmeister, Fabian Barthels, Katarzyna Swiderek, Florenci V. González, Vicent Moliner
ACS Catalysis 2023, 13, 13354-13368.

Cathepsin L (CatL) is a lysosomal cysteine protease whose activity has been related to several human pathologies. However, although preclinical trials using CatL inhibitors were promising, clinical trials have been unsuccessful up to now. We are presenting a study of two designed dipeptidyl keto Michael acceptor potential inhibitors of CatL with either a keto vinyl ester or a keto vinyl sulfone (KVS) warhead. The compounds were synthesized and experimentally assayed in vitro, and their inhibition molecular mechanism was explored based on molecular dynamics simulations at the density functional theory/molecular mechanics level. The results confirm that both compounds inhibit CatL in the nanomolar range and show a time-dependent inhibition. Interestingly, despite both presenting almost equivalent equilibrium constants for the reversible formation of the noncovalent enzyme/inhibitor complex, differences are observed in the chemical step corresponding to the enzyme–inhibitor covalent bond formation, results that are mirrored by the computer simulations. Theoretically determined kinetic and thermodynamic results, which are in very good agreement with the experiments, afford a detailed explanation of the relevance of the different structural features of both compounds having a significant impact on enzyme inhibition. The unprecedented binding interactions of both inhibitors in the P1′ site of CatL represent valuable information for the design of inhibitors. In particular, the peptidyl KVS can be used as a starting lead compound in the development of drugs with medical applications for the treatment of cancerous pathologies since sulfone warheads have previously shown promising cell stability compared to other functions such as carboxylic esters. Future improvements can be guided by the atomistic description of the enzyme–inhibitor interactions established along the inhibition reaction derived from computer simulations.

Investigation of the Compatibility between Warheads and Peptidomimetic Sequences of Protease Inhibitors - A Comprehensive Reactivity and Selectivity Study
Patrick Müller, Mergim Meta, Jan Laurenz Meidner, Marvin Schwickert, Jessica Meyr, Kevin Schwickert, Christian Kersten, Collin Zimmer, Stefan Josef Hammerschmidt, Ariane Frey, Albin Lahu, Sergio de la Hoz-Rodríguez, Laura Agost-Beltrán, Santiago Rodríguez, Kira Diemer, Wilhelm Neumann, Florenci V. González, Bernd Engels, Tanja Schirmeister
Internacional Journal of Molecular Sciences 2023, 24(8), 7226.

Covalent peptidomimetic protease inhibitors have gained a lot of attention in drug development in recent years. They are designed to covalently bind the catalytically active amino acids through electrophilic groups called warheads. Covalent inhibition has an advantage in terms of pharmacodynamic properties but can also bear toxicity risks due to non-selective off-target protein binding. Therefore, the right combination of a reactive warhead with a well-suited peptidomimetic sequence is of great importance. Herein, the selectivities of well-known warheads combined with peptidomimetic sequences suited for five different proteases were investigated, highlighting the impact of both structure parts (warhead and peptidomimetic sequence) for affinity and selectivity. Molecular docking gave insights into the predicted binding modes of the inhibitors inside the binding pockets of the different enzymes. Moreover, the warheads were investigated by NMR and LC-MS reactivity assays against serine/threonine and cysteine nucleophile models, as well as by quantum mechanics simulations.