Abstract The tumor suppressor p53 is inactivated in approximately 50% of human cancers. In about 1% of solid tumors, a specific Y220C point mutation induces p53 misfolding, leading to its inactivation. This mutation also creates a unique, druggable pocket on the protein's surface, making it a promising target for cancer therapy. The first-generation small molecule reactivator (PC14586, Rezatapopt), designed to bind to this pocket, to refold p53 and to restore its tumor-suppressive functions, has shown clinical efficacy in patients harboring the Y220C mutations. However, this first-generation compound is limited by modest potency, necessitating high dosing in patients. This high-dose requirement increases the risk of adverse events and narrows the therapeutic window, as monotherapy and even more so in combination regimens. As a result, this compound fails to deliver the drug pressure needed to reach full reactivation of this critical tumor suppressor. Thus, there is a clear need to discover and develop a highly potent p53 small molecule reactivator to fully unlock the therapeutic potential of this promising target. At Onco3R Therapeutics, our patient centric approach, integrating deep translational science with rational, structure-based and AI-augmented drug design, has led to the identification of best-in-class series of p53 Y220C reactivators. Our lead series exhibits more than 200-fold superior potency compared to the clinical compound PC14586 (Rezatapopt) across diverse preclinical assays, including biochemical p53 refolding, cellular p53 refolding, and cellular p21 induction. Importantly, our lead series, characterized by strong potency and long residence times in vitro, significantly sustains target engagement and P53 reactivation in cells. This profile enables robust activation of the pro-apoptotic protein PUMA and induces potent, selective cytotoxicity in p53 Y220C mutant cells and in an isogenic cell model. In line with their superior cellular activity and in combination with optimized key ADME, safety parameters, and favorable in vivo PK profiles across species, our leads reach similar efficacy in vivo as PC145586 at much lower exposure. In conclusion, we have identified unique p53 Y220C small molecule reactivators with clear best-in-class cellular potency and favorable drug-like properties. We are currently further profiling these leads as potential drug candidates to achieve superior efficacy at substantially lower doses, maximizing the safety window to ultimately deliver better outcomes to cancer patients with p53 Y220C mutations. Citation Format: Ruben Boon, Jo Alen, Nihed Draoui, Koen Vandyck, Elke Behaeghel, Godelieve Lammens, Sandrine Vendeville, Lijs Beke, Pieter Peeters, Stéphane De Cesco, Bart Stoops, Pierre Raboisson, Francois Gonzalvez, Carla De Legher. Discovery of a Best-in-Class small molecule p53 Y220C reactivator: Breaking through the potency ceiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4578.

Orthoflaviviruses, such as dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV), constitute a significant public health concern with billions of people at risk of infection. Climate change and the expanding geographical distribution of mosquito vectors transmitting orthoflaviviruses have increased their potential to cause large-scale disease outbreaks. The frequency and severity of disease outbreaks highlight the urgent need for a broad-spectrum antiviral agent targeting orthoflaviviruses. In this work, we conducted a comprehensive morphological profiling of approximately 200,000 small molecules through a fluorescence-based high-content imaging platform, which led to the identification of a singular small molecule exhibiting broad-spectrum activity against orthoflaviviruses. Subsequent hit deconvolution against DENV serotype 2 (DENV-2) revealed NS2A protein as a novel therapeutic target. Mechanistically, JNJ-1953 inhibits viral RNA synthesis, as demonstrated by robust reductions in intracellular viral RNA and infectious virus production. Additional experiments show that JNJ-1953 further impacts viral RNA packaging and interferes with the interaction between NS2A and prM, rendering the molecule a multimodal inhibitor.

Influenza continues to be a major threat to global health and a substantial economic burden. Innovative strategies are needed to tackle the growing resistance to established influenza therapeutics and to develop new therapeutics with novel mechanisms of action. Previous peptide and small molecule designs have been successful only against influenza group 1 hemagglutinin (HA). Here, we report on a CLIPS (Chemical Linkage of Peptides onto Scaffolds)-based approach to design potent peptidic inhibitors of influenza A viruses that now extend to both group 1 and group 2 HAs. This approach merges features of antibodies and small molecules to design constrained bicyclic peptides that engage the highly conserved HA stem. The heavy-chain complementarity-determining region 3 (HCDR3) of human broadly neutralizing antibody FI6v3 was grafted onto functionalized small molecule scaffolds. The designed peptides exhibited in vitro heterosubtypic cross-reactivity in binding to group 1 (H1 and H5) and group 2 (H3 and H7) HAs and in neutralization of H1N1, H5N1, and H7N3 viruses. A crystal structure of the bicyclic peptide with HA from H1N1 A/Puerto Rico/8/1934 (H1/PR8) at 2.35 Å resolution revealed that the designed peptide faithfully mimics the binding mode and functionality of the parent antibody FI6v3 to the highly conserved stem epitope. These structural and functional data illustrate how both group 1 and group 2 influenza A viruses can now be targeted by constrained peptidic ligands that should aid in development of pan-influenza therapeutics.

Abstract The development of novel therapeutics for infectious diseases remains a global health priority. To accelerate the treatment development, innovative strategies through new approach methodology (NAM) are needed to bridge speed of in vitro with predictive power of in vivo studies, while reducing mammalian experiments. The zebrafish ( Danio rerio ), particularly the embryo/larva, has been established as a valuable non-mammalian in vivo model in biomedical research. We developed a standardized and streamlined workflow for the zebrafish as NAM, which consisted of 3 steps: drug selection and efficacy evaluation, internal exposure assessment, and PKPD modelling. Compounds with higher tolerated doses than minimum inhibitory concentration were selected. Drug efficacy was quantified through longitudinal individual fluorescence microscopy at baseline and 24 and 48h on treatment. Drug exposure was quantified in larval homogenates and exposure medium from 0-48h on treatment. The PKPD relationship was quantified by non-linear mixed effects modelling. For case study bedaquiline, PKPD was quantified using a one-compartment model with age-depending elimination, and an Emax concentration-response relationship on the delayed logistic bacterial growth function, with an EC50 of 26.6 µg/mL and an Emax of 1.07-1.37. In the case of clarithromycin, in contrast, negligible internal exposure after waterborne treatment were observed, illustrating the risk of false negatives without internal exposure assessments. Bactericidal efficacy was confirmed by intravenous drug injections, showing a clear dose dependent antimycobacterial effect. The standardized zebrafish NAM workflow presented here facilitates the translation of drug efficacy to higher vertebrates, reducing rodent studies to confirmatory or replacing them completely, thus accelerating drug development.

Tuberculosis (TB) remains the foremost cause of death from infectious diseases globally, prompting ongoing efforts to improve treatment options. This includes developing compounds with novel modes of action and identifying optimal treatment regimens that allow for treatment shortening. One promising strategy involves targeting cytochrome bc1 oxidase in Mycobacterium tuberculosis, a key enzyme in the respiratory chain. In this study, we evaluate the potential of cytochrome bc1 inhibitors as partner drugs in TB combination regimens. Using a relapsing mouse model, we demonstrate that these inhibitors enhance regimen sterilisation and significantly reduce the time required for effective treatment. We also propose several novel combination strategies for both multidrug-resistant and drug-sensitive TB, where cytochrome bc1 inhibitors contribute to sterilisation and improved treatment outcomes. Furthermore, M. tuberculosis clinical isolates exhibited heightened susceptibility to cytochrome bc1 inhibitors compared to laboratory-adapted strains, highlighting the importance of using clinical isolates in TB drug discovery to better reflect the diversity of TB populations. These findings emphasise the potential of cytochrome bc1 inhibition in the development of more effective and shorter treatment regimens for TB, supporting the need for further clinical investigation.

Miniaturizing biologically complex structural motifs to produce synthetic functional mimetics holds significant promise for development of new therapeutic modalities. Here, we demonstrate a unique approach using the key binding loop of the single variable domain of a heavy chain (VHH) llama antibody as a starting point for peptide design. VHH antibodies of camelids and sharks generally have longer, but more ligand-efficient complementarity determining region 3 (CDR3) loops and are relatively stable structures. We harnessed these attributes as templates for design of a series of synthetic macrocyclic peptides. The designed peptides exhibit nanomolar binding to influenza hemagglutinin (HA) and heterosubtypic in vitro neutralization breadth against influenza A viruses by inhibiting the low pH mediated HA conformational changes that lead to membrane fusion. X-ray structures of peptide-HA complexes reveal high structural mimicry with the parent VHH antibody. One such macrocycle peptide candidate is promising for further development of broad protection against influenza A group 1 viruses.

Tuberculosis remains the leading cause of death from an infectious disease1,2. Here we report the discovery of a first-in-class small-molecule inhibitor targeting PurF, the first enzyme in the mycobacterial de novo purine biosynthesis pathway. The lead candidate, JNJ-6640, exhibited nanomolar bactericidal activity in vitro. Comprehensive genetic and biochemical approaches confirmed that JNJ-6640 was highly selective for mycobacterial PurF. Single-cell-level microscopy demonstrated a downstream effect on DNA replication. We determined the physiologically relevant concentrations of nucleobases in human and mouse lung tissue, showing that these levels were insufficient to salvage PurF inhibition. Indeed, proof-of-concept studies using a long-acting injectable formulation demonstrated the in vivo efficacy of the compound. Finally, we show that inclusion of JNJ-6640 could have a crucial role in improving current treatment regimens for drug-resistant tuberculosis. Together, we demonstrate that JNJ-6640 is a promising chemical lead and that targeting de novo purine biosynthesis represents a novel strategy for tuberculosis drug development.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory tract infections in infants, young children, and the elderly. We report herein the discovery and characterization of a novel RSV polymerase (RSVpol) non-nucleoside inhibitor (NNI) chemotype that binds to a previously undescribed, highly conserved site in the palm domain of the L protein. Consistent with the observed mode of inhibition, cryogenic electron microscopy (cryo-EM) revealed the site to be adjacent to the nucleotide binding site. Minireplicon assays confirmed on-target activity against RSVpol, and cell-based antiviral assays showed that the lead compound effectively inhibited viral mRNA transcription and replication in clinically relevant A and B strains. Together, our data provides valuable insights into the molecular basis of inhibition for a novel mechanism of action and paves the way for structure-based design to deliver effective therapeutics against RSV.IMPORTANCERespiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus belonging to the family Pneumoviridae of the order Mononegavirales. Currently, monoclonal antibody treatments are only approved for infants, and vaccines are reserved for pregnant women and adults aged 60 years and older. Prophylaxis is also limited to the pediatric patient population, and there are currently no direct antiviral therapies for post-exposure treatment. Viral polymerases are considered well-validated drug targets due to their critical role in transcription and genome replication. Herein, we disclose the discovery of a spiro-indolinone series as polymerase inhibitors and describe the preliminary structure-activity relationship (SAR). A cryogenic electron microscopy (cryo-EM) structure obtained with an optimized lead revealed a novel binding site located in the palm domain, which will enable future structure-based drug design efforts. Novel RSV antivirals could be beneficial both as therapeutics following diagnosis and as a prophylactic in patients less likely to respond to vaccines.

Respiratory syncytial virus (RSV) remains a public health burden due to unmet therapeutic needs. We recently reported the discovery of a non-nucleoside inhibitor of the RSV polymerase and characterized its binding to a novel pocket within the capping domain of the polymerase. Here, we describe our strategy to diversify the chemical matter targeting this site by screening our DNA-encoded chemical libraries, leading to the discovery of a novel and potent series of molecules that inhibits RSV polymerase's biochemical activity, as well as its viral replication in cells. Structural analysis via cryo-EM revealed novel contacts made within the capping domain binding pocket. By leveraging these structural insights for preliminary SAR exploration, we generated analogues for which potency and metabolic stability were improved more than 60- and 40-fold, respectively, over the initial hit. This work provides a path forward for further advanced SAR exploration and development of therapeutics against RSV.

Recent efforts to improve tuberculosis (TB) treatment options have focused on developing molecules with novel mechanisms of action and identifying optimal treatment regimens. Inhibition of Mycobacterium tuberculosis cytochrome bc1 oxidase has emerged as a promising therapeutic target that could potentially contribute to improved TB combination regimens. Using a relapsing mouse model, we demonstrate that cytochrome bc1 inhibitors could serve as effective partner drugs, enhancing regimen sterilisation. We propose several novel regimen strategies for both multidrug-resistant TB (MDR-TB) and drug-sensitive TB (DS-TB), where cytochrome bc1 inhibitors contribute to sterilisation and treatment shortening. Additionally, we show that clinical isolates exhibit heightened susceptibility to cytochrome bc1 inhibitors compared to laboratory-adapted strains, further supporting their translational potential. These findings suggest that cytochrome bc1 inhibitors have significant potential to improve TB treatment outcomes and highlight the need for further studies to evaluate their clinical contribution to novel treatment regimens.

Despite the availability of medicines preventing respiratory syncytial virus (RSV) infection, post-exposure treatment options are needed for addressing patient's needs. RSV non-nucleoside polymerase inhibitors (NNI) have emerged as a promising asset for which our group previously disclosed JNJ-8003 with potent in vitro antiviral activity and pronounced in vivo efficacy. In this work, a structural-guided design to modify the linker vector of JNJ-8003 resulted in the identification of 2-oxacyclo pyridine-containing derivatives whose various ring closing strategies are described. In addition, bioisosteric replacement of an amide bond with triazole retained potency, and cryo-electron microscopy (cryo-EM) confirmed binding in the capping domain. Subsequent NMR conformational analysis suggested a correlation between the potency and conformations. Our efforts have fulfilled the aim of identifying linker modifications with maintained biological activity while enriching structural diversity and allowing modulations of other parameters.

Tuberculosis remains the leading cause of death from an infectious disease, responsible for 1.3 million deaths annually. Here, we report the discovery of a first-in-class small molecule inhibitor targeting PurF, the first enzyme in the mycobacterial de novo purine biosynthesis pathway. Our lead candidate, JNJ-6640, exhibited nanomolar bactericidal activity in vitro. Using an array of comprehensive genetic and biochemical approaches, we confirmed JNJ-6640 was highly selective for mycobacterial PurF compared to the human homologue. Single-cell level timelapse microscopy demonstrated PurF inhibition leads to a downstream impact on DNA replication. Proof-of-concept studies using a long-acting injectable formulation demonstrated the compound's in vivo efficacy. Finally, we show inclusion of JNJ-6640 could play a crucial role in improving the current treatment regimen for drug-resistant TB. Taken together, we demonstrate that JNJ-6640 is a promising chemical lead and show that targeting purine metabolism is a novel strategy for TB drug development.

Dengue is a global public health threat, with about half of the world's population at risk of contracting this mosquito-borne viral disease. Climate change, urbanization, and global travel accelerate the spread of dengue virus (DENV) to new areas, including southern parts of Europe and the US. Currently, no dengue-specific small-molecule antiviral for prophylaxis or treatment is available. Here, we report the discovery of JNJ-1802 as a potent, pan-serotype DENV inhibitor (EC50's ranging from 0.057 to 11 nM against the four DENV serotypes). The observed oral bioavailability of JNJ-1802 across preclinical species, its low clearance in human hepatocytes, the absence of major in vitro pharmacology safety alerts, and a dose-proportional increase in efficacy against DENV-2 infection in mice were all supportive of its selection as a development candidate against dengue. JNJ-1802 is being progressed in clinical studies for the prevention or treatment of dengue.

Dengue virus (DENV) is the most widespread mosquito-borne virus worldwide, but no antiviral therapies are available yet. The pan-serotype DENV inhibitor JNJ-A07 has shown potent activity in a mouse model. It remains unknown whether an antiviral drug ingested by mosquitoes could inhibit virus replication and thus reduce transmission to other hosts. Here, we investigated the antiviral activity of JNJ-A07 when administered in the blood meal to Aedes aegypti mosquitoes. JNJ-A07 blocked DENV-2 transmission by the mosquitoes in both pre-exposure and post-exposure settings. In addition, JNJ-A07 remained in the mosquito bodies for 7 days after blood meal. Reductions of DENV systemic infection in the mosquitoes suggested a potential for decreased proportions of DENV outbreaks in a simulated environment when the mosquitoes ingested JNJ-A07 via the blood meal.

Respiratory syncytial virus (RSV) is a major cause of hospitalization in infants, the elderly, and immune-compromised patients. While a half-life extended monoclonal antibody and 2 vaccines have recently been approved for infants and the elderly, respectively, options to prevent disease in immune-compromised patients are still needed. Here, we describe spiro-azetidine oxindoles as small molecule RSV entry inhibitors displaying favorable potency, developability attributes, and long-acting PK when injected as an aqueous suspension, suggesting their potential to prevent complications following RSV infection over a period of 3 to 6 months with 1 or 2 long-acting intramuscular (IM) or subcutaneous (SC) injections in these immune-compromised patients.

Respiratory syncytial virus (RSV) is an RNA virus infecting the upper and lower respiratory tract and is recognized as a major respiratory health threat, particularly to older adults, immunocompromised individuals, and young children. Around 64 million children and adults are infected every year worldwide. Despite two vaccines and a new generation monoclonal antibody recently approved, no effective antiviral treatment is available. In this manuscript, we present the medicinal chemistry efforts resulting in the identification of compound 28 (JNJ-8003), a novel RSV non-nucleoside inhibitor displaying subnanomolar activity in vitro as well as prominent efficacy in mice and a neonatal lamb models.

In the absence of any approved dengue-specific treatment, the discovery and development of a novel small-molecule antiviral for the prevention or treatment of dengue are critical. We previously reported the identification of a novel series of 3-acyl-indole derivatives as potent and pan-serotype dengue virus inhibitors. We herein describe our optimization efforts toward preclinical candidates 24a and 28a with improved pan-serotype coverage (EC50's against the four DENV serotypes ranging from 0.0011 to 0.24 μM for 24a and from 0.00060 to 0.084 μM for 28a), chiral stability, and oral bioavailability in preclinical species, as well as showing a dose-proportional increase in efficacy against DENV-2 infection in vivo in mice.

Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million 1 with annually around 10,000 deaths 2 . However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors 3 . Here we present JNJ-1802—a highly potent DENV inhibitor that blocks the NS3–NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated 4 . These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

In continuation of our efforts of finding novel nucleoside inhibitors for the treatment of viral diseases, we initiated a discovery research program aimed at identifying novel nucleos(t)ide inhibitors for emerging diseases like Dengue and Chikungunya. Based on the previously reported 2'-spiro-oxetane uridine derivatives active against Hepatitis C Virus (HCV), we envisaged its sulfur analogue as an interesting congener both from a synthetic as well as biological point of view. Surprisingly, we found the 2'-spirothietane uridine derivatives not only to be active against HCV and Dengue virus (DENV), viruses belonging to the flavivirus family, but also to demonstrate activity against alphaviruses like Chikungunya virus (CHIKV) and Sindbis virus (SINV).

The HBV core protein serves multiple essential functions in the viral life cycle that enable chronic HBV infection to persist, and as such, represents a promising drug target. Modulation of the HBV capsid assembly has shown efficacy in early clinical trials through use of small molecule capsid assembly modulators (CAMs). Herein is described the evolution and SAR of a novel pyrazolo piperidine lead series into advanced oxadiazepinone HBV CAMs.