Biology subjects

Counago, R. M.

Publications and source records attributed to Counago, R. M..

15 recordsLinked to original sources

bioRxiv2025

Chemical genetics reveals Leishmania KKT2 and CRK9 kinase activity is required for cell cycle progression

Protein kinases are key regulators of the eukaryotic cell cycle and have consequently emerged as attractive targets for drug development. Their well-defined active sites make them particularly amenable to inhibition by small molecules, underscoring their druggability. The Leishmania kinome, shaped by diverse evolutionary processes, harbours a unique repertoire of potential drug targets. Here, we used the cysteine-directed protein kinase probe SM1-71 to identify four essential protein kinases MPK4, MPK5, MPK7 and AEK1 as candidates for covalent kinase inhibitor development, as well as CLK1/CLK2 for which covalent inhibitors have already been identified. We leveraged the absence of natural analog-sensitive (AS) kinases in L. mexicana to establish an in vivo chemical-genetic AS kinase platform for investigating essential functions of protein kinases. Using CRISPR-Cas9-mediated precision genome editing, we endogenously engineered two kinetochore-associated protein kinases, KKT2 and KKT3, and cyclin-dependent kinase CRK9, to generate AS kinases. We show that KKT2 and CRK9 kinase activities are essential for both promastigote and intracellular amastigote survival; KKT2 kinase activity being required for progression through mitosis at a stage preceding mitotic spindle assembly, while CRK9 kinase activity is required for S phase, consistent with its role in trans-splicing. This study demonstrates the utility of AS chemical genetics in Leishmania and identifies KKT2 and CRK9 as having critical roles in Leishmania cell cycle regulation and therefore being promising drug targets.

molecular biology↗

bioRxiv2025

N-Alkyl Sulfamates as a New Class of nsP2 Cysteine Protease Inhibitors with Broad Spectrum Antialphaviral Activity

The emergence of mosquito-borne alphaviruses that cause chronic arthritis or encephalitis underscores the urgent need for broad-spectrum antiviral therapeutics. The viral nsP2 cysteine protease, which is essential for alphavirus replication, is a promising antiviral target. Vinyl sulfone-based inhibitors, such as RA-2034, potently inhibit nsP2 protease but suffer from glutathione reactivity and species-dependent systemic clearance catalyzed by glutathione S-transferase. To address these liabilities, we explored alternative electrophilic warheads and identified reverse amide inhibitors bearing N-alkyl sulfamate warheads with improved biochemical and antiviral profiles. N-methyl sulfamate acetamide 5 emerged as a lead compound with potency against both New and Old World alphaviruses, low GSH reactivity, and high proteome-wide selectivity. Despite its promising antialphaviral activity, 5 exhibited rapid clearance due to hepatic glucuronidation. Structure-activity studies revealed modifications that improve metabolic stability while retaining antiviral activity. These findings introduce sulfamate acetamides as a new class of covalent nsP2 protease inhibitors and advance the discovery of direct acting pan-alphavirus drugs.

pharmacology and toxicology↗

bioRxiv2025

Discovery and characterization of cell-permeable inhibitors of Leishmania mexicana CLK1 using an in-cell target engagement assay

Leishmaniasis is a neglected tropical disease with limited treatment options and significant unmet medical need. Here, we report the development of a bioluminescence resonance energy transfer (BRET)-based target engagement assay in live intact cells to identify and validate cell-permeable, ATP-competitive inhibitors of Leishmania mexicana (Lmx)CLK1. LmxCLK2, a closely related paralogue with an identical protein kinase domain, is also considered in our analysis. Genetic and pharmacological evidence indicates that simultaneous deletion or inhibition of both LmxCLK1/2 is lethal to the parasite. Using our newly developed assay, we screened a library of human kinase inhibitors and identified WZ8040, a third-generation EGFR inhibitor, as a potent LmxCLK1 ligand. WZ8040 demonstrated robust target engagement in both promastigotes and macrophage-internalized amastigotes, with low micromolar EC values for parasite killing and minimal toxicity to host macrophages. Biochemical assays confirmed that WZ8040 covalently binds and inhibits LmxCLK1, with mass spectrometry identifying Cys172 as the primary site of modification. Genetic validation using overexpression and knockout lines supports LmxCLK1 as the primary target of WZ8040. However, the retained activity of WZ8040 in mutant lines with the Cys172Ala substitution suggests that covalent binding is not essential for compound efficacy. Our findings highlight the utility of BRET-based assays for target validation in kinetoplastid parasites and underscore the potential of CLK1 as a druggable kinase in Leishmania. This integrated approach provides a framework for accelerating the discovery of novel anti-leishmanial agents through target engagement-guided strategies. Highlights- A live-cell NanoBRET assay enables in situ target engagement of CLK1 in Leishmania mexicana. - The covalent inhibitor WZ8040 effectively targets LmxCLK1 in both promastigote and intracellular amastigote stages. - CLK1 inhibition by WZ8040 does not depend on covalent modification of a single cysteine residue. - Combined target engagement and phenotypic data support LmxCLK1 as a druggable kinase in Leishmania.

cell biology↗

bioRxiv2025

Identification of a Broadly Acting Inhibitor of the Alphavirus Non-Structural Protein 2 Helicase

Alphaviruses are mosquito-borne viruses that have caused significant outbreaks in the 21st century. Despite multiple recent outbreaks, there are no approved antiviral drugs to treat any alphavirus infection. Therefore, developing broadly acting antiviral drugs effective against multiple alphaviruses is necessary and could provide protection from both current and emerging alphavirus threats. A critical component of the alphavirus replication complex is non-structural protein 2 (nsP2), which is a multifunctional enzyme containing a helicase domain connected to a protease domain by a flexible linker. nsP2 functions as an ATP-dependent helicase, is conserved across the alphavirus genus, and is essential for virus replication, making it a promising target for development of alphavirus broad-acting antiviral drugs. Previous studies identified an enantioselective compound RA-0025298 that inhibited nsP2 ATPase activity and chikungunya virus CHIKV replication. Antiviral testing of RA-25298. against a diverse group of alphaviruses found broad activity except for Sindbis-like viruses. Using this information along with mutational profiling of virus passaged with RA-0025298 we identified the site of RA-0025298 action and confirmed the binding site via biophysical analyses. Finally, we found that the active enantiomer of RA-0025298 (SGC-NSP2hel-1) reduced viral loads in vivo and protected mice from tissue damage and disease caused by CHIKV infection. These findings further describe the mechanism of action of a first-in-class nsP2 helicase inhibitor with the potential for development as a broad spectrum drug for treating or preventing disease caused by current and emerging alphaviruses. One Sentence SummaryThis study describes the mechanism of action and in vivo efficacy of a first in class broadly acting inhibitor of alphavirus nsP2 helicase activity.

microbiology↗

bioRxiv2025

Fragment Screening by Weak Affinity Chromatography Identifies Ligands for Neisseria gonorrhoeae Undecaprenyl Diphosphate Synthase

The rapid rise of antimicrobial resistance in Neisseria gonorrhoeae underscores the urgent need for new therapeutic strategies. Undecaprenyl diphosphate synthase (UPPS), an essential enzyme in bacterial cell wall biosynthesis, has emerged as a promising antimicrobial target. However, no structural information has been available for the gonococcal enzyme (NgUPPS), and known ligand scaffolds remain limited, hindering structure-based drug discovery efforts. Here, we present a combined weak affinity chromatography-mass spectrometry (WAC-MS) and crystallographic pipeline to identify and structurally characterize fragment binders of NgUPPS. WAC-MS screening of a diverse fragment library identified 23 putative binders, 16 of which were successfully co- crystallized with NgUPPS, primarily via soaking into preformed apo crystals. The resulting high-resolution structures mapped fragment binding across four distinct regions of the enzymes hydrophobic tunnel, encompassing the pyrophosphate- binding pockets and the distal product-binding site. Additionally, we determined the co-crystal structure of NgUPPS with BPH-1100, a known phthalic acid-based UPPS inhibitor, revealing that the enzyme adopts an open, ligand-competent conformation without significant rearrangement. Our results validate NgUPPS as a structurally tractable antibacterial target and provide a strong foundation for fragment-based optimization and the future development of novel inhibitors to combat drug-resistant N. gonorrhoeae.

microbiology↗

bioRxiv2025

An Enantioselective Chemical Probe for Chikungunya nsP2 Helicase with Antialphaviral Activity

Chikungunya virus (CHIKV) replication relies on the multifunctional nsP2 protein, making it an attractive target for antiviral drug discovery. Here, we report the resolution of oxaspiropiperidine 1, a first-in-class inhibitor of the CHIKV nsP2 RNA helicase (nsP2hel), into its constitutive enantiomers and characterization of their antiviral activity. The enantiomer (R)-1 exhibited potent inhibition of viral replication, nsP2hel ATPase activity, and dsRNA unwinding, while the (S)-1 enantiomer was >100-fold less active. The (R)-1 enantiomer also demonstrated high selectivity for CHIKV over other RNA viruses and for nsP2hel over other RNA helicases. Direct binding of (R)-1 to nsP2hel protein was confirmed by 19F NMR. Biophysical and structural studies revealed conformational polymorphism in the spirocyclic scaffold of (R)-1, suggesting a potential role of thermal mobility of the ligand in allosteric inhibition of nsP2hel. Collectively, these findings designate (R)-1 (RA-NSP2-1) as a high-quality chemical probe and (S)-1 (RA-NSP2-1N) as a negative control for probing the biology of alphavirus RNA helicases.

pharmacology and toxicology↗

bioRxiv2025

Identification of Direct-acting nsP2 Helicase Inhibitors with Anti-alphaviral Activity

Alphaviruses are mosquito-borne RNA viruses that pose a significant public health threat, with no FDA-approved antiviral therapeutics available. The non-structural protein 2 helicase (nsP2hel) is an enzyme involved in unwinding dsRNA essential for alphavirus replication. This study reports the discovery and optimization of first-in-class oxaspiropiperidine inhibitors targeting nsP2hel. Structure-activity relationship (SAR) studies identified potent cyclic sulfonamide analogs with nanomolar antiviral activity against chikungunya virus (CHIKV). Biochemical analyses of nsP2hel ATPase and RNA unwindase activities showed these compounds act by a non-competitive mode suggesting that they are allosteric inhibitors. Viral resistance mutations mapped to nsP2hel and a fluorine-labeled analog exhibited direct binding to the protein by 19F NMR. The lead inhibitor, 2o, demonstrated broad-spectrum antialphaviral activity, reducing titers of CHIKV, Mayaro virus (MAYV), and Venezuelan equine encephalitis virus (VEEV). These findings support nsP2hel as a viable target for development of broad-spectrum direct-acting antialphaviral drugs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/641060v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@856df5org.highwire.dtl.DTLVardef@1f6225borg.highwire.dtl.DTLVardef@4997d5org.highwire.dtl.DTLVardef@18f42f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2022

SGC-CAMKK2-1: A chemical probe for CAMKK2

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.

cancer biology↗

bioRxiv2022

An open source plant kinase chemogenomics set

129 protein kinases, selected to represent the diversity of the rice (Oryza sativa) kinome, were cloned and tested for expression in E. coli. 40 of these rice kinases were purified and screened using differential scanning fluorimetry (DSF) against 627 diverse kinase inhibitors, with a range of structures and activities targeting diverse human kinases. 37 active compounds were then tested for their ability to modify primary root development in Arabidopsis. Of these, 14 compounds caused a significant reduction of primary root length and two slightly increased root elongation compared with control plants. Two inhibitory compounds bind to the predicted orthologue of Arabidopsis PSKR1, one of two receptors for PSK, a small sulfated peptide that positively controls root development. Inhibition could not be rescued by the exogenous addition of the PSK peptide, suggesting that chemical treatment may inhibit both PSKR1 and its closely related receptor PSKR2. Of the compounds acting as root growth inhibitors in Arabidopsis, six conferred the same effect in rice. Compound RAF265 (CHIR-265), previously shown to bind the human kinase BRAF (B-Raf proto-oncogene, serine/threonine kinase), also binds to nine highly conserved rice kinases tested. The binding of human and rice kinases to the same compound suggests that human kinase inhibitor sets will be useful for dissecting the function of plant kinases.

plant biology↗

bioRxiv2020

In depth analysis of kinase cross screening data to identify CAMKK2 inhibitory scaffolds

The calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays a central role in many cell signaling pathways. CAMKK2 activates CAMK1, CAMK4, AMPK, and AKT leading to numerous physiological responses. Deregulation of CAMKK2 is linked to several diseases, suggesting utility of CAMKK2 inhibitors for oncological, metabolic and inflammatory indications. In this work we review the role of CAMKK2 in biology and disease. Through analysis of literature and public databases we have identified starting points for CAMKK2 inhibitor medicinal chemistry campaigns. These starting points provide an opportunity for the development of selective CAMKK2 inhibitors and will lead to tools that delineate the roles of this kinase in disease biology.

cancer biology↗

bioRxiv2019

Structural and binding studies of human CAMKK2 kinase domain bound to small molecule ligands

Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no specific small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homologue, CAMKK1, are thought to have overlapping biological roles. Here we present six novel co-structures of CAMKK2 bound to potent ligands identified from a library of ATP-competitive kinase inhibitors. Isothermal titration calorimetry (ITC) revealed that binding to some of these molecules is enthalpy driven. We expect our results to further advance current efforts to discover small molecule kinase inhibitors specific to each human CAMKK.

biochemistry↗

bioRxiv2018

Structural Analysis of Inhibitor Binding to CAMKK1 Identifies Features Necessary for Design of Specific Inhibitors

The calcium/calmodulin-dependent protein kinases (CAMKKs) are upstream activators of CAMK1 and CAMK4 signalling and have important functions in neural development, maintenance and signalling, as well as in other aspects of biology such as Ca2+ signalling in the cardiovascular system. To support the development of specific inhibitors of CAMKKs we have determined the crystal structure of CAMKK1 with two ATP-competitive inhibitors. The structures reveal small but exploitable differences between CAMKK1 and CAMKK2, despite the high sequence identity, which could be used in the generation of specific inhibitors. Screening of a kinase inhibitor library revealed molecules that bind potently to CAMKK1. Isothermal titration calorimetry revealed that the most potent inhibitors had binding energies largely dependent on favourable enthalpy. Together, the data provide a foundation for future inhibitor development activities.

biochemistry↗

bioRxiv2017

In depth analysis of kinase cross screening data to identify chemical starting points for inhibition of the Nek family of kinases

Potent, selective, and cell active small molecule kinase inhibitors are useful tools to help unravel the complexities of kinase signaling. As the biological functions of individual kinases become better understood, they can become targets of drug discovery efforts. The small molecules used to shed light on function can also then serve as chemical starting points in these drug discovery efforts. The Nek family of kinases has received very little attention, as judged by number of citations in PubMed, yet they appear to play many key roles and have been implicated in disease. Here we present our work to identify high quality chemical starting points that have emerged due to the increased incidence of broad kinome screening. We anticipate that this analysis will allow the community to progress towards the generation of chemical probes and eventually drugs that target members of the Nek family.

pharmacology and toxicology↗

bioRxiv2017

Structural characterization of human Vaccinia-Related Kinases (VRK) bound to small-molecule inhibitors identifies different P-loop conformations

The human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2.

biochemistry↗

bioRxiv2016

Development of Narrow Spectrum ATP-competitive Kinase Inhibitors as Probes for BIKE and AAK1

Understanding the structural determinants of inhibitor selectivity would facilitate the design and preparation of kinase probes. We describe a pair of matched compounds differing only by one degree of saturation but showing dramatic differential activities at select kinases. We utilized x-ray crystallography and computational analysis to rationalize the basis of the differential activity.

pharmacology and toxicology↗