Chaitanya Kumar Jaladanki

Structured RNAs are critical regulators of gene expression and represent promising targets for drug discovery. Despite this potential, structure-based virtual screening approaches─in particular, molecular docking─have been underutilized for RNA targets, largely due to the intrinsic conformational flexibility of RNA and the limited docking methods specifically optimized for RNA. In our study, we evaluated the efficacy of three widely used docking programs for RNA–ligand docking screens. The performance of these programs─Glide, GOLD, and rDock─was evaluated with the data set of 25 diverse RNA targets with 432 small-molecule RNA binders. We found that the performance was similar across docking methods, but consistent differences emerged among RNA classes. We further explored consensus scoring across both receptor conformations and docking methods, finding that selecting the average scores across docking programs and RNA structures yielded stable performance when compared to individual methods. Finally, clustering analysis of RNA binders revealed distinct scoring function preferences for different chemotypes of RNA binders, underscoring the potential for tailored scoring strategies in RNA-targeted drug discovery. Together, this work provides a comprehensive evaluation of the performance of available docking methods in different cases of RNA–ligand docking and offers practical guidance for improving virtual screening against structured RNAs.

Structured RNAs are critical regulators of gene expression and represent promising targets for drug discovery. Despite this potential, structure-based virtual screening approaches—in particular, molecular docking—have been underutilized for RNA targets, largely due to the intrinsic conformational flexibility of RNA and the limited docking methods specifically optimized for RNA. In our study, we evaluated the efficacy of three widely used docking programs for RNA-ligand docking screens. The performance of these programs — Glide, Gold, and rDock, were evaluated with the dataset of 25 diverse RNA targets with 432 small molecule RNA binders. We found that the performance was similar across docking methods, but consistent differences emerged among RNA classes. We further explored consensus scoring across both receptor conformations and docking methods, finding that selecting the average scores across docking programs and RNA structures yielded stable performance when compared to individual methods. Finally, clustering analysis of RNA binders revealed distinct scoring function preferences for different chemotypes of RNA binders, underscoring the potential for tailored scoring strategies in RNA-targeted drug discovery. Together, this work provides a comprehensive evaluation of the performance of available docking methods in different cases of RNA-ligand docking and offers practical guidance for improving virtual screening against structured RNAs.

Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in three clusters, including an IMiD degron in zinc finger cluster one (ZFC1). Surprisingly, IMiDs do not inhibit the growth of SALL4-expressing cancer cells. To overcome this limit, we focused on a non-IMiD domain, SALL4 zinc finger cluster four (ZFC4). By combining ZFC4-DNA crystal structure and an in silico docking algorithm, in conjunction with cell viability assays, we screened several chemical libraries against a potentially druggable binding pocket, leading to the discovery of SH6, a compound that selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 treatment led to a significant 87% tumor growth inhibition of SALL4+ patient-derived xenografts and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors such as SALL4 in cancer.

Structured RNAs play a crucial role in regulating gene expression, which includes both protein synthesis and RNA processing. Dysregulation of these processes is associated with various conditions, including viral and bacterial infections, as well as cancer. The unique tertiary structures of structured RNAs provide an opportunity for small molecules to directly modulate such processes, making them promising targets for drug discovery. Although small-molecule inhibitors targeting RNA have shown early success, in silico strategies like structure-based virtual screening remain underutilized for RNA-targeted drug discovery. In this study, we developed a virtual screening scheme targeting the structural ensemble of EV-A71 IRES SL II, a noncoding viral RNA element essential for viral replication. We subsequently optimized the experimentally validated hit compound IRE-03 from virtual screening through an "analog-by-catalog" search. This led to the identification of a more potent IRES inhibitor, IRE-03-3, validated through biochemical and functional assays with an EC₅₀ value of 11.96 μM against viral proliferation. Our findings demonstrate that structure-based virtual screening can be effectively applied to RNA targets, providing exciting new opportunities for future antiviral drug discovery.

Drugs containing thiazole and aminothiazole groups are known to generate reactive metabolites (RMs) catalyzed by cytochrome P450s (CYPs). These RMs can covalently modify essential cellular macromolecules and lead to toxicity and induce idiosyncratic adverse drug reactions. Molecular docking and quantum chemical hybrid DFT study were carried out to explore the molecular mechanisms involved in the biotransformation of thiazole (TZ) and aminothiazole (ATZ) groups leading to RM epoxide, S-oxide, N-oxide, and oxaziridine. The energy barrier required for the epoxidation is 13.63 kcal/mol, that is lower than that of S-oxidation, N-oxidation, and oxaziridine formation (14.56, 17.90, and 20.20, kcal/mol respectively). The presence of the amino group in ATZ further facilitates all the metabolic pathways, for example, the barrier for the epoxidation reaction is reduced by ∼2.5 kcal/mol. Some of the RMs/their isomers are highly electrophilic and tend to form covalent bonds with nucleophilic amino acids, finally leading to the formation of metabolic intermediate complexes (MICs). The energy profiles of these competitive pathways have also been explored.

Filoviruses Ebola (EBOV) and Marburg (MARV) are devastating high-priority pathogens capable of causing explosive outbreaks with high human mortality rates. The matrix proteins of EBOV and MARV, as well as eVP40 and mVP40, respectively, are the key viral proteins that drive virus assembly and egress and can bud independently from cells in the form of virus-like particles (VLPs). The matrix proteins utilize proline-rich Late (L) domain motifs (e.g., PPxY) to hijack specific host proteins that contain WW domains, such as the HECT family E3 ligases, to facilitate the last step of virus–cell separation. We identified E3 ubiquitin ligase Smad Ubiquitin Regulatory Factor 2 (SMURF2) as a novel interactor with VP40 that positively regulates VP40 VLP release. Our results show that eVP40 and mVP40 interact with the three WW domains of SMURF2 via their PPxY motifs. We provide evidence that the eVP40–SMURF2 interaction is functional as the expression of SMURF2 positively regulates VLP egress, while siRNA knockdown of endogenous SMURF2 decreases VLP budding compared to controls. In sum, our identification of novel interactor SMURF2 adds to the growing list of identified host proteins that can regulate PPxY-mediated egress of VP40 VLPs. A more comprehensive understanding of the modular interplay between filovirus VP40 and host proteins may lead to the development of new therapies to combat these deadly infections.

Ebola (EBOV) and Marburg (MARV) are members of the Filoviridae family, which continue to emerge and cause sporadic outbreaks of hemorrhagic fever with high mortality rates. Filoviruses utilize their VP40 matrix protein to drive virion assembly and budding, in part, by recruitment of specific WW-domain-bearing host proteins via its conserved PPxY Late (L) domain motif. Here, we screened an array of 115 mammalian, bacterially expressed and purified WW-domains using a PPxY-containing peptide from MARV VP40 (mVP40) to identify novel host interactors. Using this unbiased approach, we identified Yes Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ) as novel mVP40 PPxY interactors. YAP and TAZ function as downstream transcriptional effectors of the Hippo signaling pathway that regulates cell proliferation, migration and apoptosis. We demonstrate that ectopic expression of YAP or TAZ along with mVP40 leads to significant inhibition of budding of mVP40 VLPs in a WW-domain/PPxY dependent manner. Moreover, YAP colocalized with mVP40 in the cytoplasm, and inhibition of mVP40 VLP budding was more pronounced when YAP was localized predominantly in the cytoplasm rather than in the nucleus. A key regulator of YAP nuclear/cytoplasmic localization and function is angiomotin (Amot); a multi-PPxY containing protein that strongly interacts with YAP WW-domains. Interestingly, we found that expression of PPxY-containing Amot rescued mVP40 VLP egress from either YAP- or TAZ-mediated inhibition in a PPxY-dependent manner. Importantly, using a stable Amot-knockdown cell line, we found that expression of Amot was critical for efficient egress of mVP40 VLPs as well as egress and spread of authentic MARV in infected cell cultures. In sum, we identified novel negative (YAP/TAZ) and positive (Amot) regulators of MARV VP40-mediated egress, that likely function in part, via competition between host and viral PPxY motifs binding to modular host WW-domains. These findings not only impact our mechanistic understanding of virus budding and spread, but also may impact the development of new antiviral strategies. By screening an array of 115 mammalian WW-domains with the PPxY motif from MARV VP40 (mVP40), we identified YAP and TAZ, transcriptional effectors of the Hippo pathway, as mVP40 interactors, and demonstrated that ectopically expressed YAP or TAZ inhibited budding of mVP40 virus-like particles (VLPs) in a WW-domain/PPxY dependent manner. Angiomotin (Amot), a multi-PPxY containing regulator of YAP nuclear/cytoplasmic localization and function, rescued mVP40 VLP egress from either YAP- or TAZ-mediated inhibition in a PPxY-dependent manner. Indeed, endogenous Amot expression was critical for egress of mVP40 VLPs and authentic MARV. In sum, we have revealed a link between the Hippo pathway and filovirus egress by identifying negative (YAP/TAZ) and positive (Amot) regulators of MARV VP40-mediated egress.

Hydrazide group (−C(O)–NH–NH₂) is considered as a structural alert in the drug discovery process because the biotransformation chemistry of this group leads to the generation of toxic radical intermediates. The most important antitubercular drug isoniazid (INH) carries the hydrazide group. The toxicity of INH has been attributed to the protein adduct formation involving isonicotinoyl radical. However, the structures of reactive metabolites (RMs) and metabolite intermediate complexes (MICs), as well as the reaction mechanism for the formation and fate of RMs/MICs, have not been established. This report provides a detailed account of the biotransformation of INH by cytochromes using quantum chemical (QC) methods. Two cycles of cytochrome catalysis are involved in the formation of the most important RM, isonicotinoyl radical. The first cycle requires ∼11 kcal/mol barrier on the oxidation pathway involving the formation of the RM isonicotinoyldiazene. The second cycle involves a barrier of ∼7 kcal/mol for the activation of the diazene intermediate leading to isonicotinic acid via three reaction steps: (i) N–H bond activation, (ii) loss of N₂ molecule, and (iii) rebound of isonicotinoyl radical. The RMs on the pathway (diazene, isonicotinoyl radical, N-hydroxy diazene) can react with the porphyrin ring/the amino acids of the cytochrome leading to many MICs (at least nine varieties), which can cause mechanism based inhibition and drug–drug interactions. This QC, molecular docking, and QM/MM study explored all the above reaction pathways and established the 3D structures of the RMs and MICs.

A cobalt-catalyzed proton-coupled electron transfer (PCET) mediated regioselective ortho-specific nitration of aromatic C(sp²)-H bonds using chelation-assisted removable vicinal diamine directing groups was developed. The reaction proceeded under mild conditions in the presence of Co(OAc)₂·4H₂O as the catalyst with AgNO₂ utilized as the nitro source as well as terminal oxidant in the presence of O₂ as an external oxidant. No external base or additives were required for this process. Controlled experiments and mechanistic investigations with DFT calculations revealed that the reaction proceeds through a PCET promoted nitro functional group transfer pathway. Moreover, the produced compounds are valuable and pharmaceutically quite relevant.