A comprehensive study of SARS-CoV-2 main protease (Mpro) inhibitor-resistant mutants selected in a VSV-based system.

Nirmatrelvir was the first protease inhibitor specifically developed against the SARS-CoV-2 main protease (3CLpro/Mpro) and licensed for clinical use. As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate Mpro resistance mutations, we passaged a previously developed, chimeric vesicular stomatitis virus (VSV-Mpro) with increasing, yet suboptimal concentrations of nirmatrelvir. Using Wuhan-1 and Omicron Mpro variants, we selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available protease inhibitors (nirmatrelvir and ensitrelvir) with cell-based, biochemical and SARS-CoV-2 replicon assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings have implications on the development of future generation Mpro inhibitors, will help to understand SARS-CoV-2 protease inhibitor resistance mechanisms and show the relevance of specific mutations, thereby informing treatment decisions.

2-Substituted (N)-Methanocarba A3 Adenosine Receptor Agonists: In Silico, In Vitro, and In Vivo Characterization.

2-Arylethynyl (N)-methanocarba adenosine 5'-methylamides are selective A3 adenosine receptor (AR) agonists containing a preestablished receptor-preferred pseudoribose conformation. Here, we compare analogues having bulky 2-substitution, either containing or lacking an ethynyl spacer between adenine and a cyclic group. 2-Aryl compounds 9-11, 13, 14, 19, 22, 23, 27, 29, 31, and 34, lacking a spacer, had human (h) A3AR K i values of 2-30 nM, and others displayed lower affinity. Mouse (m) A3AR affinity varied, with 2-arylethynyl having a higher affinity than 2-aryl analogues (7, 8 > 3c, 3d > 3b). However, 2-aryl-4'-truncated derivatives had greatly reduced hA3AR affinity, even containing affinity-enhancing N 6-dopamine-derived substituents. Molecular modeling, including molecular dynamics simulation, predicted stable poses in the canonical A3AR agonist binding site, but 2-aryl (ECL2 interactions) and 2-arylethynyl (TM2 interactions) substituents have different conformations and environments. In a hA3AR miniGαi recruitment assay, 31 (MRS8062) was (slightly) more potent compared to a ß-arrestin2 recruitment assay, both in engineered HEK293T cells, and its maximal efficacy (E max) was much higher (165%) than reference agonist NECA's. Thus, in the 2-aryl series, A3AR affinity and selectivity were variable and generally reduced compared to the 2-arylethynyl series, with a greater dependence on the specific aryl group present. Selected compounds were studied in vivo in an ischemic model of peripheral artery disease (PAD). Rigidified 2-arylethynyl analogues 3a-3c were protective in this model of skeletal muscle ischemia-reperfusion injury/claudication, as previously shown only for moderately A3AR-selective ribosides or (N)-methanocarba derivatives. Thus, we have expanded the A3AR agonist SAR for (N)-methanocarba adenosines.

Lipid Trolling to Optimize A3 Adenosine Receptor-Positive Allosteric Modulators (PAMs).

A3 adenosine receptor (A3AR) positive allosteric modulators (PAMs) (2,4-disubstituted-1H-imidazo[4,5-c]quinolin-4-amines) allosterically increase the Emax of A3AR agonists, but not potency, due to concurrent orthosteric antagonism. Following mutagenesis/homology modeling of the proposed lipid-exposed allosteric binding site on the cytosolic side, we functionalized the scaffold, including heteroatom substitutions and exocyclic phenylamine extensions, to increase allosteric binding. Strategically appended linear alkyl-alkynyl chains with terminal amino/guanidino groups improved allosteric effects at both human and mouse A3ARs. The chain length, functionality, and attachment position were varied to modulate A3AR PAM activity. For example, 26 (MRS8247, p-alkyne-linked 8 methylenes) and homologues increased agonist Cl-IB-MECA's Emax and potency ([35S]GTPγS binding). The putative mechanism involves a flexible, terminally cationic chain penetrating the lipid environment for stable electrostatic anchoring to cytosolic phospholipid head groups, suggesting "lipid trolling", supported by molecular dynamic simulation of the active-state model. Thus, we have improved A3AR PAM activity through rational design based on an extrahelical, lipidic binding site.

Extrahelical Binding Site for a 1H-Imidazo[4,5-c]quinolin-4-amine A3 Adenosine Receptor Positive Allosteric Modulator on Helix 8 and Distal Portions of Transmembrane Domains 1 and 7.

This study describes the localization and computational prediction of a binding site for the A3 adenosine receptor (A3AR) positive allosteric modulator 2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-(3,4-dichlorophenyl)amine (LUF6000). The work reveals an extrahelical lipid-facing binding pocket disparate from the orthosteric binding site that encompasses transmembrane domain (TMD) 1, TMD7, and Helix (H) 8, which was predicted by molecular modeling and validated by mutagenesis. According to the model, the nearly planar 1H-imidazo[4,5-c]quinolinamine ring system lies parallel to the transmembrane segments, inserted into an aromatic cage formed by π-π stacking interactions with the side chains of Y2847.55 in TMD7 and Y2938.54 in H8 and by π-NH bonding between Y2847.55 and the exocyclic amine. The 2-cyclohexyl group is positioned "upward" within a small hydrophobic subpocket created by residues in TMDs 1 and 7, while the 3,4-dichlorophenyl group extends toward the lipid interface. An H-bond between the N-1 amine of the heterocycle and the carbonyl of G291.49 further stabilizes the interaction. Molecular dynamics simulations predicted two metastable intermediates, one resembling a pose determined by molecular docking and a second involving transient interactions with Y2938.54; in simulations, each of these intermediates converges into the final bound state. Structure-activity-relationships for replacement of either of the identified exocyclic or endocyclic amines with heteroatoms lacking H-bond donating ability were consistent with the hypothetical pose. Thus, we characterized an allosteric pocket for 1H-imidazo[4,5-c]quinolin-4-amines that is consistent with data generated by orthogonal methods, which will aid in the rational design of improved A3AR positive allosteric modulators. SIGNIFICANCE STATEMENT: Orthosteric A3AR agonists have advanced in clinical trials for inflammatory conditions, liver diseases, and cancer. Thus, the clinical appeal of selective receptor activation could extend to allosteric enhancers, which would induce site- and time-specific activation in the affected tissue. By identifying the allosteric site for known positive allosteric modulators, structure-based drug discovery modalities can be enabled to enhance the pharmacological properties of the 1H-imidazo[4,5-c]quinolin-4-amine class of A3AR positive allosteric modulators.

Thermal titration molecular dynamics (TTMD): shedding light on the stability of RNA-small molecule complexes.

Ribonucleic acids are gradually becoming relevant players among putative drug targets, thanks to the increasing amount of structural data exploitable for the rational design of selective and potent binders that can modulate their activity. Mainly, this information allows employing different computational techniques for predicting how well would a ribonucleic-targeting agent fit within the active site of its target macromolecule. Due to some intrinsic peculiarities of complexes involving nucleic acids, such as structural plasticity, surface charge distribution, and solvent-mediated interactions, the application of routinely adopted methodologies like molecular docking is challenged by scoring inaccuracies, while more physically rigorous methods such as molecular dynamics require long simulation times which hamper their conformational sampling capabilities. In the present work, we present the first application of Thermal Titration Molecular Dynamics (TTMD), a recently developed method for the qualitative estimation of unbinding kinetics, to characterize RNA-ligand complexes. In this article, we explored its applicability as a post-docking refinement tool on RNA in complex with small molecules, highlighting the capability of this method to identify the native binding mode among a set of decoys across various pharmaceutically relevant test cases.

A comprehensive study of SARS-CoV-2 main protease (Mpro) inhibitor-resistant mutants selected in a VSV-based system.

Nirmatrelvir was the first protease inhibitor (PI) specifically developed against the SARS-CoV-2 main protease (3CLpro/Mpro) and licensed for clinical use. As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate Mpro resistance mutations, we passaged a previously developed, chimeric vesicular stomatitis virus (VSV-Mpro) with increasing, yet suboptimal concentrations of nirmatrelvir. Using Wuhan-1 and Omicron Mpro variants, we selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available PIs (nirmatrelvir and ensitrelvir) with cell-based and biochemical assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings have implications on the development of future generation Mpro inhibitors, will help to understand SARS-CoV-2 protease-inhibitor-resistance mechanisms and show the relevance of specific mutations in the clinic, thereby informing treatment decisions.

A small molecule targeting the interaction between human papillomavirus E7 oncoprotein and cellular phosphatase PTPN14 exerts antitumoral activity in cervical cancer cells.

Human papillomavirus (HPV)-induced cancers still represent a major health issue for worldwide population and lack specific therapeutic regimens. Despite substantial advancements in anti-HPV vaccination, the incidence of HPV-related cancers remains high, thus there is an urgent need for specific anti-HPV drugs. The HPV E7 oncoprotein is a major driver of carcinogenesis that acts by inducing the degradation of several host factors. A target is represented by the cellular phosphatase PTPN14 and its E7-mediated degradation was shown to be crucial in HPV oncogenesis. Here, by exploiting the crystal structure of E7 bound to PTPN14, we performed an in silico screening of small-molecule compounds targeting the C-terminal CR3 domain of E7 involved in the interaction with PTPN14. We discovered a compound able to inhibit the E7/PTPN14 interaction in vitro and to rescue PTPN14 levels in cells, leading to a reduction in viability, proliferation, migration, and cancer-stem cell potential of HPV-positive cervical cancer cells. Mechanistically, as a consequence of PTPN14 rescue, treatment of cancer cells with this compound altered the Yes-associated protein (YAP) nuclear-cytoplasmic shuttling and downstream signaling. Notably, this compound was active against cervical cancer cells transformed by different high-risk (HR)-HPV genotypes indicating a potential broad-spectrum activity. Overall, our study reports the first-in-class inhibitor of E7/PTPN14 interaction and provides the proof-of-principle that pharmacological inhibition of this interaction by small-molecule compounds could be a feasible therapeutic strategy for the development of novel antitumoral drugs specific for HPV-associated cancers.

"Dual Anta-Inhibitors" of the A2A Adenosine Receptor and Casein Kinase CK1delta: Synthesis, Biological Evaluation, and Molecular Modeling Studies.

Based on a screening of a chemical library of A2A adenosine receptor (AR) antagonists, a series of di- and tri-substituted adenine derivatives were synthesized and tested for their ability to inhibit the activity of the enzyme casein kinase 1 delta (CK1δ) and to bind adenosine receptors (ARs). Some derivatives, here called "dual anta-inhibitors", demonstrated good CK1δ inhibitory activity combined with a high binding affinity, especially for the A2AAR. The N6-methyl-(2-benzimidazolyl)-2-dimethyamino-9-cyclopentyladenine (17, IC50 = 0.59 µM and KiA2A = 0.076 µM) showed the best balance of A2AAR affinity and CK1δ inhibitory activity. Computational studies were performed to simulate, at the molecular level, the protein-ligand interactions involving the compounds of our series. Hence, the dual anta-inhibitor 17 could be considered the lead compound of new therapeutic agents endowed with synergistic effects for the treatment of chronic neurodegenerative and cancer diseases.

Structural Manipulations of Marine Natural Products Inspire a New Library of 3-Amino-1,2,4-Triazine PDK Inhibitors Endowed with Antitumor Activity in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the main aggressive types of cancer, characterized by late prognosis and drug resistance. Among the main factors sustaining PDAC progression, the alteration of cell metabolism has emerged to have a key role in PDAC cell proliferation, invasion, and resistance to standard chemotherapeutic agents. Taking into account all these factors and the urgency in evaluating novel options to treat PDAC, in the present work we reported the synthesis of a new series of indolyl-7-azaindolyl triazine compounds inspired by marine bis-indolyl alkaloids. We first assessed the ability of the new triazine compounds to inhibit the enzymatic activity of pyruvate dehydrogenase kinases (PDKs). The results showed that most of derivatives totally inhibit PDK1 and PDK4. Molecular docking analysis was executed to predict the possible binding mode of these derivatives using ligand-based homology modeling technique. Evaluation of the capability of new triazines to inhibit the cell growth in 2D and 3D KRAS-wild-type (BxPC-3) and KRAS-mutant (PSN-1) PDAC cell line, was carried out. The results showed the capacity of the new derivatives to reduce cell growth with a major selectivity against KRAS-mutant PDAC PSN-1 on both cell models. These data demonstrated that the new triazine derivatives target PDK1 enzymatic activity and exhibit cytotoxic effects on 2D and 3D PDAC cell models, thus encouraging further structure manipulation for analogs development against PDAC.

Targeting the I7L Protease: A Rational Design for Anti-Monkeypox Drugs?

The latest monkeypox virus outbreak in 2022 showcased the potential threat of this viral zoonosis to public health. The lack of specific treatments against this infection and the success of viral protease inhibitors-based treatments against HIV, Hepatitis C, and SARS-CoV-2, brought the monkeypox virus I7L protease under the spotlight as a potential target for the development of specific and compelling drugs against this emerging disease. In the present work, the structure of the monkeypox virus I7L protease was modeled and thoroughly characterized through a dedicated computational study. Furthermore, structural information gathered in the first part of the study was exploited to virtually screen the DrugBank database, consisting of drugs approved by the Food and Drug Administration (FDA) and clinical-stage drug candidates, in search for readily repurposable compounds with similar binding features as TTP-6171, the only non-covalent I7L protease inhibitor reported in the literature. The virtual screening resulted in the identification of 14 potential inhibitors of the monkeypox I7L protease. Finally, based on data collected within the present work, some considerations on developing allosteric modulators of the I7L protease are reported.

Are Two Riboses Better Than One? The Case of the Recognition and Activation of Adenosine Receptors.

Traditionally, molecular recognition between the orthosteric site of adenosine receptors and their endogenous ligand occurs with a 1 : 1 stoichiometry. Inspired by previous mechanistic insights derived from supervised molecular dynamics (SuMD) simulations, which suggested an alternative 2 : 1 binding stoichiometry, we synthesized BRA1, a bis-ribosyl adenosine derivative, tested its ability to bind to and activate members of the adenosine receptor family, and rationalized its activity through molecular modeling.

Lessons Learnt from COVID-19: Computational Strategies for Facing Present and Future Pandemics.

Since its outbreak in December 2019, the COVID-19 pandemic has caused the death of more than 6.5 million people around the world. The high transmissibility of its causative agent, the SARS-CoV-2 virus, coupled with its potentially lethal outcome, provoked a profound global economic and social crisis. The urgency of finding suitable pharmacological tools to tame the pandemic shed light on the ever-increasing importance of computer simulations in rationalizing and speeding up the design of new drugs, further stressing the need for developing quick and reliable methods to identify novel active molecules and characterize their mechanism of action. In the present work, we aim at providing the reader with a general overview of the COVID-19 pandemic, discussing the hallmarks in its management, from the initial attempts at drug repurposing to the commercialization of Paxlovid, the first orally available COVID-19 drug. Furthermore, we analyze and discuss the role of computer-aided drug discovery (CADD) techniques, especially those that fall in the structure-based drug design (SBDD) category, in facing present and future pandemics, by showcasing several successful examples of drug discovery campaigns where commonly used methods such as docking and molecular dynamics have been employed in the rational design of effective therapeutic entities against COVID-19.

Thermal Titration Molecular Dynamics (TTMD): Not Your Usual Post-Docking Refinement.

Molecular docking is one of the most widely used computational approaches in the field of rational drug design, thanks to its favorable balance between the rapidity of execution and the accuracy of provided results. Although very efficient in exploring the conformational degrees of freedom available to the ligand, docking programs can sometimes suffer from inaccurate scoring and ranking of generated poses. To address this issue, several post-docking filters and refinement protocols have been proposed throughout the years, including pharmacophore models and molecular dynamics simulations. In this work, we present the first application of Thermal Titration Molecular Dynamics (TTMD), a recently developed method for the qualitative estimation of protein-ligand unbinding kinetics, to the refinement of docking results. TTMD evaluates the conservation of the native binding mode throughout a series of molecular dynamics simulations performed at progressively increasing temperatures with a scoring function based on protein-ligand interaction fingerprints. The protocol was successfully applied to retrieve the native-like binding pose among a set of decoy poses of drug-like ligands generated on four different pharmaceutically relevant biological targets, including casein kinase 1δ, casein kinase 2, pyruvate dehydrogenase kinase 2, and SARS-CoV-2 main protease.

Discovery of the 3-Amino-1,2,4-triazine-Based Library as Selective PDK1 Inhibitors with Therapeutic Potential in Highly Aggressive Pancreatic Ductal Adenocarcinoma.

Pyruvate dehydrogenase kinases (PDKs) are serine/threonine kinases, that are directly involved in altered cancer cell metabolism, resulting in cancer aggressiveness and resistance. Dichloroacetic acid (DCA) is the first PDK inhibitor that has entered phase II clinical; however, several side effects associated with weak anticancer activity and excessive drug dose (100 mg/kg) have led to its limitation in clinical application. Building upon a molecular hybridization approach, a small library of 3-amino-1,2,4-triazine derivatives has been designed, synthesized, and characterized for their PDK inhibitory activity using in silico, in vitro, and in vivo assays. Biochemical screenings showed that all synthesized compounds are potent and subtype-selective inhibitors of PDK. Accordingly, molecular modeling studies revealed that a lot of ligands can be properly placed inside the ATP-binding site of PDK1. Interestingly, 2D and 3D cell studies revealed their ability to induce cancer cell death at low micromolar doses, being extremely effective against human pancreatic KRAS mutated cancer cells. Cellular mechanistic studies confirm their ability to hamper the PDK/PDH axis, thus leading to metabolic/redox cellular impairment, and to ultimately trigger apoptotic cancer cell death. Remarkably, preliminary in vivo studies performed on a highly aggressive and metastatic Kras-mutant solid tumor model confirm the ability of the most representative compound 5i to target the PDH/PDK axis in vivo and highlighted its equal efficacy and better tolerability profile with respect to those elicited by the reference FDA approved drugs, cisplatin and gemcitabine. Collectively, the data highlights the promising anticancer potential of these novel PDK-targeting derivatives toward obtaining clinical candidates for combatting highly aggressive KRAS-mutant pancreatic ductal adenocarcinomas.

1,2,4-Amino-triazine derivatives as pyruvate dehydrogenase kinase inhibitors: Synthesis and pharmacological evaluation.

Among the different hallmarks of cancer, deregulation of cellular metabolism turned out to be an essential mechanism in promoting cancer resistance and progression. The pyruvate dehydrogenase kinases (PDKs) are well known as key regulators in cells metabolic process and their activity was found to be overexpressed in different metabolic alerted types of cancer, including the high aggressive pancreatic ductal adenocarcinoma (PDAC). To date few PDK inhibitors have been reported, and the different molecules developed are characterized by structural chemical diversity. In an attempt to find novel classes of potential PDK inhibitors, the molecular hybridization approach, which combine two or more active scaffolds in a single structure, was employed. Herein we report the synthesis and the pharmacological evaluation of the novel hybrid molecules, characterized by the fusion of three different pharmacophoric sub-units such as 1,2,4-amino triazines, 7-azaindoles and indoles, in a single structure. The synthesized derivatives demonstrated a promising ability in hampering the enzymatic activity of PDK1 and 4, further confirmed by docking studies. Interestingly, these derivatives retained a strong antiproliferative activity against pancreatic cancer cells either in 2D and 3D models. Mechanistic studies in highly aggressive PDAC cells confirmed their ability to hamper PDK axis and to induce cancer cell death by apoptosis. Moreover, in vivo translational studies in a murine syngeneic solid tumor model confirmed the ability of the most representative compounds to target the PDK system and highlight the ability to reduce the tumor growth without inducing substantial body weight changes in the treated mice.

SARS-CoV-2 3CLpro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376.

Protease inhibitors are among the most powerful antiviral drugs. Nirmatrelvir is the first protease inhibitor specifically developed against the SARS-CoV-2 protease 3CLpro that has been licensed for clinical use. To identify mutations that confer resistance to this protease inhibitor, we engineered a chimeric vesicular stomatitis virus (VSV) that expressed a polyprotein composed of the VSV glycoprotein (G), the SARS-CoV-2 3CLpro, and the VSV polymerase (L). Viral replication was thus dependent on the autocatalytic processing of this precursor protein by 3CLpro and release of the functional viral proteins G and L, and replication of this chimeric VSV was effectively inhibited by nirmatrelvir. Using this system, we applied nirmatrelvir to select for resistance mutations. Resistance was confirmed by retesting nirmatrelvir against the selected mutations in additional VSV-based systems, in an independently developed cellular system, in a biochemical assay, and in a recombinant SARS-CoV-2 system. We demonstrate that some mutants are cross-resistant to ensitrelvir and GC376, whereas others are less resistant to these compounds. Furthermore, we found that most of these resistance mutations already existed in SARS-CoV-2 sequences that have been deposited in the NCBI and GISAID databases, indicating that these mutations were present in circulating SARS-CoV-2 strains.

Streptogramin A derivatives as mitochondrial translation inhibitors to suppress glioblastoma stem cell growth.

New therapeutic strategies for glioblastoma treatment, especially tackling the tumour's glioblastoma stem cell (GSC) component, are an urgent medical need. Recently, mitochondrial translation inhibition has been shown to affect GSC growth, clonogenicity, and self-renewal capability, therefore becoming an attractive therapeutic target. The combination of streptogramins B and A antibiotics quinupristin/dalfopristin (Q/D), which inhibits mitochondrial ribosome function, affects GSCs more effectively in vitro than the standard of care temozolomide. Here, docking calculations based on the cryo-EM structure of the Q/D-bound mitochondrial ribosome have been used to develop a series of streptogramin A derivatives. We obtained twenty-two new and known molecules starting from the dalfopristin and virginiamycin M1 scaffolds. A structure-activity relationship refinement was performed to evaluate the capability of these compounds to suppress GSC growth and inhibit mitochondrial translation, either alone or in combination with quinupristin. Finally, quantitative ultra HPLC-mass spectrometry allowed us to assess the cell penetration of some of these derivatives. Among all, the fluorine derivatives of dalfopristin and virginiamycin M1, (16R)-1e and (16R)-2e, respectively, and flopristin resulted in being more potent than the corresponding lead compounds and penetrating to a greater extent into the cells. We, therefore, propose these three compounds for further evaluation in vivo as antineoplastic agents.

Investigating RNA-protein recognition mechanisms through supervised molecular dynamics (SuMD) simulations.

Ribonucleic acid (RNA) plays a key regulatory role within the cell, cooperating with proteins to control the genome expression and several biological processes. Due to its characteristic structural features, this polymer can mold itself into different three-dimensional structures able to recognize target biomolecules with high affinity and specificity, thereby attracting the interest of drug developers and medicinal chemists. One successful example of the exploitation of RNA's structural and functional peculiarities is represented by aptamers, a class of therapeutic and diagnostic tools that can recognize and tightly bind several pharmaceutically relevant targets, ranging from small molecules to proteins, making use of the available structural and conformational freedom to maximize the complementarity with their interacting counterparts. In this scientific work, we present the first application of Supervised Molecular Dynamics (SuMD), an enhanced sampling Molecular Dynamics-based method for the study of receptor-ligand association processes in the nanoseconds timescale, to the study of recognition pathways between RNA aptamers and proteins, elucidating the main advantages and limitations of the technique while discussing its possible role in the rational design of RNA-based therapeutics.

Qualitative Estimation of Protein-Ligand Complex Stability through Thermal Titration Molecular Dynamics Simulations.

The prediction of ligand efficacy has long been linked to thermodynamic properties such as the equilibrium dissociation constant, which considers both the association and the dissociation rates of a defined protein-ligand complex. In the last 15 years, there has been a paradigm shift, with an increased interest in the determination of kinetic properties such as the drug-target residence time since they better correlate with ligand efficacy compared to other parameters. In this article, we present thermal titration molecular dynamics (TTMD), an alternative computational method that combines a series of molecular dynamics simulations performed at progressively increasing temperatures with a scoring function based on protein-ligand interaction fingerprints for the qualitative estimation of protein-ligand-binding stability. The protocol has been applied to four different pharmaceutically relevant test cases, including protein kinase CK1δ, protein kinase CK2, pyruvate dehydrogenase kinase 2, and SARS-CoV-2 main protease, on a variety of ligands with different sizes, structures, and experimentally determined affinity values. In all four cases, TTMD was successfully able to distinguish between high-affinity compounds (low nanomolar range) and low-affinity ones (micromolar), proving to be a useful screening tool for the prioritization of compounds in a drug discovery campaign.

Implementing a Scoring Function Based on Interaction Fingerprint for Autogrow4: Protein Kinase CK1δ as a Case Study.

In the last 20 years, fragment-based drug discovery (FBDD) has become a popular and consolidated approach within the drug discovery pipeline, due to its ability to bring several drug candidates to clinical trials, some of them even being approved and introduced to the market. A class of targets that have proven to be particularly suitable for this method is represented by kinases, as demonstrated by the approval of BRAF inhibitor vemurafenib. Within this wide and diverse set of proteins, protein kinase CK1δ is a particularly interesting target for the treatment of several widespread neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Computational methodologies, such as molecular docking, are already routinely and successfully applied in FBDD campaigns alongside experimental techniques, both in the hit-discovery and in the hit-optimization stage. Concerning this, the open-source software Autogrow, developed by the Durrant lab, is a semi-automated computational protocol that exploits a combination between a genetic algorithm and a molecular docking software for de novo drug design and lead optimization. In the current work, we present and discuss a modified version of the Autogrow code that implements a custom scoring function based on the similarity between the interaction fingerprint of investigated compounds and a crystal reference. To validate its performance, we performed both a de novo and a lead-optimization run (as described in the original publication), evaluating the ability of our fingerprint-based protocol to generate compounds similar to known CK1δ inhibitors based on both the predicted binding mode and the electrostatic and shape similarity in comparison with the standard Autogrow protocol.