Structure-activity Relationship Research Articles

A Review on Indole as a Cardinal Scaffold for Anticancer Drugs Development

Abstract: Chemotherapy is the mainstay of therapeutic cancer therapy; however, the development of resistance typically makes it less effective. There are continuous efforts by researchers to find novel lead compounds with potent anti-cancer activity. Generally, synthetic or natural heterocyclic compounds have been investigated in detail as a scaffold for cancer therapeutics. Among them, indole, owing to its unique physiochemical and biological properties, provides a promising platform for the development of pharmacophores for drug development against cancer, acting via various mechanisms. Till now, several indole-based derivatives have been identified as anti-cancer agents, which are either being used in clinics or are in various phases of clinical trials, suggesting their importance in anti-cancer drug development. These anti-cancer drugs have been classified into different classes depending on their mechanism of action. For example, histone deacetylase inhibitors (HDAC inhibitors), silent mating type information regulation 2 homolog (SIRT) inhibitors, tubulin inhibitors, proviral insertion site in Moloney murine leukemia virus (Pim) inhibitors, DNA Topoisomerase inhibitors, and kinase inhibitors. In this review, the author's approach is to compile the recent developments on indole-based anti-cancer drugs and provide insight into the respective structureactivity relationships (SARs) of the compounds. We hope the review will provide a thorough understanding to the reader and guide to developing novel and potent indole-based anticancer agents against drug-sensitive and drug-resistant cancer in the future.

Insight into the Synthesis, Biological Activity, and Structure-activity Relationship of 1,2,4-Oxadiazole and Analogs: A Comprehensive Review

Abstract: A five-membered heterocyclic compound known as 1,2,4-oxadiazole has one oxygen, two carbon, and two nitrogen atoms within a ring. Numerous studies have shown that 1,2,4-oxadiazole has the potential to be an essential moiety in many harmful pharmacological conditions. 1,2,4-oxadiazole and its derivatives have been used as an antiviral, cough suppressant, vasodilator, anxiolytic, etc. To synthesize a single molecule with potent pharmacological action, two or more pharmacophores are combined currently. This enhances pharmacological effects, facilitates interaction possible with many targets, and reduces the adverse effects related to them. It has been reported that synthesis of 1,2,4-oxadiazole and its analog is possible using a variety of methods. In this present review, we emphasized recently accepted synthetic methods for 1,2,4-oxadiazole and its analogs synthesis. Additionally, the structure-activity relationship concerning several pharmacological effects, interactions with various targets, and the utility of different techniques for the analysis and purification has been described. On the behalf of the above study, the researchers can use this review study to better understand their 1,2,4-oxadiazole research in the future.

Thiazolidinedione as a Promising Medicinal Scaffold for the Treatment of Type 2 Diabetes.

Thiazolidinediones, also known as glitazones, are considered as biologically active scaffold and a well-established class of anti-diabetic agents for the treatment of type 2 diabetes mellitus. Thiazolidinediones act by reducing insulin resistance through elevated peripheral glucose disposal and glucose production. These molecules activate peroxisome proliferated activated receptor (PPARγ), one of the sub-types of PPARs, and a diverse group of its hybrid have also shown numerous therapeutic activities along with antidiabetic activity. The objective of this review was to collect and summarize the research related to the medicinal potential, structure-activity relationship and safety aspects of thiazolidinedione analogues designed and investigated in type 2 diabetes during the last two decades. The mentioned objective was achieved by collecting and reviewing the research manuscripts, review articles, and patents from PubMed, Science Direct, Embase, google scholar and journals related to the topic from different publishers like Wiley, Springer, Elsevier, Taylor and Francis, Indian and International government patent sites etc. Results: The thiazolidinedione scaffold has been a focus of research in the design and synthesis of novel derivatives for the management of type 2 diabetes, specifically in the case of insulin resistance. The complications like fluid retention, idiosyncratic hepatotoxicity, weight gain and congestive heart failure in the case of trosiglitazone, and pioglitazone have restricted their use. The newer analogues have been synthesized by different research groups to attain better efficacy and less side effects. Thus, the potential of thiazolidinediones in terms of their chemical evolution, action on nuclear receptors, aldose reductase and free fatty acid receptor 1 is well established. The newer TZD analogues with better safety profiles and tolerability will soon be available in the market for common use without further delay.

Heparin Oligosaccharides as Vasoactive Intestinal Peptide Inhibitors via their Binding Process Characterization.

It has been proven that vasoactive intestinal peptide (VIP) was involved in the pathogenesis of prostate cancer. Cardin et al. found that by an alanine scan, the heparin- binding site on VIP was exactly the same sequence in VIP and its receptor. Therefore, heparin could competitively block the binding of VIP and its receptor. However, the structure-activity relationship between heparin and VIP has not been reported, especially in terms of the sequence and sulfation patterns of heparin oligosaccharides upon binding to VIP. A variety of experiments were designed to study the binding process and structure-activity relationship between heparin oligosaccharides and VIP. Heparin was enzymatically digested and purified to produce heparin oligosaccharides, and the structures were characterized by NMR. The binding capacity between heparin oligosaccharides and VIP was analyzed by GMSA and ITC experiments. The binding between heparin oligosaccharides and VIP was simulated using a molecular docking program to show the complex. ELISA assay was used to investigate the effect of non-anticoagulant heparin oligosaccharides on the VIP-mediated cAMP/PKA signaling pathway in vitro. The results indicated that both the length and the sulfation pattern of heparin oligosaccharides affected its binding to VIP. VIP could induce the expression of cAMP at a higher level in PC3 cells, which could be regulated by the interaction of heparin oligosaccharides and VIP. The binding between heparin oligosaccharides and VIP could block the binding between VIP and its receptor on tumor cells. Downloading the regulation of the expression level of cAMP could possibly further affect the subsequent activation of PKA. These non-anticoagulant heparin oligosaccharides may block the VIP-mediated cAMP/PKA signaling pathway and thus exert their antitumor activity.

QSAR Studies on a Series of Pyrazole Azabicyclo [3.2.1] Octane Sulfonamides N-acylethanolamine-hydrolyzing Acid Amidase Inhibitors

Background: Inflammation is a common and intractable disease for humans. Current antiinflammatory drugs have a lot of side effects, which cause irreversible damage to the body. Objective: We predict the activity of the N-acylethanolamine-hydrolyzing acid amidase (NAAA) inhibitor to find more effective compounds. Methods: We established a quantitative structure-activity relationship (QSAR) model by gene expression programming to predict the IC50 values of natural compounds. The NAAA inhibitor, as a cysteine enzyme, plays an important role in the therapy of pain, anti-inflammatory effects and application of other diseases. A total of 36 NAAA inhibitors were optimized by the heuristic method in the CODESSA program to build a linear model. The 27 compounds and 9 compounds were in train and test sets. On this basis, we selected three descriptors and used them to build nonlinear models in gene expression programming. Results: The best model in the gene expression programming method was found, the square of correlation coefficients of R2 and mean square error for the training set were 0.79 and 0.14, testing set was 0.78 and 0.20, respectively. Conclusion: From this method, the activity of molecules could be predicted, and the best method was found. Therefore, this model has a stronger predictive ability to develop NAAA inhibitors.

On Certain Degree Based and Bond-additive Topological Indices of Dodeca-benzo-circumcorenene

Background: Chemical graph theory has been used to mathematically model the various physical and biological aspects of chemical substances. A mathematical formulation that may be applied to any graph and can characterise a molecule structure is known as a topological index or molecular descriptor. Objective: It is convenient and efficient to analyse the mathematical values and further research on various physical properties of a molecule based on these molecular descriptors. They provide useful alternatives to lengthy, expensive, and labour-intensive laboratory experiments. The topological indices can be used to predict the chemical structures, physicochemical properties, and biological activities using quantitative structure-activity relationships (QSARs) and quantitative structure-property relationships (QSPRs). Result: In this study, the molecular descriptors of the Dodeca-benzo-circumcorenene compounds are derived based on their corresponding molecular structures. Conclusion: The computed indices are then compared graphically to study their relationship with the molecular structure and with each other.

Research progress in Estrogen-related receptor gamma (ERRγ) agonists and inverse agonists.

Estrogen-related receptor gamma (ERRγ), one of three members of the ERR family, is an inducible transcription factor. ERRγ has dual functions in different tissues. The decreased expression of ERRγ in the brain, stomach, prostate, and fat cells can cause neuropsychological dysfunction, gastric cancer, prostate cancer, and obesity. However, when ERRγ is present in the liver, pancreas, and thyroid follicular cells, ERRγ overexpression is related to liver cancer, type II diabetes, oxidative liver injury, and anaplastic thyroid carcinoma. Signaling pathway studies have confirmed that ERRγ agonists or inverse agonists can regulate ERRγ expression to treat related diseases. The collision between residue Phe435 and the modulator is a key factor determining the activation or inhibition of ERRγ. Although more than 20 agonists and inverse agonists of ERRγ have been reported, no clinical studies have been found in the literature. This review summarizes the important relationship between ERRγ-related signaling pathways and diseases, research progress, and the structure-activity relationship of modulators. These findings provide guidance for further study on new ERRγ modulators.

Calcium Modulating Effect of Polycyclic Cages: A Suitable Therapeutic Approach Against Excitotoxic-induced Neurodegeneration.

Neurodegenerative disorders pose a significant challenge to global healthcare systems due to their progressive nature and the resulting loss of neuronal cells and functions. Excitotoxicity, characterized by calcium overload, plays a critical role in the pathophysiology of these disorders. In this review article, we explore the involvement of calcium dysregulation in neurodegeneration and neurodegenerative disorders. A promising therapeutic strategy to counter calcium dysregulation involves the use of calcium modulators, particularly polycyclic cage compounds. These compounds, structurally related to amantadine and memantine, exhibit neuroprotective properties by attenuating calcium influx into neuronal cells. Notably, the pentacycloundecylamine NGP1-01, a cage-like structure, has shown efficacy in inhibiting both N-methyl-D-aspartate (NMDA) receptors and voltage-gated calcium channels (VGCCs), making it a potential candidate for neuroprotection against excitotoxic-induced neurodegenerative disorders. The structure-activity relationship of polycyclic cage compounds is discussed in detail, highlighting their calcium-inhibitory activities. Various closed, open, and rearranged cage compounds have demonstrated inhibitory effects on calcium influx through NMDA receptors and VGCCs. Additionally, these compounds have exhibited neuroprotective properties, including free radical scavenging, attenuation of neurotoxicities, and reduction of neuroinflammation. Although the calcium modulatory activities of polycyclic cage compounds have been extensively studied, apart from amantadine and memantine, none have undergone clinical trials. Further in vitro and in vivo studies and subsequent clinical trials are required to establish the efficacy and safety of these compounds. The development of polycyclic cages as potential multifunctional agents for treating complex neurodegenerative diseases holds great promise.

Bridged Pt-OH-Mn Mediator in N-coordinated Mn Single Atoms and Pt Nanoparticles for Electrochemical Biomolecule Oxidation and Discrimination.

The rational design of efficient catalysts for uric acid (UA) electrooxidation, as well as the establishment of structure-activity relationships, remains a critical bottleneck in the field of electrochemical sensing. To address these challenges, herein, a hybrid catalyst that integrates carbon-supported Pt nanoparticles and nitrogen-coordinated Mn single atoms (PtNPs/MnNC) is developed. The metal-metal interaction during annealing affords the construction of metallic-bonded Pt-Mn pairs between PtNPs and Mn single atoms, facilitating the electron transfer from PtNPs to the support and thereby optimizing the electronic structure of catalysts. More importantly, experiments and theoretical calculations provide visual proof for the 'incipient hydrous oxide adatom mediator' mechanism for UA oxidation. The Pt-Mn pairs first adsorb OH* to construct the bridged Pt-OH-Mn mediators to serve as a highly active intermediate for N-H bond dissociation and proton transfer. Benefiting from the unique electronic and geometric structure of the catalytic center and reactive intermediates, PtNPs/MnNC exhibits superior electrooxidation performance. The electrochemical sensor based on PtNPs/MnNC enables sensitive detection and discrimination of UA and dopamine in serum samples. This work offers new insights into the construction of novel electrocatalysts for sensitive sensing platforms.

Total Synthesis of (-)-Piericone D.

The total synthesis of (-)-piericone D, a potential antithrombotic dihydrochalcone featuring an [3.3.0] octane core, is reported. Salient features of our synthesis include a stereoselective β-O-glycosylation to install the asebogenin aglycone and a late-stage global deprotection followed by simultaneous lactonization. The convergent synthesis paved the way for further structure-activity relationship (SAR) studies of (-)-piericone D.

Progressive Optimization of Lanthanide Nanoparticle Scintillators for Enhanced Triple‐Activated Radioluminescence Imaging

AbstractLanthanide nanoparticle (LnNP) scintillators exhibit huge potential in achieving radionuclide‐activated luminescence (radioluminescence, RL). However, their structure–activity relationship remains largely unexplored. Herein, progressive optimization of LnNP scintillators is presented to unveil their structure‐dependent RL property and enhance their RL output efficiency. Benefiting from the favorable host matrix and the luminescence‐protective effect of core–shell engineering, NaGdF4 : 15 %Eu@NaLuF4 nanoparticle scintillators with tailored structures emerged as the top candidates. Living imaging experiments based on optimal LnNP scintillators validated the feasibility of laser‐free continuous RL activated by clinical radiopharmaceuticals for tumor multiplex visualization. This research provides unprecedented insights into the rational design of LnNP scintillators, which would enable efficient energy conversion from Cerenkov luminescence, γ‐radiation, and β‐electrons into visible photon signals, thus establishing a robust nanotechnology‐aided approach for tumor‐directed radio‐phototheranostics.

Progressive Optimization of Lanthanide Nanoparticle Scintillators for Enhanced Triple-Activated Radioluminescence Imaging.

Lanthanide nanoparticle (LnNP) scintillators exhibit huge potential in achieving radionuclide-activated luminescence (radioluminescence, RL). However, their structure-activity relationship remains largely unexplored. Herein, progressive optimization of LnNP scintillators is presented to unveil their structure-dependent RL property and enhance their RL output efficiency. Benefiting from the favorable host matrix and the luminescence-protective effect of core-shell engineering, NaGdF4 : 15 %Eu@NaLuF4 nanoparticle scintillators with tailored structures emerged as the top candidates. Living imaging experiments based on optimal LnNP scintillators validated the feasibility of laser-free continuous RL activated by clinical radiopharmaceuticals for tumor multiplex visualization. This research provides unprecedented insights into the rational design of LnNP scintillators, which would enable efficient energy conversion from Cerenkov luminescence, γ-radiation, and β-electrons into visible photon signals, thus establishing a robust nanotechnology-aided approach for tumor-directed radio-phototheranostics.

Anticancer Potential of the S-Heterocyclic Ring Containing Drugs and its Bioactivation to Reactive Metabolites.

Sulfur-containing heterocyclic derivatives have been disclosed for binding with a wide range of cancer-specific protein targets. Various interesting derivatives of sulfur-containing heterocyclics such as benzothiazole, thiazole, thiophene, thiazolidinedione, benzothiophene, and phenothiazine, etc have been shown to inhibit diverse signaling pathways implicated in cancer. Significant progress has also been made in molecular targeted therapy against specific enzymes such as kinase receptors due to potential binding interactions inside the ATP pocket. Sulfur-containing heterocyclic ring metal complexes i. e., benzothiazole, thiazole, thiophene, benzothiophene and phenothiazines are among the most promising active anticancer compounds. However, sulfur heteroaromatic rings, particularly thiophene, are of high structural alert due to their metabolism to reactive metabolites. The mere presence of a structural alert itself does not determine compound toxicity therefore, this review focuses on some specific findings that shed light on factors influencing the toxicity. In the current review, synthetic strategies of introducing the sulfur core ring in the synthesized derivatives are discussed with their structure-activity relationships to enhance our understanding of toxicity mechanisms and develop safer therapeutic options. The sulfur-containing marketed anticancer drugs included in this review direct the synthesis of novel compounds and will help in the development of potent, safer sulfur-based anticancer drugs in near future.

Task-Similarity is a Crucial Factor for Few-Shot Meta-Learning of Structure-Activity Relationships.

Machine learning models support computer-aided molecular design and compound optimization. However, the initial phases of drug discovery often face a scarcity of training data for these models. Meta-learning has emerged as a potentially promising strategy, harnessing the wealth of structure-activity data available for known targets to facilitate efficient few-shot model training for the specific target of interest. In this study, we assessed the effectiveness of two different meta-learning methods, namely model-agnostic meta-learning (MAML) and adaptive deep kernel fitting (ADKF), specifically in the regression setting. We investigated how factors such as dataset size and the similarity of training tasks impact predictability. The results indicate that ADKF significantly outperformed both MAML and a single-task baseline model on the inhibition data. However, the performance of ADKF varied across different test tasks. Our findings suggest that considerable enhancements in performance can be anticipated primarily when the task of interest is similar to the tasks incorporated in the meta-learning process.

Fluorescent small molecule donors.

Small molecule donors (SMDs) play subtle roles in the signaling mechanism and disease treatments. While many excellent SMDs have been developed, dosage control, targeted delivery, spatiotemporal feedback, as well as the efficiency evaluation of small molecules are still key challenges. Accordingly, fluorescent small molecule donors (FSMDs) have emerged to meet these challenges. FSMDs enable controllable release and non-invasive real-time monitoring, providing significant advantages for drug development and clinical diagnosis. Integration of FSMDs with chemotherapeutic, photodynamic or photothermal properties can take full advantage of each mode to enhance therapeutic efficacy. Given the remarkable properties and the thriving development of FSMDs, we believe a review is needed to summarize the design, triggering strategies and tracking mechanisms of FSMDs. With this review, we compiled FSMDs for most small molecules (nitric oxide, carbon monoxide, hydrogen sulfide, sulfur dioxide, reactive oxygen species and formaldehyde), and discuss recent progress concerning their molecular design, structural classification, mechanisms of generation, triggered release, structure-activity relationships, and the fluorescence response mechanism. Firstly, from the large number of fluorescent small molecular donors available, we have organized the common structures for producing different types of small molecules, providing a general strategy for the development of FSMDs. Secondly, we have classified FSMDs in terms of the respective donor types and fluorophore structures. Thirdly, we discuss the mechanisms and factors associated with the controlled release of small molecules and the regulation of the fluorescence responses, from which universal guidelines for optical properties and structure rearrangement were established, mainly involving light-controlled, enzyme-activated, reactive oxygen species-triggered, biothiol-triggered, single-electron reduction, click chemistry, and other triggering mechanisms. Fourthly, representative applications of FSMDs for trackable release, and evaluation monitoring, as well as for visible in vivo treatment are outlined, to illustrate the potential of FSMDs in drug screening and precision medicine. Finally, we discuss the opportunities and remaining challenges for the development of FSMDs for practical and clinical applications, which we anticipate will stimulate the attention of researchers in the diverse fields of chemistry, pharmacology, chemical biology and clinical chemistry. With this review, we hope to impart new understanding thereby enabling the rapid development of the next generation of FSMDs.

In Silico Prediction of Skin Sensitization for Compounds via Flexible Evidence Combination Based on Machine Learning and Dempster-Shafer Theory.

Skin sensitization is increasingly becoming a significant concern in the development of drugs and cosmetics due to consumer safety and occupational health problems. In silico methods have emerged as alternatives to traditional in vivo animal testing due to ethical and economic considerations. In this study, machine learning methods were used to build quantitative structure-activity relationship (QSAR) models on five skin sensitization data sets (GPMT, LLNA, DPRA, KeratinoSens, and h-CLAT), achieving effective predictive accuracies (correct classification rates of 0.688-0.764 on test sets). To address the complex mechanisms of human skin sensitization, the Dempster-Shafer theory was applied to merge multiple QSAR models, resulting in an evidence-based integrated decision model. Various evidence combinations and combination rules were explored, with the self-defined Q3 rule showing superior balance. The combination of evidence such as GPMT and KeratinoSens and h-CLAT achieved a correct classification rate (CCR) of 0.880 and coverage of 0.893 while maintaining the competitiveness of other combinations. Additionally, the Shapley additive explanations (SHAP) method was used to interpret important features and substructures related to skin sensitization. A comparative analysis of an external human test set demonstrated the superior performance of the proposed method. Finally, to enhance accessibility, the workflow was implemented into a user-friendly software named HSkinSensDS.

Bicyclic Pyrrolidine Inhibitors of Toxoplasma gondii Phenylalanine t-RNA Synthetase with Antiparasitic Potency In Vitro and Brain Exposure.

Previous studies have shown that bicyclic azetidines are potent and selective inhibitors of apicomplexan phenylalanine tRNA synthetase (PheRS), leading to parasite growth inhibition in vitro and in vivo, including in models of Toxoplasma infection. Despite these useful properties, additional optimization is required for the development of efficacious treatments of toxoplasmosis from this inhibitor series, in particular, to achieve optimal exposure in the brain. Here, we describe a series of PheRS inhibitors built on a new bicyclic pyrrolidine core scaffold designed to retain the exit-vector geometry of the isomeric bicyclic azetidine core scaffold while offering avenues to sample diverse chemical space. Relative to the parent series, bicyclic pyrrolidines retain reasonable potency and target selectivity for parasite PheRS vs host. Further structure-activity relationship studies revealed that the introduction of aliphatic groups improved potency and ADME and PK properties, including brain exposure. The identification of this new scaffold provides potential opportunities to extend the analogue series to further improve selectivity and potency and ultimately deliver a novel, efficacious treatment of toxoplasmosis.

Systematic Structure-Activity Relationship Study of Nalfurafine Analogues toward Development of Potentially Nonaddictive Pain Management Treatments.

Despite the availability of numerous pain medications, the current array of Food and Drug Administration-approved options falls short in adequately addressing pain states for numerous patients and consequently worsens the opioid crisis. Thus, it is imperative for basic research to develop novel and nonaddictive pain medications. Toward addressing this clinical goal, nalfurafine (NLF) was chosen as a lead and its structure-activity relationship (SAR) systematically studied through design, syntheses, and in vivo characterization of 24 analogues. Two analogues, 21 and 23, showed longer durations of action than NLF in a warm-water tail immersion assay, produced in vivo effects primarily mediated by KOR and DOR, penetrated the blood-brain barrier, and did not function as reinforcers. Additionally, 21 produced fewer sedative effects than NLF. Taken together, these results aid the understanding of NLF SAR and provide insights for future endeavors in developing novel nonaddictive therapeutics to treat pain.

Design, Synthesis, and Structure-Activity Relationships of Novel Peptide Derivatives of the Severe Acute Respiratory Syndrome-Coronavirus-2 Spike-Protein that Potently Inhibit Nicotinic Acetylcholine Receptors.

The spike-protein of SARS-CoV-2 has a distinctive amino-acid sequence (682RRARS686) that forms a cleavage site for the enzyme furin. Strikingly, the structure of the spike-protein loop containing the furin cleavage site bears substantial similarity to neurotoxin peptides found in the venoms of certain snakes and marine cone snails. Leveraging this relationship, we designed and synthesized disulfide-constrained peptides with amino-acid sequences corresponding to the furin cleavage-sites of wild-type (B.1 variant) SARS-CoV-2 or the Alpha, Delta, and Omicron variants. Remarkably, some of these peptides potently inhibited α7 and α9α10 nicotinic acetylcholine receptors (nAChR) with nM affinity and showed SARS-CoV-2 variant and nAChR subtype-dependent potencies. Nuclear magnetic resonance spectroscopy and molecular dynamics were used to rationalize structure-activity relationships between peptides and their cognate receptors. These findings delineate nAChR subtypes that can serve as high-affinity spike-protein targets in tissues central to COVID-19 pathophysiology and identify ligands and target receptors to inform the development of novel SARS-CoV-2 therapeutics.

Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CLpro main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

SARS-CoV-2 3C-like main protease (3CLpro) is essential for protein excision from the viral polyprotein. 3CLpro inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6-P6' specificity for 3CLpro from >800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS). Cleavage occurred after the canonical P1-Gln and non-canonical P1-His and P1-Met residues. Moreover, P3 showed a preference for Arg/Lys and P3' for His. Essential H-bonds between the N-terminal Ser1 of protomer-B in 3CLpro dimers form with P1-His, but not with P1-Met. Nonetheless, cleavage occurs at P1-Met456 in native MAP4K5. Elevated reactive oxygen species in SARS-CoV-2 infection oxidize methionines. Molecular simulations revealed P1-MetOX forms an H-bond with Ser1 and notably, strong positive cooperativity between P1-Met with P3'-His was revealed, which enhanced peptide-cleavage rates. The highly plastic S3' subsite accommodates P3'-His that displays stabilizing backbone H-bonds with Thr25 lying central in a "'threonine trio" (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CLpro-substrate interactions, and the role of these structural determinants was confirmed by MALDI-TOF-MS cleavage assays of P1'- and P3'-positional scanning peptide libraries carrying a 2nd optimal cut-site as an internal positive control. These data informed the design of two new and highly soluble 3CLproquenched-fluorescent peptide substrates for improved FRET monitoring of 3CLpro activity with 15× improved sensitivity over current assays.IMPORTANCEFrom global proteomics identification of >800 cleavage sites, we characterized the P6-P6' active site specificity of SARS-CoV-2 3CLpro using proteome-derived peptide library screens, molecular modeling simulations, and focussed positional peptide libraries. In P1', we show that alanine and serine are cleaved 3× faster than glycine and the hydrophobic small amino acids Leu, Ile, or Val prevent cleavage of otherwise optimal non-prime sequences. In characterizing non-canonical non-prime P1 specificity, we explored the unusual P1-Met specificity, discovering enhanced cleavage when in the oxidized state (P1-MetOX). We unveiled unexpected amino acid cooperativity at P1-Met with P3'-His and noncanonical P1-His with P2-Phe, and the importance of the threonine trio (Thr24-Thr25-Thr26) in the prime side binding domain I in defining prime side binding in SARS-CoV-2 3CLpro. From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with >15× improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs.