Modest Affinity Research Articles

The identification of insect specific iAANAT inhibitors.

An important aspect of food security is the development of innovative insecticides, particularly ones that specifically target insect pests and exhibit minimal toxicity to mammals. The insect arylalkylamine N-acyltransferases (iAANATs) could serve as targets for novel insecticides that satisfy these criteria. There exists a wealth of structural and biochemical information for the iAANATs and iAANAT knockdown experiments show that these enzymes are critical to insect health. Herein, we have expressed, purified, and characterized two new iAANATs, one from Apis mellifera (honey bee, AmNAT1) and another from Diaphorina citri (Asian citrus psyllid, DcNAT). We discovered that diminazene, a compound used to treat livestock for trypanosomiasis and babesiosis, inhibits AmNAT1, DcNAT, and D. melanogaster DmAgmNAT with modest affinity, Ki values ranging from 0.8μM to 200μM. We found a series of guanidines, amidines, and a hydroxamate, structurally related to diminazene, also inhibit the iAANATs, including camostat, gabexate, nafamostate, and panobinostat. Significantly, we found DmAgmNAT is far more susceptible to inhibition by four of these five of these compounds. In particular, camostat, nafamostat, and gabexate inhibit DmAgmNAT with Ki values of 0.2-30μM, but no inhibition of AmNAT1 and DcNAT was observed at 500μM for any of the three. These results show that a species-specific inhibitor targeted against an iAANAT is a real possibility. Also, we report that adipoyl-CoA is a substrate for AmNAT1 and DcNAT and that succinoyl-CoA is a substrate for DcNAT. These results contribute to a growing body of data suggesting that N-dicarboxyacyl-amines are metabolites in insects and other organisms.

Single-chain nanobody inhibition of Notch and avidity enhancement utilizing the β-pore forming toxin Aerolysin.

Notch plays critical roles in developmental processes and disease pathogenesis, which has led to numerous efforts to modulate its function with small molecules and antibodies. Here we present a nanobody inhibitor of Notch signaling, derived from a synthetic phage-display library targeting the notch Negative Regulatory Region (NRR). The nanobody inhibits Notch signaling in a luciferase reporter assay and in Notch-dependent hematopoietic progenitor cell differentiation assay, despite a modest 19uM affinity for Notch. We addressed the low affinity by fusion to a membrane-associating domain derived from the β-Pore forming toxin Aerolysin, resulting in a significantly improved IC50 for Notch inhibition. The nanobody-aerolysin fusion inhibits proliferation of T-ALL cell lines with similar efficacy to other Notch pathway inhibitors. Overall, this study reports the development of a Notch inhibitory antibody, and demonstrates a proof-of-concept for a generalizable strategy to increase the efficacy and potency of low-affinity antibody binders.

Structure-Based Design of CBP/EP300 Degraders: When Cooperativity Overcomes Affinity.

We present the development of dCE-2, a structurally novel PROTAC targeting the CREB-binding protein (CBP) and E1A-associated protein (EP300)-two homologous multidomain enzymes crucial for enhancer-mediated transcription. The design of dCE-2 was based on the crystal structure of an in-house bromodomain (BRD) inhibitor featuring a 3-methyl-cinnoline acetyl-lysine mimic acetyl-lysine mimic discovered by high-throughput fragment docking. Our study shows that, despite its modest binding affinity to CBP/EP300-BRD, dCE-2's remarkable protein degradation activity stems from its good cooperativity, which we demonstrate by the characterization of its ternary complex formation both in vitro and in cellulo. Molecular dynamics simulations indicate that in aqueous solvents, this active degrader populates both folded and extended conformations, which are likely to promote cell permeability and ternary complex formation, respectively.

Identification of small-molecule ligand-binding sites on and in the ARNT PAS-B domain

Transcription factors are challenging to target with small molecule inhibitors due to their structural plasticity and lack of catalytic sites. Notable exceptions include naturally ligand-regulated transcription factors, including our prior work with the HIF-2 transcription factor, showing that small molecule binding within an internal pocket of the HIF-2α PAS-B domain can disrupt its interactions with its dimerization partner, ARNT. Here, we explore the feasibility of targeting small molecules to the analogous ARNT PAS-B domain itself, potentially opening a promising route to modulate several ARNT-mediated signaling pathways. Using solution NMR fragment screening, we previously identified several compounds that bind ARNT PAS-B and, in certain cases, antagonize ARNT association with the TACC3 transcriptional coactivator. However, these ligands have only modest binding affinities, complicating characterization of their binding sites. We address this challenge by combining NMR, MD simulations, and ensemble docking to identify ligand-binding 'hotspots' on and within the ARNT PAS-B domain. Our data indicate that the two ARNT/TACC3 inhibitors, KG-548 and KG-655, bind to a β-sheet surface implicated in both HIF-2 dimerization and coactivator recruitment. Furthermore, while KG-548 binds exclusively to the β-sheet surface, KG-655 can additionally bind within a water-accessible internal cavity in ARNT PAS-B. Finally, KG-279, while not a coactivator inhibitor, exemplifies ligands that preferentially bind only to the internal cavity. All three ligands promoted ARNT PAS-B homodimerization, albeit to varying degrees. Taken together, our findings provide a comprehensive overview of ARNT PAS-B ligand-binding sites and may guide the development of more potent coactivator inhibitors for cellular and functional studies.

Transcriptome-wide mapping of small-molecule RNA-binding sites in live cells.

Small molecules targeting RNA can be valuable chemical probes and potential therapeutics. The interactions between small molecules, particularly fragments, and RNA, however, can be difficult to detect due to their modest affinities and short residence times. Here, we describe the procedures for mapping the molecular fingerprints of small molecules in vitro and throughout the human transcriptome in live cells, identifying both the targets bound by the small molecule and the sites of binding therein. For complete details on the use and execution of this protocol, please refer to 1.

LiF-MS+, a revised technique for mapping peptide-protein interactions.

Short linear motifs are sequences of amino acids present in unstructured polypeptide regions that function as ligands for specific sites on folded protein domains. These interactions, which often occur with low to modest affinity, modulate dynamic biological processes such as signal transduction and membrane trafficking. We recently described Ligand Footprinting-Mass Spectrometry (LiF-MS), a technique that rapidly and precisely maps sites at which short peptide ligands bind their biologically relevant recognition sites on folded protein domains. This approach marks the binding location of a peptide ligand on a structured protein using a cleavable crosslinker appended to the ligand that leaves behind a stable chemical modification following cleavage. This modification serves as a mass tag detectable by mass spectrometry, pinpointing sites of peptide ligand binding. Here we present LiF-MS+, an improved version of the footprinting technique that replaces the butanol mass tag with 1-butylpyrrolidine, which is positively charged at neutral pH and thus aids in ionization of the crosslinked peptide for analysis by mass spectrometry. We show ligand-mediated butylpyrrolidine footprinting effectively maps the well characterized binding interaction of the p38α mitogen-activated protein kinase (MAPK) with a MKK6 D-motif short linear motif peptide ligand, uncovering additional binding site information not observed in our original experiment. LiF-MS+ is thus a straightforward improvement of our previously published methodology for mapping the binding of short linear motifs to folded protein domains.

Aromatic linker variations in novel dopamine D2 and D3 receptor ligands.

Dopamine D2-like receptors, especially D2 and D3 receptor subtypes, are important targets of antipsychotic agents. Many of these antipsychotics share an aliphatic linker element between a protonable amine group and an acyl-like moiety. Here, we have modified this aliphatic linker into phenylmethyl and phenylethyl linkers substituted in different positions. The design, synthesis, and in vitro evaluation of 18 dopamine D2 and D3 receptor ligands were performed in this study. Using a radioligand displacement assay, all ligands were found to have modest nanomolar affinity to D2R and D3R. N-(4-{2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl}phenyl)acetamide (6c) demonstrates the highest D3R and D2R affinity values (pKi values of 7.83 [D2R] and 8.04 [D3R]), featuring a slight preference to D3R. This derivative can be taken as a reference structure for the development of a new class of D2R and D3R ligands.

Phage-Displayed SH2 Domain Libraries: From Ultrasensitive Tyrosine Phosphoproteome Probes to Translational Research.

Tyrosine phosphorylation is a critical regulator of cell signaling. A large fraction of the tyrosine phosphoproteome, however, remains uncharacterized, largely due to a lack of robust and scalable methods. The Src homology 2 (SH2) domain, a structurally conserved protein domain present in many intracellular signal-transducing proteins, naturally binds phosphorylated tyrosine (pTyr) residues, providing an ideal scaffold for the development of sensitive pTyr probes. Its modest affinity, however, has greatly limited its application. Phage display is an in vitro technique used for identifying ligands for proteins and other macromolecules. Using this technique, researchers have been able to engineer SH2 domains to increase their affinity and customize their specificity. Indeed, highly diverse phage display libraries have enabled the engineering of SH2 domains as affinity-purification (AP) tools for proteomic analysis as well as probes for aberrant tyrosine signaling detection and rewiring, and represent a promising class of novel diagnostics and therapeutics. This review describes the unique structure-function characteristics of SH2 domains, highlights the fundamental contribution of phage display in the development of technologies for the dissection of the tyrosine phosphoproteome, and highlights prospective uses of SH2 domains in basic and translational research.

A Lipopeptidomimetic of Transcriptional Activation Domains Selectively Disrupts the Coactivator Med25 Protein-Protein Interactions.

Short amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium-chain, branched fatty acid to the N-terminus of one such heptameric lipopeptidomimetic (LPPM-8) increases the affinity for the coactivator Med25 >20-fold (Ki >100 μM to 4 μM), rendering it an effective inhibitor of Med25 protein-protein interactions (PPIs). The lipid structure, the peptide sequence, and the C-terminal functionalization of the lipopeptidomimetic each influence the structural propensity of LPPM-8 and its effectiveness as an inhibitor. LPPM-8 engages Med25 through interaction with the H2 face of its activator interaction domain and in doing so stabilizes full-length protein in the cellular proteome. Further, genes regulated by Med25-activator PPIs are inhibited in a cell model of triple-negative breast cancer. Thus, LPPM-8 is a useful tool for studying Med25 and mediator complex biology and the results indicate that lipopeptidomimetics may be a robust source of inhibitors for activator-coactivator complexes.

A Lipopeptidomimetic of Transcriptional Activation Domains Selectively Disrupts the Coactivator Med25 Protein–Protein Interactions

AbstractShort amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium‐chain, branched fatty acid to the N‐terminus of one such heptameric lipopeptidomimetic (LPPM‐8) increases the affinity for the coactivator Med25 >20‐fold (Ki >100 μM to 4 μM), rendering it an effective inhibitor of Med25 protein–protein interactions (PPIs). The lipid structure, the peptide sequence, and the C‐terminal functionalization of the lipopeptidomimetic each influence the structural propensity of LPPM‐8 and its effectiveness as an inhibitor. LPPM‐8 engages Med25 through interaction with the H2 face of its activator interaction domain and in doing so stabilizes full‐length protein in the cellular proteome. Further, genes regulated by Med25‐activator PPIs are inhibited in a cell model of triple‐negative breast cancer. Thus, LPPM‐8 is a useful tool for studying Med25 and mediator complex biology and the results indicate that lipopeptidomimetics may be a robust source of inhibitors for activator‐coactivator complexes.

The Cyclic Imine Core Common to the Marine Macrocyclic Toxins Is Sufficient to Dictate Nicotinic Acetylcholine Receptor Antagonism.

Macrocyclic imine phycotoxins are an emerging class of chemical compounds associated with harmful algal blooms and shellfish toxicity. Earlier binding and electrophysiology experiments on nAChR subtypes and their soluble AChBP surrogates evidenced common trends for substantial antagonism, binding affinities, and receptor-subtype selectivity. Earlier, complementary crystal structures of AChBP complexes showed that common determinants within the binding nest at each subunit interface confer high-affinity toxin binding, while distinctive determinants from the flexible loop C, and either capping the nest or extending toward peripheral subsites, dictate broad versus narrow receptor subtype selectivity. From these data, small spiroimine enantiomers mimicking the functional core motif of phycotoxins were chemically synthesized and characterized. Voltage-clamp analyses involving three nAChR subtypes revealed preserved antagonism for both enantiomers, despite lower subtype specificity and binding affinities associated with faster reversibility compared with their macrocyclic relatives. Binding and structural analyses involving two AChBPs pointed to modest affinities and positional variability of the spiroimines, along with a range of AChBP loop-C conformations denoting a prevalence of antagonistic properties. These data highlight the major contribution of the spiroimine core to binding within the nAChR nest and confirm the need for an extended interaction network as established by the macrocyclic toxins to define high affinities and marked subtype specificity. This study identifies a minimal set of functional pharmacophores and binding determinants as templates for designing new antagonists targeting disease-associated nAChR subtypes.

Exploring Product Release from Yeast Cytosine Deaminase with Metadynamics.

The yeast cytosine deaminase (yCD) enzyme/5-fluorocytosine prodrug system is a promising candidate for targeted chemotherapeutics. After conversion of the prodrug into the toxic chemotherapeutic drug, 5-fluorouracil (5-FU), the slow product release from the enzyme limits the overall catalytic efficiency of the enzyme/prodrug system. Here, we present a computational study of the product release of the anticancer drug, 5-FU, from yCD using metadynamics. We present a comparison of the 5-FU drug to the natural enzyme product, uracil. We use volume-based metadynamics to compute the free energy landscape for product release and show a modest binding affinity for the product to the enzyme, consistent with experiments. Next, we use infrequent metadynamics to estimate the unbiased release rate from Kramers time-dependent rate theory and find a favorable comparison to experiment with a slower rate of product release for the 5-FU system. Our work demonstrates how adaptive sampling methods can be used to study the protein-ligand unbinding process for engineering enzyme/prodrug systems and gives insights into the molecular mechanism of product release for the yCD/5-FU system.

Aptamer-Antibody Chimera Sensors for Sensitive, Rapid, and Reversible Molecular Detection in Complex Samples.

The development of receptors suitable for the continuous detection of analytes in complex, interferent-rich samples remains challenging. Antibodies are highly sensitive but difficult to engineer in order to introduce signaling functionality, while aptamer switches are easy to construct but often yield only a modest target sensitivity. We present here a programmable antibody and DNA aptamer switch (PANDAS), which combines the desirable properties of both receptors by using a nucleic acid tether to link an analyte-specific antibody to an internal strand-displacement (ISD)-based aptamer switch that recognizes the same target through different epitopes. The antibody increases PANDAS analyte binding due to its high affinity, and the effective concentration between the two receptors further enhances two-epitope binding and fluorescent aptamer signaling. We developed a PANDAS sensor for the clotting protein thrombin and show that a tuned design achieves a greater than 300-fold enhanced sensitivity compared to that of using an aptamer alone. This design also exhibits reversible binding, enabling repeated measurements with a temporal resolution of ∼10 min, and retains excellent sensitivity even in interferent-rich samples. With future development, this PANDAS approach could enable the adaptation of existing protein-binding aptamers with modest affinity to sensors that deliver excellent sensitivity and minute-scale resolution in minimally prepared biological specimens.

Improving Genomic Predictions in Multi-Breed Cattle Populations: A Comparative Analysis of BayesR and GBLUP Models.

Numerous studies have shown that combining populations from similar or closely related genetic breeds improves the accuracy of genomic predictions (GP). Extensive experimentation with diverse Bayesian and genomic best linear unbiased prediction (GBLUP) models have been developed to explore multi-breed genomic selection (GS) in livestock, ultimately establishing them as successful approaches for predicting genomic estimated breeding value (GEBV). This study aimed to assess the effectiveness of using BayesR and GBLUP models with linkage disequilibrium (LD)-weighted genomic relationship matrices (GRMs) for genomic prediction in three different beef cattle breeds to identify the best approach for enhancing the accuracy of multi-breed genomic selection in beef cattle. Additionally, a comparison was conducted to evaluate the predictive precision of different marker densities and genetic correlations among the three breeds of beef cattle. The GRM between Yunling cattle (YL) and other breeds demonstrated modest affinity and highlighted a notable genetic concordance of 0.87 between Chinese Wagyu (WG) and Huaxi (HX) cattle. In the within-breed GS, BayesR demonstrated an advantage over GBLUP. The prediction accuracies for HX cattle using the BayesR model were 0.52 with BovineHD BeadChip data (HD) and 0.46 with whole-genome sequencing data (WGS). In comparison to the GBLUP model, the accuracy increased by 26.8% for HD data and 9.5% for WGS data. For WG and YL, BayesR doubled the within-breed prediction accuracy to 14.3% from 7.1%, outperforming GBLUP across both HD and WGS datasets. Moreover, analyzing multiple breeds using genomic selection showed that BayesR consistently outperformed GBLUP in terms of predictive accuracy, especially when using WGS. For instance, in a mixed reference population of HX and WG, BayesR achieved a significant accuracy of 0.53 using WGS for HX, which was a substantial enhancement over the accuracies obtained with GBLUP models. The research further highlights the benefit of including various breeds in the reference group, leading to enhanced accuracy in predictions and emphasizing the importance of comprehensive genomic selection methods. Our research findings indicate that BayesR exhibits superior performance compared to GBLUP in multi-breed genomic prediction accuracy, achieving a maximum improvement of 33.3%, especially in genetically diverse breeds. The improvement can be attributed to the effective utilization of higher single nucleotide polymorphism (SNP) marker density by BayesR, resulting in enhanced prediction accuracy. This evidence conclusively demonstrates the significant impact of BayesR on enhancing genomic predictions in diverse cattle populations, underscoring the crucial role of genetic relatedness in selection methodologies. In parallel, subsequent studies should focus on refining GRM and exploring alternative models for GP.

The RNA-Binding Domain of hnRNP U Extends beyond the RGG/RG Motifs.

Heterogeneous nuclear ribonucleoprotein U (hnRNP U) is a ubiquitously expressed protein that regulates chromatin architecture through its interactions with numerous DNA, protein, and RNA partners. The RNA-binding domain (RBD) of hnRNP U was previously mapped to an RGG/RG motif within its disordered C-terminal region, but little is understood about its binding mode and potential for selective RNA recognition. Analysis of publicly available hnRNP U enhanced UV cross-linking and immunoprecipitation (eCLIP) data identified high-confidence binding sites within human RNAs. We synthesized a set of diverse RNAs encompassing 11 of these identified cross-link sites for biochemical characterization using a combination of fluorescence anisotropy and electrophoretic mobility shift assays. These in vitro binding experiments with a rationally designed set of RNAs and hnRNP U domains revealed that the RGG/RG motif is a small part of a more expansive RBD that encompasses most of the disordered C-terminal region. This RBD contains a second, previously experimentally uncharacterized RGG/RG motif with RNA-binding properties comparable to those of the canonical RGG/RG motif. These RGG/RG motifs serve redundant functions, with neither serving as the primary RBD. While in isolation, each RGG/RG motif has modest affinity for RNA, together they significantly enhance the association of hnRNP U with RNA, enabling the binding of most of the designed RNA set with low to midnanomolar binding affinities. Identification and characterization of the complete hnRNP U RBD highlight the perils of a reductionist approach to defining biochemical activities in this system and pave the way for a detailed investigation of its RNA-binding specificity.

Facile Synthesis of Homodimeric Protein Ligands.

Many proteins exist as oligomers (homodimers, homotrimers, etc.). A proven strategy for the development of high affinity ligands for such targets is to link together two modest affinity ligands that allows the formation of a 2 : 2 (or higher-order) protein-ligand complex. We report here the discovery of a convenient, "click-like" reaction for the homodimerization of protein ligands that is efficient, operationally simple to carry out, and tolerant of many functional groups. This chemistry reduces the synthetic burden inherent in the creation of homodimeric ligands since only a single precursor is required. The utility of this strategy is demonstrated by the synthesis of homodimeric inhibitors, including PROTACs.

Phenolic compounds as potential adenosine deaminase inhibitors: molecular docking and dynamics simulation coupled with MM-GBSA calculations.

Adenosine deaminase (ADA) is a Zn2+-containing enzyme that catalyzes the irreversible deamination of adenosine to inosine or deoxyadenosine to deoxyinosine. In addition to this enzymatic function, ADA mediates cell-to-cell interactions involved in lymphocyte co-stimulation or endothelial activation. ADA is implicated in cardiovascular pathologies such as atherosclerosis and certain types of cancers, including lymphoma and leukemia. To date, only two drugs (pentostatin and cladribine) have been approved for the treatment of hairy cell leukemia. In search of natural ADA inhibitors, we demonstrated the binding of selected phenolic compounds to the active site of ADA using molecular docking and molecular dynamics simulation. Our results show that phenolic compounds (chlorogenic acid, quercetin, and hyperoside) stabilized the ADA complex by forming persistent interactions with the catalytically essential Zn2+ ion. Furthermore, MM-GBSA ligand binding affinity calculations revealed that hyperoside had a comparable binding energy score (ΔG = -46.56 ± 8.26kcal/mol) to that of the cocrystal ligand in the ADA crystal structure (PDB ID: 1O5R) (ΔG = -51.97 ± 4.70kcal/mol). Similarly, chlorogenic acid exhibited a binding energy score (ΔG = -18.76 ± 4.60kcal/mol) comparable to those of the two approved ADA inhibitor drugs pentostatin (ΔG = -14.54 ± 2.25kcal/mol) and cladribine (ΔG = - 25.52 ± 4.10kcal/mol) while quercetin was found to have modest binding affinity (ΔG = - 8.85 ± 7.32kcal/mol). This study provides insights into the possible inhibitory potential of these phenolic compounds against ADA.

Substantia nigral dopamine transporter uptake in dementia with Lewy bodies

Nigrostriatal dopaminergic degeneration is a pathological hallmark of dementia with Lewy bodies (DLB). To identify the subregional dopamine transporter (DAT) uptake patterns that improve the diagnostic accuracy of DLB, we analyzed N-(3-[18F] fluoropropyl)-2β-carbomethoxy-3β-(4-iodophenyl)-nortropane (FP-CIT) PET in 51 patients with DLB, in 36 patients with mild cognitive impairment with Lewy body (MCI-LB), and in 40 healthy controls (HCs). In addition to a high affinity for DAT, FP-CIT show a modest affinity to serotonin or norepinephrine transporters. Specific binding ratios (SBRs) of the nigrostriatal subregions were transformed to age-adjusted z-scores (zSBR) based on HCs. The diagnostic accuracy of subregional zSBRs were tested using receiver operating characteristic (ROC) curve analyses separately for MCI-LB and DLB versus HCs. Then, the effect of subregional zSBRs on the presence of clinical features and gray matter (GM) density were evaluated in all patients with MCI-LB or DLB as a group. ROC curve analyses showed that the diagnostic accuracy of DLB based on the zSBR of substantia nigra (area under the curve [AUC], 0.90) or those for MCI-LB (AUC, 0.87) were significantly higher than that based on the zSBR of posterior putamen for DLB (AUC, 0.72) or MCI-LB (AUC, 0.65). Lower zSBRs in nigrostriatal regions were associated with visual hallucination, severe parkinsonism, and cognitive dysfunction, while lower zSBR of substantia nigra was associated with widespread GM atrophy in DLB and MCI-LB patients. Taken together, our results suggest that evaluation of nigral DAT uptake may increase the diagnostic accuracy of DLB and MCI-LB than other striatal regions.

Synthesis and Fluorescence Properties of 4-Cyano and 4-Formyl Melatonin as Putative Melatoninergic Ligands.

Fluorescent ligands are imperative to many facets of chemical biology and medicinal chemistry. Herein, we report the syntheses of two fluorescent melatonin-based derivatives as potential ligands of melatonin receptors. The two compounds, namely, 4-cyano and 4-formyl melatonin (4CN-MLT and 4CHO-MLT, respectively), which differ from melatonin by only two/three atoms that are very compact in size, were prepared using the selective C3-alkylation of indoles with N-acetyl ethanolamines involving the "borrowing hydrogen" strategy. These compounds exhibit absorption/emission spectra that are red-shifted from those of melatonin. Binding studies on two melatonin receptor subtypes showed that these derivatives have a modest affinity and selectivity ratio.

Use of computational and wet lab techniques to examine the molecular association between a potent hepatitis C virus inhibitor, PSI-6206 and human serum albumin.

This study explores the plausible molecular interaction between a potent hepatitis C virus inhibitor, PSI-6206 (PSI), and human serum albumin (HSA), a primary transporter in blood plasma. Results obtained from both computational viz. molecular docking and molecular dynamics (MD) simulation and wet lab techniques such as UV absorption, fluorescence, circular dichroism (CD), and atomic force microscopy (AFM) complemented each other. While docking results identified PSI binding to subdomain IIA (Site I) of HSA by forming six hydrogen bonds, MD simulations signified the complex stability throughout the 50,000ps. A consistent cutback in the Stern-Volmer quenching constant (Ksv) along with rising temperatures supported the static mode of fluorescence quenching in response to PSI addition and implied the development of the PSI-HSA complex. This discovery was backed by the alteration of the HSA UV absorption spectrum, a larger value (>1010 M-1.s-1) of the bimolecular quenching rate constant (kq) and the AFM-guided swelling of the HSA molecule, in the presence of PSI. Moreover, the fluorescence titration results revealed a modest binding affinity (4.27-6.25×103 M-1) in the PSI-HSA system, involving hydrogen bonds, van der Waals and hydrophobic interactions, as inferred from ΔS = + 22.77Jmol-1 K-1 and ΔH = - 11.02 KJ mol-1values. CD and 3D fluorescence spectra reminded significant adjustment in the 2° and 3° structures and modification in the Tyr/Trp microenvironment of the protein in the PSI-bound state. The results obtained from drug competing experiments also advocated the binding location of PSI in HSA as Site I.