New Hybrid Tetrahydropyrrolo[3,2,1-ij]quinolin-1-ylidene-2-thioxothiazolidin-4-ones as New Inhibitors of Factor Xa and Factor XIa: Design, Synthesis, and In Silico and Experimental Evaluation.

Despite extensive research in the field of thrombotic diseases, the prevention of blood clots remains an important area of study. Therefore, the development of new anticoagulant drugs with better therapeutic profiles and fewer side effects to combat thrombus formation is still needed. Herein, we report the synthesis and evaluation of novel pyrroloquinolinedione-based rhodanine derivatives, which were chosen from 24 developed derivatives by docking as potential molecules to inhibit the clotting factors Xa and XIa. For the synthesis of new hybrid derivatives of pyrrolo[3,2,1-ij]quinoline-2-one, we used a convenient structural modification of the tetrahydroquinoline fragment by varying the substituents in positions 2, 4, and 6. In addition, the design of target molecules was achieved by alkylating the amino group of the rhodanine fragment with propargyl bromide or by replacing the rhodanine fragment with 2-thioxoimidazolidin-4-one. The in vitro testing showed that eight derivatives are capable of inhibiting both coagulation factors, two compounds are selective inhibitors of factor Xa, and two compounds are selective inhibitors of factor XIa. Overall, these data indicate the potential anticoagulant activity of these molecules through the inhibition of the coagulation factors Xa and XIa.

Bifunctional fusion protein targeting both FXIIa and FXIa displays potent anticoagulation effects.

AIMS: Anticoagulation in disease treatment has been wildly studied in recent years. The intrinsic coagulation pathway is attracting attention of research community due to its low bleeding risk, and inhibitors against intrinsic coagulation factor XIIa (FXIIa) or XIa (FXIa) have been extensively studied. However, studies to develop anticoagulant inhibitors simultaneous targeting FXIIa and FXIa have not been reported. Our study aimed to evaluate the anticoagulation effect of the dual targeting of FXIIa and FXIa. MAIN METHODS: A fusion protein Infestin-PN2KPI (IP) was designed by linking FXIIa inhibitor Infesin4 and FXIa inhibitor PN2KPI through a rigid linker, and was cloned, expressed and characterized. The binding of IP to FXIIa and FXIa was verified by SPR, and inhibitory ability of IP against FXIIa and FXIa was verified by chromogenic substrate method. And then, the anticoagulation and antithrombotic functions of IP were extensively evaluated by aPTT assay, FeCl3-induced carotid artery thrombosis model and transient occlusion of the middle cerebral artery model. KEY FINDINGS: IP significantly prolonged aPTT, inhibited thrombosis and prevented stroke at a dose of at least 1/2 lower than the effective dose of its component Infestin4 or PN2KPI, and did not cause bleed risk. SIGNIFICANCE: The bifunctional fusion protein IP showed good anticoagulation effects, and simultaneous targeting FXIIa and FXIa is a promising strategy for anticoagulation drug development.

Sulfonated non-saccharide molecules and human factor XIa: Enzyme inhibition and computational studies.

Human factor XIa (FXIa) is a serine protease in the intrinsic coagulation pathway. FXIa has been actively targeted to develop new anticoagulants that are associated with a reduced risk of bleeding. Thousands of FXIa inhibitors have been reported, yet none has reached the clinic thus far. We describe here a novel class of sulfonated molecules that allosterically inhibit FXIa with moderate potency. A library of 18 sulfonated molecules was evaluated for the inhibition of FXIa using a chromogenic substrate hydrolysis assay. Only six molecules inhibited FXIa with IC50 values of 4.6-29.5 µM. Michaelis-Menten kinetics indicated that sulfonated molecules are allosteric inhibitors of FXIa. Inhibition of FXIa by these molecules was reversed by protamine. The molecules also showed moderate anticoagulant effects in human plasma with preference to prolong activated partial thromboplastin time. Their binding to an allosteric site in the catalytic domain of FXIa was modeled to illustrate potential binding mode and potential important Arg/Lys residues. Particularly, inhibitor 16 (IC50  = 4.6 µM) demonstrated good selectivity over a panel of serine proteases including those in the coagulation process. Inhibitor 16 did not significantly compromise the viability of three cell lines. Overall, the reported sulfonated molecules serve as a new platform to design selective, potent, and allosteric inhibitors of FXIa for therapeutic applications.

Structural insights into the activation of blood coagulation factor XI zymogen by thrombin: A computational molecular dynamics study.

Activation of human blood coagulation factor XI zymogen to factor XIa plays a significant role in the upstream coagulation pathway, in which factor XIa activates factor IX zymogen. The mechanistic details of the proteolytic activation of factor XI by the activating enzyme thrombin are not well-understood at atomic level. In this study, we employed a combination of molecular docking and microsecond time-scale molecular dynamics simulations to identify the key regions of interaction between fXI and thrombin. The activating complex between the substrate and enzyme was modeled to represent the initial acylation step of the serine-protease hydrolysis mechanism. The proposed solution structural complex, fIX:fIIa, obtained from 3 microseconds of MD refinement, suggests that the activation of factor XI is mediated by thrombin's anion binding exosite-II interactions with A3 and A4 domains. We predict that the two positively charged arginine residues (Arg409 and Arg413) in the exosite-2 region, the ß- and γ-insertion loops of thrombin play an important structural role in the initial activating complex between fXI and thrombin.

The effect of water on the large-scale supercritical fluid chromatography purification of two factor XIa active pharmaceutical ingredients.

BMS-962212, a parenteral Factor XIa inhibitor, was scaled-up for toxicity studies. Two steps of supercritical fluid chromatography (SFC) were developed for the chiral resolution of the penultimate and achiral purification of final active pharmaceutical ingredient (API), BMS-962212. A robust SFC process using Chiralcel OD-H with methanol-acetonitrile as modifier in CO2 was established to achieve a stable and uninterrupted operation with reduced mobile phase viscosity and system pressure drop. More than 230 g of the racemic penultimate was chirally resolved to reach >99% chiral purity, ready for final tert-butyl ester deprotection to provide the API. There were a significant number of impurities in BMS-962212 generated from the final step that needed to be removed. In contrast to conventional SFC conditions, an SFC method exploiting water and ammonia as additives in both the mobile phase and sample solution was developed to accomplish purification and desalting (i.e. removing TFA) of the zwitterionic API in one step. Water as an additive eliminated salt precipitation and improved the resolution while ammonia contributed to the desalting, details of which will be discussed in this article. A throughput of 2 g/h was achieved, and >80 g of the crude API was purified. The same strategy was applied to another Factor XIa API (compound A) and its penultimate.

Identification of an alpha-1 antitrypsin variant with enhanced specificity for factor XIa by phage display, bacterial expression, and combinatorial mutagenesis.

Coagulation Factor XIa (FXIa) is an emerging target for antithrombotic agent development. The M358R variant of the serpin alpha-1 antitrypsin (AAT) inhibits both FXIa and other proteases. Our aim was to enhance the specificity of AAT M358R for FXIa. We randomized two AAT M358R phage display libraries at reactive centre loop positions P13-P8 and P7-P3 and biopanned them with FXIa. A bacterial expression library randomized at P2'-P3' was also probed. Resulting novel variants were expressed as recombinant proteins in E. coli and their kinetics of FXIa inhibition determined. The most potent FXIa-inhibitory motifs were: P13-P8, HASTGQ; P7-P3, CLEVE; and P2-P3', PRSTE (respectively, novel residues bolded). Selectivity for FXIa over thrombin was increased up to 34-fold versus AAT M358R for these single motif variants. Combining CLEVE and PRSTE motifs in AAT-RC increased FXIa selectivity for thrombin, factors XIIa, Xa, activated protein C, and kallikrein by 279-, 143-, 63-, 58-, and 36-fold, respectively, versus AAT M358R. AAT-RC lengthened human plasma clotting times less than AAT M358R. AAT-RC rapidly and selectively inhibits FXIa and is worthy of testing in vivo. AAT specificity can be focused on one target protease by selection in phage and bacterial systems coupled with combinatorial mutagenesis.

Discovery of Benzyl Tetraphosphonate Derivative as Inhibitor of Human Factor Xia.

The inhibition of factor XIa (FXIa) is a trending paradigm for the development of new generations of anticoagulants without a substantial risk of bleeding. In this report, we present the discovery of a benzyl tetra-phosphonate derivative as a potent and selective inhibitor of human FXIa. Biochemical screening of four phosphonate/phosphate derivatives has led to the identification of the molecule that inhibited human FXIa with an IC50 value of ∼7.4 µM and a submaximal efficacy of ∼68 %. The inhibitor was at least 14-fold more selective to FXIa over thrombin, factor IXa, factor Xa, and factor XIIIa. It also inhibited FXIa-mediated activation of factor IX and prolonged the activated partial thromboplastin time of human plasma. In Michaelis-Menten kinetics experiment, inhibitor 1 reduced the VMAX of FXIa hydrolysis of a chromogenic substrate without significantly affecting its KM suggesting an allosteric mechanism of inhibition. The inhibitor also disrupted the formation of FXIa - antithrombin complex and inhibited thrombin-mediated and factor XIIa-mediated formation of FXIa from its zymogen factor XI. Inhibitor 1 has been proposed to bind to or near the heparin/polyphosphate-binding site in the catalytic domain of FXIa. Overall, inhibitor 1 is the first benzyl tetraphosphonate small molecule that allosterically inhibits human FXIa, blocks its physiological function, and prevents its zymogen activation by other clotting factors under in vitro conditions. Thus, we put forward benzyl tetra-phosphonate 1 as a novel lead inhibitor of human FXIa to guide future efforts in the development of allosteric anticoagulants.

Role of Factor XIa and Plasma Kallikrein in Arterial and Venous Thrombosis.

Cardiovascular disease, including stroke, myocardial infarction, and venous thromboembolism, is one of the leading causes of morbidity and mortality worldwide. Excessive coagulation may cause vascular occlusion in arteries and veins eventually leading to thrombotic diseases. Studies in recent years suggest that coagulation factors are involved in these pathological mechanisms. Factors XIa (FXIa), XIIa (FXIIa), and plasma kallikrein (PKa) of the contact system of coagulation appear to contribute to thrombosis while playing a limited role in hemostasis. Contact activation is initiated upon autoactivation of FXII on negatively charged surfaces. FXIIa activates plasma prekallikrein (PK) to PKa, which in turn activates FXII and initiates the kallikrein-kinin pathway. FXI is also activated by FXIIa, leading to activation of FIX and finally to thrombin formation, which in turn activates FXI in an amplification loop. Animal studies have shown that arterial and venous thrombosis can be reduced by the inhibition of FXI(a) or PKa. Furthermore, data from human studies suggest that these enzymes may be valuable targets to reduce thrombosis risk. In this review, we discuss the structure and function of FXI(a) and PK(a), their involvement in the development of venous and arterial thrombosis in animal models and human studies, and current therapeutic strategies.

Discovery of a High Affinity, Orally Bioavailable Macrocyclic FXIa Inhibitor with Antithrombotic Activity in Preclinical Species.

Oral factor XIa (FXIa) inhibitors may provide a promising new antithrombotic therapy with an improved benefit to bleeding risk profile over existing antithrombotic agents. Herein, we report application of a previously disclosed cyclic carbamate P1 linker which provided improved oral bioavailability in the imidazole-based 13-membered macrocycle to the 12-membered macrocycle. This resulted in identification of compound 4 with desired FXIa inhibitory potency and good oral bioavailability but high in vivo clearance. Further structure-activity relationship (SAR) studies of heterocyclic core modifications to replace the imidazole core as well as various linkers to the P1 group led to the discovery of compound 6f, a potent FXIa inhibitor with selectivity against most of the relevant serine proteases. Compound 6f also demonstrated excellent pharmacokinetics (PK) profile (high oral bioavailability and low clearance) in multiple preclinical species. Compound 6f achieved robust antithrombotic efficacy in a rabbit efficacy model at doses which preserved hemostasis.

Synthesis, Docking, and In Vitro Anticoagulant Activity Assay of Hybrid Derivatives of Pyrrolo[3,2,1-ij]Quinolin-2(1H)-one as New Inhibitors of Factor Xa and Factor XIa.

Coagulation factor Xa and factor XIa are proven to be convenient and crucial protein targets for treatment for thrombotic disorders and thereby their inhibitors can serve as effective anticoagulant drugs. In the present work, we focused on the structure-activity relationships of derivatives of pyrrolo[3,2,1-ij]quinolin-2(1H)-one and an evaluation of their activity against factor Xa and factor XIa. For this, docking-guided synthesis of nine compounds based on pyrrolo[3,2,1-ij]quinolin-2(1H)-one was carried out. For the synthesis of new hybrid hydropyrrolo[3,2,1-ij]quinolin-2(1H)-one derivatives, we used convenient structural modification of both the tetrahydro- and dihydroquinoline moiety by varying the substituents at the C6,8,9 positions. In vitro testing revealed that four derivatives were able to inhibit both coagulation factors and three compounds were selective factor XIa inhibitors. An IC50 value of 3.68 µM for was found for the best factor Xa inhibitor and 2 µM for the best factor XIa inhibitor.

Allosteric Inhibition as a New Mode of Action for BAY 1213790, a Neutralizing Antibody Targeting the Activated Form of Coagulation Factor XI.

Factor XI (FXI), the zymogen of activated FXI (FXIa), is an attractive target for novel anticoagulants because FXI inhibition offers the potential to reduce thrombosis risk while minimizing the risk of bleeding. BAY 1213790, a novel anti-FXIa antibody, was generated using phage display technology. Crystal structure analysis of the FXIa-BAY 1213790 complex demonstrated that the tyrosine-rich complementarity-determining region 3 loop of the heavy chain of BAY 1213790 penetrated deepest into the FXIa binding epitope, forming a network of favorable interactions including a direct hydrogen bond from Tyr102 to the Gln451 sidechain (2.9 Å). The newly discovered binding epitope caused a structural rearrangement of the FXIa active site, revealing a novel allosteric mechanism of FXIa inhibition by BAY 1213790. BAY 1213790 specifically inhibited FXIa with a binding affinity of 2.4 nM, and in human plasma, prolonged activated partial thromboplastin time and inhibited thrombin generation in a concentration-dependent manner.

Hydrogen-deuterium exchange mass spectrometry highlights conformational changes induced by factor XI activation and binding of factor IX to factor XIa.

BACKGROUND: Factor XI (FXI) is a zymogen in the coagulation pathway that, once activated, promotes haemostasis by activating factor IX (FIX). Substitution studies using apple domains of the homologous protein prekallikrein have identified that FIX binds to the apple 3 domain of FXI. However, the molecular changes upon activation of FXI or binding of FIX to FXIa have remained largely unresolved. OBJECTIVES: This study aimed to gain more insight in the FXI activation mechanism by identifying the molecular differences between FXI and FXIa, and in the conformational changes in FXIa induced by binding of FIX. METHODS: Hydrogen-deuterium exchange mass spectrometry was performed on FXI, FXIa, and FXIa in complex with FIX. RESULTS: Both activation and binding to FIX induced conformational changes at the interface between the catalytic domain and the apple domains of FXI(a)-more specifically at the loops connecting the apple domains. Moreover, introduction of FIX uniquely induced a reduction of deuterium uptake in the beginning of the apple 3 domain. CONCLUSIONS: We propose that the conformational changes of the catalytic domain upon activation increase the accessibility to the apple 3 domain to enable FIX binding. Moreover, our HDX MS results support the location of the proposed FIX binding site at the beginning of the apple 3 domain and suggest a mediating role in FIX binding for both loops adjacent to the apple 3 domain.

Endothelial PAI-1 (Plasminogen Activator Inhibitor-1) Blocks the Intrinsic Pathway of Coagulation, Inducing the Clearance and Degradation of FXIa (Activated Factor XI).

Objective- Activation of coagulation FXI (factor XI) by FXIIa (activated factor XII) is a prothrombotic process. The endothelium is known to play an antithrombotic role by limiting thrombin generation and platelet activation. It is unknown whether the antithrombotic role of the endothelium includes sequestration of FXIa (activated factor XI) activity. This study aims to determine the role of endothelial cells (ECs) in the regulation of the intrinsic pathway of coagulation. Approach and Results- Using a chromogenic assay, we observed that human umbilical veins ECs selectively blocked FXIa yet supported kallikrein and FXIIa activity. Western blotting and mass spectrometry analyses revealed that FXIa formed a complex with endothelial PAI-1 (plasminogen activator inhibitor-1). Blocking endothelial PAI-1 increased the cleavage of a chromogenic substrate by FXIa and the capacity of FXIa to promote fibrin formation in plasma. Western blot and immunofluorescence analyses showed that FXIa-PAI-1 complexes were either released into the media or trafficked to the early and late endosomes and lysosomes of ECs. When baboons were challenged with Staphylococcus aureus to induce a prothrombotic phenotype, an increase in circulating FXIa-PAI-1 complex levels was detected by ELISA within 2 to 8 hours postchallenge. Conclusions- PAI-1 forms a complex with FXIa on ECs, blocking its activity and inducing the clearance and degradation of FXIa. Circulating FXIa-PAI-1 complexes were detected in a baboon model of S. aureus sepsis. Although ECs support kallikrein and FXIIa activity, inhibition of FXIa by ECs may promote the clearance of intravascular FXIa. Visual Overview- An online visual overview is available for this article.

A mechanism for hereditary angioedema with normal C1 inhibitor: an inhibitory regulatory role for the factor XII heavy chain.

The plasma proteins factor XII (FXII) and prekallikrein (PK) undergo reciprocal activation to the proteases FXIIa and kallikrein by a process that is enhanced by surfaces (contact activation) and regulated by the serpin C1 inhibitor. Kallikrein cleaves high-molecular-weight kininogen (HK), releasing the vasoactive peptide bradykinin. Patients with hereditary angioedema (HAE) experience episodes of soft tissue swelling as a consequence of unregulated kallikrein activity or increased prekallikrein activation. Although most HAE cases are caused by reduced plasma C1-inhibitor activity, HAE has been linked to lysine/arginine substitutions for Thr309 in FXII (FXII-Lys/Arg309). Here, we show that FXII-Lys/Arg309 is susceptible to cleavage after residue 309 by coagulation proteases (thrombin and FXIa), resulting in generation of a truncated form of FXII (δFXII). The catalytic efficiency of δFXII activation by kallikrein is 15-fold greater than for full-length FXII. The enhanced rate of reciprocal activation of PK and δFXII in human plasma and in mice appears to overwhelm the normal inhibitory function of C1 inhibitor, leading to increased HK cleavage. In mice given human FXII-Lys/Arg309, induction of thrombin generation by infusion of tissue factor results in enhanced HK cleavage as a consequence of δFXII formation. The effects of δFXII in vitro and in vivo are reproduced when wild-type FXII is bound by an antibody to the FXII heavy chain (HC; 15H8). The results contribute to our understanding of the predisposition of patients carrying FXII-Lys/Arg309 to angioedema after trauma, and reveal a regulatory function for the FXII HC that normally limits PK activation in plasma.

Engineering varied serine protease inhibitors by converting P1 site of BF9, a weakly active Kunitz-type animal toxin.

Although there were a lot of weakly active animal toxins in the venoms, their values and applications are still mysterious, such as BF9, which is a Kunitz-type toxin isolated from the venom of the elapid snake Bungarus fasciatus. Here, we used BF9 to be a molecular scaffold, and engineered eight BF9-derived peptides by changing P1 site Asn17 of BF9, such as BF9-N17Y and BF9-N17T designed from the polar subfamily, BF9-N17L and BF9-N17G designed from the Non-polar subfamily, BF9-N17D designed from acidic subfamily, and BF9-N17H, BF9-N17K and BF9-N17R designed from basic subfamily. Through enzyme inhibitor experiment assays, we found a potent and selective chymotrypsin inhibitor BF9-N17Y, a potent and selective coagulation factor XIa inhibitor BF9-N17H, and two highly potent coagulation factor XIa inhibitors BF9-N17K and BF9-N17. APTT and PT assays further showed that BF9-N17H, BF9-N17K and BF9-N17R were three novel anticoagulants with selectively intrinsic coagulation pathway inhibitory activity. Considering that natural weakly active animal toxins are also a huge peptide resource, our present work might open a new window about pharmacological applications of weakly active animal toxins, which might be good templates for potent and selective molecular probe and lead drug designs.

Fragment-Based Lead Generation of 5-Phenyl-1H-pyrazole-3-carboxamide Derivatives as Leads for Potent Factor Xia Inhibitors.

FXIa is suggested as a major target for anticoagulant drug discovery because of reduced risk of bleeding. In this paper, we defined 5-phenyl-1H-pyrazole-3-carboxylic acid derivatives as privileged fragments for FXIa inhibitors' lead discovery. After replacing the (E)-3-(5-chloro-2-(1H-tetrazol-1-yl)phenyl)acrylamide moiety in compound 3 with 5-(3-chlorophenyl)-1H-pyrazole-3-carboxamide, we traveled from FXIa inhibitor 3 to a scaffold that fused the privileged fragments into a pharmacophore for FXIa inhibitors. Subsequently, we synthesized and assessed the FXIa inhibitory potency of a series of 5-phenyl-1H-pyrazole-3-carboxamide derivatives with different P1, P1' and P2'moiety. Finally, the SAR of them was systematically investigated to afford the lead compound 7za (FXIa Ki = 90.37 nM, 1.5× aPTT in rabbit plasma = 43.33 µM) which exhibited good in vitro inhibitory potency against FXIa and excellent in vitro coagulation activities. Furthermore, the binding mode of 7za with FXIa was studied and the results suggest that the 2-methylcyclopropanecarboxamide group of 7za makes 2 direct hydrogen bonds with Tyr58B and Thr35 in the FXIa backbone, making 7za binds to FXIa in a highly efficient manner.

Factor XIa Inhibitors as New Anticoagulants.

With the introduction of thrombin and factor Xa inhibitors to the oral anticoagulant market, significant improvements in both efficacy and safety have been achieved. Early clinical and preclinical data suggest that inhibitors of factor XIa can provide a still safer alternative, with expanded efficacy for arterial indications. This Perspective provides an overview of target rationale and details of the discovery and development of inhibitors of factor XIa as next generation antithrombotic agents.

Chemical Footprinting Reveals Conformational Changes Following Activation of Factor XI.

Coagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.

Discovery of a Parenteral Small Molecule Coagulation Factor XIa Inhibitor Clinical Candidate (BMS-962212).

Factor XIa (FXIa) is a blood coagulation enzyme that is involved in the amplification of thrombin generation. Mounting evidence suggests that direct inhibition of FXIa can block pathologic thrombus formation while preserving normal hemostasis. Preclinical studies using a variety of approaches to reduce FXIa activity, including direct inhibitors of FXIa, have demonstrated good antithrombotic efficacy without increasing bleeding. On the basis of this potential, we targeted our efforts at identifying potent inhibitors of FXIa with a focus on discovering an acute antithrombotic agent for use in a hospital setting. Herein we describe the discovery of a potent FXIa clinical candidate, 55 (FXIa Ki = 0.7 nM), with excellent preclinical efficacy in thrombosis models and aqueous solubility suitable for intravenous administration. BMS-962212 is a reversible, direct, and highly selective small molecule inhibitor of FXIa.

A receptor dependent-4D QSAR approach to predict the activity of mutated enzymes.

Screening and selection tools to obtain focused libraries play a key role in successfully engineering enzymes of desired qualities. The quality of screening depends on efficient assays; however, a focused library generated with a priori information plays a major role in effectively identifying the right enzyme. As a proof of concept, for the first time, receptor dependent - 4D Quantitative Structure Activity Relationship (RD-4D-QSAR) has been implemented to predict kinetic properties of an enzyme. The novelty of this study is that the mutated enzymes also form a part of the training data set. The mutations were modeled in a serine protease and molecular dynamics simulations were conducted to derive enzyme-substrate (E-S) conformations. The E-S conformations were enclosed in a high resolution grid consisting of 156,250 grid points that stores interaction energies to generate QSAR models to predict the enzyme activity. The QSAR predictions showed similar results as reported in the kinetic studies with >80% specificity and >50% sensitivity revealing that the top ranked models unambiguously differentiated enzymes with high and low activity. The interaction energy descriptors of the best QSAR model were used to identify residues responsible for enzymatic activity and substrate specificity.