Discovery of Nirmatrelvir Resistance Mutations in SARS-CoV-2 3CLpro: A Computational-Experimental Approach.

The COVID-19 pandemic has emphasized the urgency for effective antiviral therapies against SARS-CoV-2. Targeting the main protease (3CLpro) of the virus has emerged as a promising approach, and nirmatrelvir (PF-07321332), the active component of Pfizer's oral drug Paxlovid, has demonstrated remarkable clinical efficacy. However, the emergence of resistance mutations poses a challenge to its continued success. In this study, we employed alchemical free energy perturbation (FEP) alanine scanning to identify nirmatrelvir-resistance mutations within SARS-CoV-2 3CLpro. FEP identified several mutations, which were validated through in vitro IC50 experiments and found to result in 8- and 72-fold increases in nirmatrelvir IC50 values. Additionally, we constructed SARS-CoV-2 omicron replicons containing these mutations, and one of the mutants (S144A/E166A) displayed a 20-fold increase in EC50, confirming the role of FEP in identifying drug-resistance mutations. Our findings suggest that FEP can be a valuable tool in proactively monitoring the emergence of resistant strains and guiding the design of future inhibitors with reduced susceptibility to drug resistance. As nirmatrelvir is currently widely used for treating COVID-19, this research has important implications for surveillance efforts and antiviral development.

In-silico screening and microsecond molecular dynamics simulations to identify single point mutations that destabilize ß-hexosaminidase A causing Tay-Sachs disease.

ß-hexosaminidase A (HexA) protein is responsible for the degradation of GM2 gangliosides in the central and peripheral nervous systems. Tay-Sachs disease occurs when HexA within Hexosaminidase does not properly function and harmful GM2 gangliosides begin to build up within the neurons. In this study, in silico methods such as SIFT, PolyPhen-2, PhD-SNP, and MutPred were utilized to analyze the effects of nonsynonymous single nucleotide polymorphisms (nsSNPs) on HexA in order to identify possible pathogenetic and deleterious variants. Molecular dynamics (MD) simulations showed that two mutants, P25S and W485R, experienced an increase in structural flexibility compared to the native protein. Particularly, there was a decrease in the overall number and frequencies of hydrogen bonds for the mutants compared to the wildtype. MM/GBSA calculations were performed to help assess the change in binding affinity between the wildtype and mutant structures and a mechanism-based inhibitor, NGT, which is known to help increase the residual activity of HexA. Both of the mutants experienced a decrease in the binding affinity from -23.8 kcal/mol in wildtype to -20.9 and -18.7 kcal/mol for the P25S and W485R variants of HexA, respectively.

In silico screening and molecular dynamics simulation of deleterious PAH mutations responsible for phenylketonuria genetic disorder.

Phenylketonuria (PKU) is a genetic disorder that if left untreated can lead to behavioral problems, epilepsy, and even mental retardation. PKU results from mutations within the phenylalanine-4-hydroxylase (PAH) gene that encodes for the PAH protein. The study of all PAH causing mutations is improbable using experimental techniques. In this study, a collection of in silico resources, sorting intolerant from tolerant, Polyphen-2, PhD-SNP, and MutPred were used to identify possible pathogenetic and deleterious PAH non-synonymous single nucleotide polymorphisms (nsSNPs). We identified two variants of PAH, I65N and L311P, to be the most deleterious and disease causing nsSNPs. Molecular dynamics (MD) simulations were carried out to characterize these point mutations on the atomic level. MD simulations revealed increased flexibility and a decrease in the hydrogen bond network for both mutants compared to the native protein. Free energy calculations using the MM/GBSA approach found that BH4 , a drug-based therapy for PKU patients, had a higher binding affinity for I65N and L311P mutants compared to the wildtype protein. We also identify important residues in the BH4 binding pocket that may be of interest for the rational drug design of other PAH drug-based therapies. Lastly, free energy calculations also determined that the I65N mutation may impair the dimerization of the N-terminal regulatory domain of PAH.

Computationally Designed ACE2 Decoy Receptor Binds SARS-CoV-2 Spike (S) Protein with Tight Nanomolar Affinity.

Even with the availability of vaccines, therapeutic options for COVID-19 still remain highly desirable, especially in hospitalized patients with moderate or severe disease. Soluble ACE2 (sACE2) is a promising therapeutic candidate that neutralizes SARS CoV-2 infection by acting as a decoy. Using computational mutagenesis, we designed a number of sACE2 derivatives carrying three to four mutations. The top-predicted sACE2 decoy based on the in silico mutagenesis scan was subjected to molecular dynamics and free-energy calculations for further validation. After illuminating the mechanism of increased binding for our designed sACE2 derivative, the design was verified experimentally by flow cytometry and BLI-binding experiments. The computationally designed sACE2 decoy (ACE2-FFWF) bound the receptor-binding domain of SARS-CoV-2 tightly with low nanomolar affinity and ninefold affinity enhancement over the wild type. Furthermore, cell surface expression was slightly greater than wild-type ACE2, suggesting that the design is well-folded and stable. Having an arsenal of high-affinity sACE2 derivatives will help to buffer against the emergence of SARS CoV-2 variants. Here, we show that computational methods have become sufficiently accurate for the design of therapeutics for current and future viral pandemics.

Nitrone and Alkyne Cascade Reactions for Regio- and Diastereoselective 1-Pyrroline Synthesis.

The synthesis of 1-pyrrolines from N-alkenylnitrones and alkynes has been explored as a retrosynthetic alternative to traditional approaches. These cascade reactions are formal [4+1] cycloadditions that proceed through a proposed dipolar cycloaddition and N-alkenylisoxazoline [3,3']-sigmatropic rearrangement. A variety of cyclic alkynes and terminal alkynes have been shown to undergo the transformation with N-alkenylnitrones under mild conditions to provide the corresponding spirocyclic and densely substituted 1-pyrrolines with high regio- and diastereoselectivity. Mechanistic studies provide insight into the balance of steric and electronic effects that promote the cascade process and control the diastereo- and regioisomeric preferences of the 1-pyrroline products. Diastereoselective derivatization of the 1-pyrrolines prepared by the cascade reaction demonstrate the divergent synthetic utility of the new method.

CHARMM-GUI DEER facilitator for spin-pair distance distribution calculations and preparation of restrained-ensemble molecular dynamics simulations.

The double electron-electron resonance (DEER) is a powerful structural biology technique to obtain distance information in the range of 18 to 80 å by measuring the dipolar coupling between two unpaired electron spins. The distance distributions obtained from the experiment provide valuable structural information about the protein in its native environment that can be exploited using restrained ensemble molecular dynamics (reMD) simulations. We present a new tool DEER Facilitator in CHARMM-GUI that consists of two modules Spin-Pair Distributor and reMD Prepper to setup simulations that utilize information from DEER experiments. Spin-Pair Distributor provides a web-based interface to calculate the spin-pair distance distribution of labeled sites in a protein using MD simulations. The calculated distribution can be used to guide the selection of the labeling sites in experiments as well as validate different protein structure models. reMD Prepper facilities the setup of reMD simulations using different types of spin labels in four different environments including vacuum, solution, micelle, and bilayer. The applications of these two modules are demonstrated with several test cases. Spin-Pair Distributor and reMD Prepper are available at http://www.charmm-gui.org/input/deer and http://www.charmm-gui.org/input/deerre. DEER Facilitator is expected to facilitate advanced biomolecular modeling and simulation, thereby leading to an improved understanding of the structure and dynamics of complex biomolecular systems based on experimental DEER data. © 2019 Wiley Periodicals, Inc.

N2 O Reductase Activity of a [Cu4 S] Cluster in the 4CuI Redox State Modulated by Hydrogen Bond Donors and Proton Relays in the Secondary Coordination Sphere.

The model complex [Cu4 (µ4 -S)(dppa)4 ]2+ (1, dppa=µ2 -(Ph2 P)2 NH) has N2 O reductase activity in methanol solvent, mediating 2 H+ /2 e- reduction of N2 O to N2 +H2 O in the presence of an exogenous electron donor (CoCp2 ). A stoichiometric product with two deprotonated dppa ligands was characterized, indicating a key role of second-sphere N-H residues as proton donors during N2 O reduction. The activity of 1 towards N2 O was suppressed in solvents that are unable to provide hydrogen bonding to the second-sphere N-H groups. Structural and computational data indicate that second-sphere hydrogen bonding induces structural distortion of the [Cu4 S] active site, accessing a strained geometry with enhanced reactivity due to localization of electron density along a dicopper edge site. The behavior of 1 mimics aspects of the CuZ catalytic site of nitrous oxide reductase: activity in the 4CuI :1S redox state, use of a second-sphere proton donor, and reactivity dependence on both primary and secondary sphere effects.

Synthesis of Spirocyclic 1-Pyrrolines from Nitrones and Arynes through a Dearomative [3,3']-Sigmatropic Rearrangement.

A dearomative [3,3']-sigmatropic rearrangement that converts N-alkenylbenzisoxazolines into spirocyclic pyrroline cyclohexadienones has been developed by using the dipolar cycloaddition of an N-alkenylnitrone and an aryne to access these unusual transient rearrangement precursors. This cascade reaction affords spirocyclic pyrrolines that are inaccessible through dipolar cycloadditions of exocyclic cyclohexenones and provides a fundamentally new approach to novel spirocyclic pyrroline and pyrrolidine motifs that are common scaffolds in biologically-active molecules. Diastereoselective functionalization processes have also been explored to demonstrate the divergent synthetic utility of the unsaturated spirocyclic products.

Synthesis of Allylic Alcohols via Cu-Catalyzed Hydrocarbonylative Coupling of Alkynes with Alkyl Halides.

We have developed a modular procedure to synthesize allylic alcohols from tertiary, secondary, and primary alkyl halides and alkynes via a Cu-catalyzed hydrocarbonylative coupling and 1,2-reduction tandem sequence. The use of tertiary alkyl halides as electrophiles was found to enable the synthesis of various allylic alcohols bearing α-quaternary carbon centers in good yield with high 1,2-reduction selectivity. Mechanistic studies that suggested a different pathway was operative with tertiary alkyl halides compared with primary and secondary alkyl halides for generating the key copper(III) oxidative adduct. For tertiary electrophiles, an acyl halide likely forms via radical atom transfer carbonylation. The preference for 1,2-reduction over 1,4-reduction of α,ß-unsaturated ketones bearing tertiary substituents was rationalized using density functional theory transition state analysis. On the basis of this computational model, the coupling method was extended to primary and secondary alkyl iodide electrophiles by using internal alkynes with aryl substituents, providing trisubstituted allylic alcohols in high yield with good regioselectivity.

Dicopper(I) Complexes Incorporating Acetylide-Functionalized Pyridinyl-Based Ligands: Synthesis, Structural, and Photovoltaic Studies.

Heteroaryl incorporated acetylide-functionalized pyridinyl ligands (L1-L6) with the general formula Py-C≡C-Ar (Py = pyridine and Ar = thiophene-2-yl, 2,2' -bithiophene]-5-yl, 2,2' :5',2″ -terthiophene]-5-yl, thieno[2,3- b]thiophen-2-yl, quinoline-5-yl, benzo[c][1,2,5]thiadiazole-5-yl) have been synthesized by Pd(0)/Cu(I)-catalyzed cross-coupling reaction of 4-ethynylpyridine and the respective heteroaryl halide. Ligands L1-L6 were isolated in respectable yields and characterized by microanalysis, IR spectroscopy, 1H NMR spectroscopy, and ESI-MS mass spectrometry. A series of dinuclear Cu(I) complexes 1-10 have been synthesized by reacting L1-L6 with CuI and triphenylphosphine (PPh3) (R1) or with an anchored phosphine derivative, 4-(diphenylphosphino) benzoic acid (R2)/2-(diphenylphosphino)benzenesulfonic acid (R3), in a stoichiometric ratio. The complexes are soluble in common organic solvents and have been characterized by analytical, spectroscopic, and computational methods. Single-crystal X-ray structure analysis confirmed rhomboid dimeric structures for complexes 1, 2, 4, and 5, and a polymeric structure for 6. Complexes 1-6 showed oxidation potential responses close to 0.9 V vs Fc0/+, which were chemically irreversible and are likely to be associated with multiple steps and core oxidation. Preliminary photovoltaic (PV) results of these new materials indicated moderate power conversion efficiency (PCE) in the range of 0.15-1.56% in dye-sensitized solar cells (DSSCs). The highest PCE was achieved with complex 10 bearing the sulfonic acid anchoring functionality.

Dynamics transitions at the outer vestibule of the KcsA potassium channel during gating.

In K(+) channels, the selectivity filter, pore helix, and outer vestibule play a crucial role in gating mechanisms. The outer vestibule is an important structurally extended region of KcsA in which toxins, blockers, and metal ions bind and modulate the gating behavior of K(+) channels. Despite its functional significance, the gating-related structural dynamics at the outer vestibule are not well understood. Under steady-state conditions, inactivating WT and noninactivating E71A KcsA stabilize the nonconductive and conductive filter conformations upon opening the activation gate. Site-directed fluorescence polarization of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled outer vestibule residues shows that the outer vestibule of open/conductive conformation is highly dynamic compared with the motional restriction experienced by the outer vestibule during inactivation gating. A wavelength-selective fluorescence approach shows a change in hydration dynamics in inactivated and noninactivated conformations, and supports a possible role of restricted/bound water molecules in C-type inactivation gating. Using a unique restrained ensemble simulation method, along with distance measurements by EPR, we show that, on average, the outer vestibule undergoes a modest backbone conformational change during its transition to various functional states, although the structural dynamics of the outer vestibule are significantly altered during activation and inactivation gating. Taken together, our results support the role of a hydrogen bond network behind the selectivity filter, side-chain conformational dynamics, and water molecules in the gating mechanisms of K(+) channels.

Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1.

Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuT-fold has been captured in outward-facing, occluded, and inward-facing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na(+)-coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion- and substrate-dependent conformational equilibria. In contrast to the Na(+)/leucine transporter LeuT, our results suggest that Na(+) binding at the conserved second Na(+) binding site does not change the energetics of the inward- and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class.

Structural Refinement of Proteins by Restrained Molecular Dynamics Simulations with Non-interacting Molecular Fragments.

The knowledge of multiple conformational states is a prerequisite to understand the function of membrane transport proteins. Unfortunately, the determination of detailed atomic structures for all these functionally important conformational states with conventional high-resolution approaches is often difficult and unsuccessful. In some cases, biophysical and biochemical approaches can provide important complementary structural information that can be exploited with the help of advanced computational methods to derive structural models of specific conformational states. In particular, functional and spectroscopic measurements in combination with site-directed mutations constitute one important source of information to obtain these mixed-resolution structural models. A very common problem with this strategy, however, is the difficulty to simultaneously integrate all the information from multiple independent experiments involving different mutations or chemical labels to derive a unique structural model consistent with the data. To resolve this issue, a novel restrained molecular dynamics structural refinement method is developed to simultaneously incorporate multiple experimentally determined constraints (e.g., engineered metal bridges or spin-labels), each treated as an individual molecular fragment with all atomic details. The internal structure of each of the molecular fragments is treated realistically, while there is no interaction between different molecular fragments to avoid unphysical steric clashes. The information from all the molecular fragments is exploited simultaneously to constrain the backbone to refine a three-dimensional model of the conformational state of the protein. The method is illustrated by refining the structure of the voltage-sensing domain (VSD) of the Kv1.2 potassium channel in the resting state and by exploring the distance histograms between spin-labels attached to T4 lysozyme. The resulting VSD structures are in good agreement with the consensus model of the resting state VSD and the spin-spin distance histograms from ESR/DEER experiments on T4 lysozyme are accurately reproduced.

Exploring Cannabinoids as Potential Inhibitors of SARS-CoV-2 Papain-like Protease: Insights from Computational Analysis and Molecular Dynamics Simulations.

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global COVID-19 pandemic, challenging healthcare systems worldwide. Effective therapeutic strategies against this novel coronavirus remain limited, underscoring the urgent need for innovative approaches. The present research investigates the potential of cannabis compounds as therapeutic agents against SARS-CoV-2 through their interaction with the virus's papain-like protease (PLpro) protein, a crucial element in viral replication and immune evasion. Computational methods, including molecular docking and molecular dynamics (MD) simulations, were employed to screen cannabis compounds against PLpro and analyze their binding mechanisms and interaction patterns. The results showed cannabinoids with binding affinities ranging from -6.1 kcal/mol to -4.6 kcal/mol, forming interactions with PLpro. Notably, Cannabigerolic and Cannabidiolic acids exhibited strong binding contacts with critical residues in PLpro's active region, indicating their potential as viral replication inhibitors. MD simulations revealed the dynamic behavior of cannabinoid-PLpro complexes, highlighting stable binding conformations and conformational changes over time. These findings shed light on the mechanisms underlying cannabis interaction with SARS-CoV-2 PLpro, aiding in the rational design of antiviral therapies. Future research will focus on experimental validation, optimizing binding affinity and selectivity, and preclinical assessments to develop effective treatments against COVID-19.

Photophysical Insights of Halogenated Dipyrrolonaphthyridine-Diones as Potential Photodynamic Therapy Agents†.

We report the synthesis and photophysical characterization of novel halogenated dipyrrolonaphthyridine-diones (X2 -DPNDs, X = Cl, Br, and I), as candidates for photodynamic therapy (PDT) application. Apart from the heavy atom-induced spin-orbit coupling (SOC) dynamics in the investigated X2 -DPNDs, it was found that the position of the halogen atom (relative to the nitrogen of the pyrrole ring) also influenced the triplet excited state behavior. Interestingly, the faster/efficiency sensitization of 3 O2 to 1 O2 using X2 -DPND correlates with the rate of triplet population, kISC >1.6 × 108 s-1 for I2 -DPND vs kISC >2.9 × 109 s-1 for Cl2 -DPND and Br2 -DPND (where τISC  = 343 ± 3 ps for I2 -DPND and τISC  = 5-6 ns for Cl2 -DPND and Br2 -DPND are the lowest time constants/values for ISC). Furthermore, the heavy atom-induced SOC in Cl2 -DPND and Br2 -DPND did not lead to a reduction of the corresponding fluorescence (ca 75% vs 67% for the parent DPND). The attractive photophysical characteristics of Cl2 /Br2 -DPND put them on the landscape as not only promising PDT agents but also as fluorescence probes. The present study is a stepping stone in the development of novel organic photosystems for synergistic photomedicinal applications.

Synthesis and Characterization of the Donor-Acceptor Conjugated Polymer PBDB-T Implementing Group IV Element Germanium.

Here, we synthesized and characterized a novel two-dimensional (2D) conjugated electron donor-acceptor (D-A) copolymer (PBDB-T-Ge), wherein the substituent of triethyl germanium was added to the electron donor unit of the polymer. The Turbo-Grignard reaction was used to implement the group IV element into the polymer, resulting in a yield of 86%. This corresponding polymer, PBDB-T-Ge, exhibited a down-shift in the highest occupied molecular orbital (HOMO) level to -5.45 eV while the lowest unoccupied molecular orbital (LUMO) level was -3.64 eV. The peaks in UV-Vis absorption and the PL emission of PBDB-T-Ge were observed at 484 nm and 615 nm, respectively.

A computationally designed ACE2 decoy has broad efficacy against SARS-CoV-2 omicron variants and related viruses in vitro and in vivo.

SARS-CoV-2, especially B.1.1.529/omicron and its sublineages, continues to mutate to evade monoclonal antibodies and antibodies elicited by vaccination. Affinity-enhanced soluble ACE2 (sACE2) is an alternative strategy that works by binding the SARS-CoV-2 S protein, acting as a 'decoy' to block the interaction between the S and human ACE2. Using a computational design strategy, we designed an affinity-enhanced ACE2 decoy, FLIF, that exhibited tight binding to SARS-CoV-2 delta and omicron variants. Our computationally calculated absolute binding free energies (ABFE) between sACE2:SARS-CoV-2 S proteins and their variants showed excellent agreement to binding experiments. FLIF displayed robust therapeutic utility against a broad range of SARS-CoV-2 variants and sarbecoviruses, and neutralized omicron BA.5 in vitro and in vivo. Furthermore, we directly compared the in vivo therapeutic efficacy of wild-type ACE2 (non-affinity enhanced ACE2) against FLIF. A few wild-type sACE2 decoys have shown to be effective against early circulating variants such as Wuhan in vivo. Our data suggest that moving forward, affinity-enhanced ACE2 decoys like FLIF may be required to combat evolving SARS-CoV-2 variants. The approach described herein emphasizes how computational methods have become sufficiently accurate for the design of therapeutics against viral protein targets. Affinity-enhanced ACE2 decoys remain highly effective at neutralizing omicron subvariants.

Draxin inhibits axonal outgrowth through the netrin receptor DCC.

Draxin, a recently identified axon guidance protein, is essential for the formation of forebrain commissures, and can mediate repulsion of netrin-stimulated spinal commissural axons. Here, we report that draxin binds multiple netrin receptors: DCC (deleted in colorectal cancer), Neogenin, UNC5s (H1, H2, H3), and DSCAM (Down's syndrome cell adhesion molecule). Since draxin and Dcc knockouts showed similar phenotype in forebrain commissures formation, we show here the functional importance of draxin/DCC interaction. Draxin interacts with subnanomolar affinity to the netrin receptor DCC, in a region of DCC distinct from its netrin-binding domain. In vitro, neurite outgrowth from cortical and olfactory bulb explants of Dcc knock-out mice is significantly less inhibited by draxin, when compared with neurites from explants of wild-type mice. Furthermore, in comparison with wild-type mice, the growth cone collapse in response to draxin is largely abolished in Dcc-deficient cortical neurons. In vivo, double heteros of draxin/Dcc mice show markedly higher frequency of complete agenesis of corpus callosum than either of the single hetero. These results identify DCC as a convergent receptor for netrin and draxin in axon growth and guidance.

Draxin is involved in the proper development of the dI3 interneuron in chick spinal cord.

Generation of the appropriate types, numbers and distribution of neurons during the development of the nervous system requires the careful coordination of proliferation, differentiation, and patterning. In this work, we analyzed the roles of a repulsive axon guidance protein, draxin, on the development of chick spinal cord dI3 interneuron. draxin mRNA and/or protein were detected in the roof plate at first and then the boundary region between the ventricular and the mantle zones in chick spinal cord and dorsal basement membrane of the chick spinal cord. Overexpression of draxin caused the decreased and delayed migration of the dI3 interneuron, the reduction of progenitor cell proliferation, and abnormal localization of some ectopic progenitor-like cells in the mantle zone of the spinal cord. Our data reveal that draxin may be involved in the proper development of the dI3 interneuron in chick spinal cord.

Prediction and evaluation of deleterious and disease causing non-synonymous SNPs (nsSNPs) in human NF2 gene responsible for neurofibromatosis type 2 (NF2).

The majority of genetic variations in the human genome that lead to variety of different diseases are caused by non-synonymous single nucleotide polymorphisms (nsSNPs). Neurofibromatosis type 2 (NF2) is a deadly disease caused by nsSNPs in the NF2 gene that encodes for a protein called merlin. This study used various in silico methods, SIFT, Polyphen-2, PhD-SNP and MutPred, to investigate the pathogenic effect of 14 nsSNPs in the merlin FERM domain. The G197C and L234R mutations were found to be two deleterious and disease mutations associated with the mild and severe forms of NF2, respectively. Molecular dynamics (MD) simulations were conducted to understand the stability, structure and dynamics of these mutations. Both mutant structures experienced larger flexibility compared to the wildtype. The L234R mutant suffered from more prominent structural instability, which may help to explain why it is associated with the more severe form of NF2. The intramolecular hydrogen bonding in L234R mutation decreased from the wildtype, while intermolecular hydrogen bonding of L234R mutation with solvent greatly increased. The native contacts were also found to be important. Protein-protein docking revealed that L234R mutation decreased the binding complementarity and binding affinity of LATS2 to merlin, which may have an impact on merlin's ability to regulate the Hippo signaling pathway. The calculated binding affinity of the LATS2 to L234R mutant and wildtype merlin protein is found to be 21.73 and -11 kcal/mol, respectively. The binding affinity of the wildtype merlin agreed very well with the experimental value, -8 kcal/mol.Communicated by Ramaswamy H. Sarma.