A Comparative Molecular Dynamics, MM−PBSA and Thermodynamic Integration Study of Saquinavir Complexes with Wild-Type HIV‑1 PR and L10I, G48V, L63P, A71V, G73S, V82A and I84V Single Mutants

A great challenge toward Acquired Immunodeficiency Syndrome (AIDS) treatment is to combat the HIV-1 virus. The major problem of drug resistance has kept the virus one step ahead of the medical community, and the call for more effective drugs remains as urgent as ever. Saquinavir, the first inhibitor against HIV-1 protease, offers the most extensive clinical data regarding resistance mutations. In this work, we examine L10I, G48V, L63P, A71V, G73S, V82A, and I84V single mutant HIV-1 PR strains in complexes with saquinavir to elucidate drug–protease interactions and dynamics. A comparative analysis of these mutations at the molecular level may lead to a deeper understanding of saquinavir resistance. The G48V mutation induces structural changes to the protease that reflect upon the drug’s binding affinity, as shown by MM–PBSA and thermodynamic integration (TI) calculations (ΔΔGTI = 0.3 kcal/mol; ΔΔGMM–PBSA = 1.2 kcal/mol). It was shown that mutations, which increase the flexibility of the flaps (G48V, L63P, L10I) diminish binding. The preservation of hydrogen bonds of saquinavir with both the active site and flap residues in the wild-type and certain single mutants (A71V, V82A) is also crucial for effective inhibition. It was shown that mutations conferring major resistance (G48V, L63P, I84V) did not present these interactions. Finally, it was indicated that a water-mediated hydrogen bond between saquinavir and Asp29 in the active site (wild-type, A71V, G73S) facilitates a proper placement of the drug into the binding cavity that favors binding. Mutants lacking this interaction (G48V, V82A, I84V) demonstrated reduced binding affinities. This systematic and comparative study is a contribution to the elucidation of the drug resistance mechanism in HIV-1 PR

Refinement of the gonadotropin releasing hormone receptor I homology model by applying molecular dynamics

Sexual maturation of human cells in ovaries and prostate is linked to the biochemical cascade initiated by the activation of cell receptors through the binding of Gonadotropin Releasing Hormone (GnRH). The GnRH receptors (GnRHR) are part of the rhodopsin G-protein coupled receptor (GPCR) family and consist of seven trans–membrane helical domains connected via extra– and intra–cellular segments. The GnRH–GnRHR complex has been implicated in various forms of prostate and ovarian cancer. The lack of any structural data about the GnRH receptor impedes the design of antagonists for use in cancer treatment. The aim of the study is to devise a model of GnRHR to be used further for the design of improved peptide/non-peptide GnRH analogues and, to our knowledge provide new structural information regarding the extracellular loop 2 (ECL2) that acts a regulator of ligand entry to GnRHR. The common structural characteristics, of the members of the rhodopsin family of GPCRs, have been employed for the construction of a homology model for GnRHR. Structural information from the human β2–adrenergic receptor, as well as rhodopsins have been used in order to create a theoretical model for GnRHR. Furthermore, molecular dynamics (MD) simulations have been employed for the refinement of the model and to explore the impact of the bilayer membrane in GnRHR conformation

Molecular dynamics at the receptor level of immunodominant myelin oligodendrocyte glycoprotein 35-55 epitope implicated in multiple sclerosis

Multiple Sclerosis (MS) is a common autoimmune disease whereby myelin is destroyed by the immune system. The disease is triggered by the stimulation of encephalitogenic T-cells via the formation of a trimolecular complex between the Human Leukocyte Antigen (HLA), an immunodominant epitope of myelin proteins and T-cell Receptor (TCR). Myelin Oligodendrocyte Glycoprotein (MOG) is located on the external surface of myelin and has been implicated in MS induction. The immunodominant 35–55 epitope of MOG is widely used for in vivo biological evaluation and immunological studies that are related with chronic Experimental Autoimmune Encephalomyelitis (EAE, animal model of MS), inflammatory diseases and MS. In this report, Molecular Dynamics (MD) simulations were used to explore the interactions of MOG35–55 at the receptor level. A detailed mapping of the developed interactions during the creation of the trimolecular complex is reported. This is the first attempt to gain an understanding of the molecular recognition of the MOG35–55 epitope by the HLA and TCR receptors. During the formation of the trimolecular complex, the residues Arg41 and Arg46 of MOG35–55 have been confirmed to serve as TCR anchors while Tyr40 interacts with HLA. The present structural findings indicate that the Arg at positions 41 and 46 is a key residue for the stimulation of the encephalitogenic T-cells

Design of novel inhibitors for renin and HIV-1 protease

Acquired immunodeficiency syndrome (AIDS) and hypertension present a public health challenge worldwide. Hitherto, more than 27 million people have died from HIV-1 infection (UNAIDS). Also, up to 1 billion people have developed some form of hypertension. Studies have identified HIV-1 protease (HIV-1 PR) and renin as the primary targets for drug design against AIDS and hypertension, respectively. The viral protease plays a crucial role in viral replication and renin induces the production of angiotensin I (AT-I), that is involved in the renin-angiotensin-aldosterone system (RAAS). Both enzymes belong to the family of aspartic proteases. Understanding the binding mechanism of different commercially available drugs offers valuable information for the design of novel inhibitors. The aim of this study is to analyze the different interactions between ligands and the enzymes and thus propose new inhibitors. Also, an important part of the present project is dedicated to the identification of common functional aspects (hydrogen bond interactions) in both proteins. Different computational techniques (e.g. 3D-QSAR and molecular dynamics) have been used for this purpose. The binding energy in the different renin and HIV-1 PR complexes has been calculated with the MM–PBSA method. Thermodynamic integration has been implemented for the analysis of the effect of mutations on the binding of saquinavir in HIV-1 PR. Of great importance is the dual inhibition of the drugs darunavir (AIDS) and aliskiren (hypertension) in both enzymes. This is further supported by the inhibitory action of canagliflozin –an anti-diabetic agent– in renin and in HIV-1 PR as it is depicted by the favoured ΔG binding values.Το σύνδρομο επίκτητης ανοσοποιητικής ανεπάρκειας (AIDS) και η υπέρταση, αποτελούν δύο από τα σημαντικότερα προβλήματα της παγκόσμιας υγείας. Μέχρι σήμερα, 27 εκατ. άτομα έχουν αποβιώσει από τον ιό HIV-1 (στοιχεία UNAIDS). Επίσης, πάνω από 1 δισεκατομμύρια άτομα πάσχουν από υπέρταση. Μελέτες έχουν υποδείξει την HIV-1 πρωτεάση και τη ρενίνη, ως βασικούς φαρμακευτικούς στόχους, κατά του AIDS και της υπέρτασης, αντίστοιχα. Η HIV-1 πρωτεάση έχει σημαντικό ρόλο στην αναπαραγωγή του ιού, ενώ η ρενίνη έχει συνδεθεί με την παραγωγή της αγγειοτασίνης Ι (ΑΤ-Ι), στο σύστημα ρενίνης-αγγειοτασίνης-αλδοστερόνης (RAAS). Και τα δύο ένζυμα ανήκουν στην οικογένεια των ασπαρτικών πρωτεασών. Η κατανόηση του μηχανισμού πρόσδεσης διαφορετικών εμπορικά διαθέσιμων φαρμάκων αποτελεί βασικό στοιχείο για το σχεδιασμό νέων καινοτόμων αναστολέων. Σκοπός της παρούσας εργασίας είναι η ανάλυση των αλληλεπιδράσεων μεταξύ των ενζύμων και των προσδετών με στόχο την πρόταση νέων φαρμακευτικών μορίων. Επίσης σημαντικό τμήμα της εργασίας είναι η αναγνώριση κοινών λειτουργικών στοιχείων (δημιουργία δεσμών υδρογόνου) στις δύο πρωτεΐνες. Η μελέτη στηρίχθηκε στη χρήση διαφορετικών υπολογιστικών τεχνικών (πχ 3D-QSAR και μοριακή δυναμική). Ο υπολογισμός της ενέργειας πρόσδεσης στα διάφορα σύμπλοκα της ρενίνης και της HIV-1 πρωετάσης έγινε με τη χρήση της μεθόδου MM–PBSA ενώ η μέθοδος της θερμοδυναμικής ολοκλήρωσης χρησιμοποιήθηκε για την ανάλυση της επίδρασης μεταλλάξεων στην πρόσδεση της σακουιναβίρης στην HIV-1 πρωτεάση. Ένα από τα σημαντικότερα ευρήματα αποτέλεσε η αναγνώριση κοινών λειτουργικών στοιχείων των ασπαρτικών πρωτεασών μέσω της ανάλυσης της επίδρασης των φαρμάκων νταρουναβίρης (AIDS) και αλισκιρένης (υπέρταση) στα δύο ένζυμα. Αυτό υποστηρίζεται περαιτέρω από τη διττή ανασταλτική δράση του αντιδιαβητικού φαρμάκου καναφλιφλοζίνης στη ρενίνη και στην HIV-1 πρωτεάση όπως προκύπτει από την ευνοϊκή μεταβολή ελεύθερης ενέργειας στην πρόσδεση

Systematic molecular dynamics, MM-PBSA, and ab initio approaches to the saquinavir resistance mechanism in HIV-1 PR Due to 11 double and multiple mutations

Mutations in the human immunodeficiency virus (HIV) enable virus replication even when appropriate antiretroviral therapy is followed, thus leading to the emergence of drug resistance. In a previous work, we systematically examined seven single mutations that are associated with saquinavir (SQV) resistance in HIV-1 protease (Tzoupis, H.; Leonis, G.; Mavromoustakos, T.; Papadopoulos, M. G. J. Chem. Theory Comput. 2013, 9, 1754−1764). Herein, we extend our analysis, which includes seven double (G48VV82A, L10I-G48V, G48V-L90M, I84V-L90M, L10I-V82A, L10I-L63P, A71V-G73S) and four multiple (L10I-L63PA71V, L10I-G48V-V82A, G73S-I84V-L90M, L10I-L63PA71V-G73S-I84V-L90M) SQV−HIV-1 PR mutant complexes, in an attempt to generalize our findings and formulate the main elements of the SQV resistance mechanism in the protease. On the basis of molecular dynamics (MD), molecular mechanics Poisson−Boltzmann surface area (MM−PBSA), and ab initio computational approaches, we identified specific features that constitute the HIV-1 PR mechanism of resistance at the molecular level: the low flexibility of SQV in the binding cavity and the preservation of hydrogen bonding (HB) and van der Waals interactions between SQV and several active-site (Gly27/27′, Asp29/29′/30/30′, especially Asp25/25′) and flap (Ile50/50′, Gly48/48′) residues of the protease contribute significantly to efficient binding. The total enthalpy loss in all mutants is mostly due to the loss in enthalpy of the active-site region. Furthermore, it was observed that mutation accumulation may induce stabilization to SQV and to the flaps through enhanced HB interactions that lead to improved inhibition (e.g., accumulation of mutations in complexes containing L10I, G48V, L63P, I84V, or L90M single mutations). It was also concluded that permanent flap closure is obtained independently of mutations and SQV binding is mostly driven by van der Waals, nonpolar, and exchangeenergy contributions. Importantly, it was indicated that the optimal positioning of SQV and the structure of the binding cavity are tightly coupled, since small changes in geometry may affect the binding energy greatly. The results of our theoretical approaches are in agreement with experimental evidence and provide a reliable description of SQV resistance in HIV-1 PR

Systematic Molecular Dynamics, MM–PBSA, and Ab Initio Approaches to the Saquinavir Resistance Mechanism in HIV‑1 PR Due to 11 Double and Multiple Mutations

Mutations in the human immunodeficiency virus (HIV) enable virus replication even when appropriate antiretroviral therapy is followed, thus leading to the emergence of drug resistance. In a previous work, we systematically examined seven single mutations that are associated with saquinavir (SQV) resistance in HIV-1 protease (Tzoupis, H.; Leonis, G.; Mavromoustakos, T.; Papadopoulos, M. G. <i>J. Chem. Theory Comput.</i> <b>2013</b>, <i>9</i>, 1754–1764). Herein, we extend our analysis, which includes seven double (G48V-V82A, L10I-G48V, G48V-L90M, I84V-L90M, L10I-V82A, L10I-L63P, A71V-G73S) and four multiple (L10I-L63P-A71V, L10I-G48V-V82A, G73S-I84V-L90M, L10I-L63P-A71V-G73S-I84V-L90M) SQV–HIV-1 PR mutant complexes, in an attempt to generalize our findings and formulate the main elements of the SQV resistance mechanism in the protease. On the basis of molecular dynamics (MD), molecular mechanics Poisson–Boltzmann surface area (MM–PBSA), and ab initio computational approaches, we identified specific features that constitute the HIV-1 PR mechanism of resistance at the molecular level: the low flexibility of SQV in the binding cavity and the preservation of hydrogen bonding (HB) and van der Waals interactions between SQV and several active-site (Gly27/27′, Asp29/29′/30/30′, especially Asp25/25′) and flap (Ile50/50′, Gly48/48′) residues of the protease contribute significantly to efficient binding. The total enthalpy loss in all mutants is mostly due to the loss in enthalpy of the active-site region. Furthermore, it was observed that mutation accumulation may induce stabilization to SQV and to the flaps through enhanced HB interactions that lead to improved inhibition (e.g., accumulation of mutations in complexes containing L10I, G48V, L63P, I84V, or L90M single mutations). It was also concluded that permanent flap closure is obtained independently of mutations and SQV binding is mostly driven by van der Waals, nonpolar, and exchange-energy contributions. Importantly, it was indicated that the optimal positioning of SQV and the structure of the binding cavity are tightly coupled, since small changes in geometry may affect the binding energy greatly. The results of our theoretical approaches are in agreement with experimental evidence and provide a reliable description of SQV resistance in HIV-1 PR

Dual Inhibitors for Aspartic Proteases HIV-1 PR and Renin: Advancements in AIDS–Hypertension–Diabetes Linkage via Molecular Dynamics, Inhibition Assays, and Binding Free Energy Calculations

Human immunodeficiency virus type 1 protease (HIV-1 PR) and renin are primary targets toward AIDS and hypertension therapies, respectively. Molecular mechanics Poisson–Boltzmann surface area (MM–PBSA) free-energy calculations and inhibition assays for canagliflozin, an antidiabetic agent verified its effective binding to both proteins (Δ<i>G</i>_pred = −9.1 kcal mol^–1 for canagliflozin–renin; <i>K</i>_i,exp= 628 nM for canagliflozin–HIV-1 PR). Moreover, drugs aliskiren (a renin inhibitor) and darunavir (an HIV-1 PR inhibitor) showed high affinity for HIV-1 PR (<i>K</i>_i,exp= 76.5 nM) and renin (<i>K</i>_i,pred= 261 nM), respectively. Importantly, a high correlation was observed between experimental and predicted binding energies (<i>r</i>² = 0.92). This study suggests that canagliflozin, aliskiren, and darunavir may induce profound effects toward dual HIV-1 PR and renin inhibition. Since patients on highly active antiretroviral therapy (HAART) have a high risk of developing hypertension and diabetes, aliskiren-based or canagliflozin-based drug design against HIV-1 PR may eliminate these side-effects and also facilitate AIDS therapy