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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
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  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses

Definitions

• the invention relates to systems and methods for obtaining inhibitors of human immunodeficiency virus- 1 (HIV-1) protease by designing compounds that resemble the charge and geometry of the HIV-1 protease transition state.
• HIV-1 human immunodeficiency virus- 1
• HIV-1 protease is an essential enzyme for the human immunodeficiency virus- 1 (HIV-1) viral life cycle and is the target of nine drugs approved by the FDA for the treatment of HIV/AIDS (1). HIV-1 protease inhibitors are administered as part of a drug combination in a treatment termed highly active antiretroviral therapy (HAART), which has become the most effective therapeutic strategy since the discovery of the virus. Nevertheless, mutations in viral enzymes that reduce drug affinity but not catalytic activity continue to breed resistance to HAART. Several mutations in HIV-1 protease have been identified and resistance to all nine FDA approved drugs has been reported (1, 2). Accordingly, attempts to improve inhibitor quality continue with a common goal of increasing the specificity and affinity of enzyme-drug interactions.
• HIV-1 protease is an essential enzyme for the human immunodeficiency virus- 1 (HIV-1) viral life cycle and is the target of nine drugs approved by the FDA for the treatment of HIV/AIDS (1). HIV-1 protease inhibitors are administered as part of a drug combination in
• the present invention address the need for new inhibitors for HIV-1 protease, particularly ones that will be effective against viral strains that have developed resistance to current drugs.
• the invention provides methods of obtaining inhibitors of human immunodeficiency virus- 1 (HIV-1) protease comprising designing a chemically stable compound that resembles the charge and geometry of the HIV-1 protease transition state.
• HIV-1 human immunodeficiency virus- 1
• the invention also provides systems for obtaining a putative inhibitor of a human immunodeficiency virus- 1 (HIV-1) protease comprising one or more data processing apparatus and a computer-readable medium coupled to the one or more data processing apparatus having instructions stored thereon that are configured to perform a method comprising designing a chemically stable compound that resembles the charge and geometry of the HIV- 1 protease transition state, wherein the compound is a putative inhibitor of HIV- 1 protease.
• HAV-1 human immunodeficiency virus- 1
• the invention further provides methods for screening for a compound that is an inhibitor of a human immunodeficiency virus- 1 (HIV-1) protease, the method comprising the steps of:
• compound comprises an -NH group and two hydroxyl groups bound to the same carbon atom
• the compound comprises three photons with a natural bond orbital charge between +0.500 and +0.600;
• transition structure 4 the r ⁇ .n) is defined as the bond distance between the nitrogen on the proline and the proton on the catalytic aspartate and r(H-o) is defined as the bond distance between the oxygen and proton on the catalytic aspartate. Finally, in transition structure 6, rfC-N) is the bond distance of the scissile bond of the peptide.
• Figure 2A-2B Kinetic isotope effects measured for native and I84V HIV-1 protease.
• A Peptide structure indicating the location of isotopic substitutions used for kinetic isotope effect (KIE) measurements. KIE values are listed for each isotope (values for I84V are in bold).
• KIE kinetic isotope effect
• Figure 3 Theoretical structures calculated for each step of the HIV-1 protease chemical mechanism using ONIOM(aml :M06-2X/6-31+G**). Calculated structures include: (8) TS for attack of water on the carbonyl; (9) gem-diol intermediate; (10) proton transfer from Asp-25 to proline; (11) protonated amide intermediate; (12) scissile bond cleavage. Distances are shown in Angstroms.
• Figure 4 Theoretical structure for proton transfer from Asp-25 to proline calculated by fixing the r ⁇ N-H) bond length at 1.20 A using ONIOM(aml :M06-2X/6-31+G**). Predicted KIEs derived from this transition structure (13) most closely match the experimentally measured values.
• FIG. 5A-5C Electrostatic potential, NBO charges, and inhibitor scaffolds.
• A Structure of the clinical inhibitor indinavir bound to the HIV-1 protease active site obtained from the PDB structure 2AVO (left) and the proton transfer transition structure 13 (right).
• NBO charges are indicated in parentheses.
• FIG. 6 Synthesis of L-[2- 3 H]-phenylalanine.
• 3 H is transferred from [1- 3 H]- glucose to [2- 3 H]-Phe-OH via a sequence of 3 H-transfer reactions.
• ATP-dependent hexokinase catalyzes conversion of [1- 3 H] -glucose to [l- 3 H]-glucose-6-phosphate.
• 3 H is then transferred from [l- 3 H]-glucose-6-phosphate to NAD + by glucose-6-phosphate dehydrogenase (G6PDH), reducing it to [(5)-4- 3 H]-NADH and forming 6-phosphogluconate.
• G6PDH glucose-6-phosphate dehydrogenase
• phenylalanine dehydrogenase (PheDH) is used to produce 2- 3 H-Phe-OH from [(S)-4- 3 H]-NADH and N3 ⁇ 4 by reductive amination of phenylpyruvate.
• the invention provides a method of obtaining a putative inhibitor of a human immunodeficiency virus- 1 (HIV-1) protease, the method comprising designing a chemically stable compound that resembles the charge and geometry of the HIV-1 protease transition state, wherein the compound is a putative inhibitor of HIV- 1 protease.
• HIV-1 human immunodeficiency virus- 1
• the invention also provides a system for obtaining a putative inhibitor of a human immunodeficiency virus- 1 (HIV-1) protease comprising one or more data processing apparatus and a computer-readable medium coupled to the one or more data processing apparatus having instructions stored thereon that are configured to perform a method comprising designing a chemically stable compound that resembles the charge and geometry of the HIV- 1 protease transition state, wherein the compound is a putative inhibitor of HIV- 1 protease.
• HAV-1 human immunodeficiency virus- 1
• the method can include the steps of:
• the HIV-1 protease transition state structure can comprise three protons with natural bond orbital charges of +0.549, +0.564 (diol OHs) and +0.504 ( roline N).
• the HIV-1 protease transition state structure can comprise
• the method can also comprise synthesizing the putative inhibitor compound and/or testing the compound for inhibitory activity to HIV-1 protease.
• the compound comprises two hydroxyl groups bound to the same carbon atom and an -NH group.
• the compound comprises three photons with a natural bond orbital charge between +0.500 and +0.600.
• the invention further provides a method for screening for a compound that is an inhibitor of a human immunodeficiency virus- 1 (HIV-1) protease, the method comprising the steps of:
• compound comprises an -NH group and two hydroxyl groups bound to the same carbon atom
• the compound comprises three photons with a natural bond orbital charge between +0.500 and +0.600;
• the HIV-1 protease can be native HIV-1 protease or a mutant form of HIV-1 protease.
• the mutation can be of the form that causes resistance to currently approved (2012) drugs for treating HIV-1.
• the mutant form of HIV-1 protease can have valine
• the invention also provides methods of inhibiting HIV-1 protease comprising obtaining a HIV-1 protease inhibitor by any of the methods disclosed herein or by using the system disclosed herein, and contacting the HIV-1 protease with the compound.
• the invention further provides methods of treating a subject having human immunodeficiency virus- 1 (HIV-1) comprising obtaining a HIV-1 protease inhibitor by any of the methods disclosed herein or by using the system disclosed herein, and administering the compound to the subject in an amount effective to inhibit HIV-1 protease.
• HIV-1 human immunodeficiency virus- 1
• the invention still further provides compounds obtained by any of the methods disclosed herein or by using the system disclosed herein.
• a compound resembles the HIV-1 protease transition state molecular electrostatic potential at the van der Waals surface computed from the wave function of the transition state and the geometric atomic volume if that compound has an S e and S g >0.5, where S e and S g are determined as in Formulas (1) and (2) on page 8831 of Bagdassarian, Schramm and Schwartz, 1996 (55).
• ⁇ * is the electrostatic potential at surface point i of molecule A
• J defines point j of molecule B
• in the numerator is the spatial distance squared between point i on A and j on B.
• nA and « ⁇ refer to the number of surface points on each molecule. The double summation is therefore over all possible interactions between points on the two molecules, and a is the length scale for the interaction between i and j.
• the numerator compares A to B for a particular orientation of molecule B relative to molecule A.
• the denominator serves as a normalization factor for the comparison of A to itself and for B to itself.
• ⁇ refers to the distance between i and j on the same molecule. The distance between points is squared to decrease computation time.
• HIV-1 protease is a homodimer of 99 amino acid subunits, each of which contribute a catalytic aspartate to the enzyme's active site (Asp-25 and Asp- 125) (4, 5).
• the catalytic mechanism and structural details of the HIV-1 protease reaction are illustrated in Fig. 1 (structures 1-7). The cycle initiates with water activation by hydrogen-bonding interactions with the active site aspartate residues (structure 1) (6, 7).
• a powerful technique in determining the chemical details of a reaction mechanism is to use experimentally measured kinetic isotope effects (KIEs) to constrain theoretical predictions (33).
• KIEs kinetic isotope effects
• This method for elucidating enzymatic transition structures has provided sufficiently robust transition-state knowledge to permit design and synthesis of powerful transition-state mimics (34).
• transition-state analysis is applied to the study of the chemical mechanism of HIV-1 protease.
• Experimentally measured intrinsic KIEs are used to evaluate candidate transition-state structures originating from the distinct chemical barriers and intermediates in the reaction cycle of HIV-1 protease.
• the availability of a geometrically accurate transition structure provides chemical resolution of the reaction and allows development of transition-state inhibitors of the enzyme. Understanding transition- state features for native and resistant enzymes also provides insights into the mechanism of resistance.
• Peptide substrates (Fig 2A, Table 1) were synthesized by sequentially coupling FMOC-protected amino acids onto a CLEAR- amide resin (100-200 mesh 0.43 mmol/g) followed by N-acetylation, resin cleavage, precipitation, and purification.
• Isotopic labels were incorporated at the remote positions by acetylation with either [ 3 H 3 ]Ac 2 0 (purchased) or [l- 14 C]Ac 2 0 (purchased), and isotopic labels at the scissile positions were incorporated by coupling the appropriate labeled amino acid from the following: FMOC-[l- 14 C]Phe-OH (purchased), FMOC-[ 15 N]Pro-OH (purchased), FMOC-[a- 3 H]Phe-OH (synthesized), or FMOC-[ 18 0 2 ]Phe-OH (synthesized).
• Peptides were purified by semi-preparative reverse-phase HPLC using a linear gradient (3- 40% acetonitrile in water with 0.1% TFA over 40 min).
• the purified non-radioactive peptides were characterized by MS-ESI, and radioactive peptides were confirmed by co- elution with the non-radioactive peptide using analytical reverse-phase HPLC over the same linear gradient.
• FMOC-[l- 14 C]Phe-OH, [ 3 H]Ac 2 0, and [l- 14 C]Ac 2 0 were purchased from American Radiolabeled Chemicals, Inc., and FMOC-[ 15 N]-proline was purchased from Cambridge Isotope Laboratories, Inc.
• a reaction mixture of 4 mM [l- 3 H]glucose, 50 mM ATP, 160 ⁇ NAD, 250 mM NH 4 C1, 10 mM MgCl 2 , and 10 mM phenylpyruvate in 50 mM Tris-HCl pH 8.5 was prepared and the following enzymes were added sequentially (0.5 units each): 1) phenylalanine dehydrogenase (Sigma), 2) glucose-6-phosphate dehydrogenase (Sigma), 3) hexokinase (Sigma). The reaction was completed in approximately 45 minutes at room temperature.
• HIV-1 protease (PR) constructs (native and I84V) bearing the five background mutations Q7K, L33I, L63I (for restricted auto-proteolysis), and C67A and C95A (for restricted Cys thiol oxidation) were provided in pET-l la vectors.
• PR constructs native and I84V bearing the five background mutations Q7K, L33I, L63I (for restricted auto-proteolysis), and C67A and C95A (for restricted Cys thiol oxidation) were provided in pET-l la vectors.
• the constructs were transformed into BL21(DE3) E. Coli cells and expressed and purified from inclusion bodies according to an established protocol (46).
• KIEs Kinetic isotope effects
• the KIE on V/K was determined by the relative change in the ratio of light and heavy peptides (each bearing either 14 C or 3 H radiolabels) in the unreacted substrate vs. remaining substrate after multiple reaction cycles.
• Peptides were radiolabeled with either 3 H or 14 C as shown in Table 1.
• KIEs were measured by mixing the heavy and light peptides such that the cpm ratio of 3 H: 14 C was 3 : 1 (150,000 cpm:50,000 cpm), with a total peptide concentration kept at 0.5 mM in a 200 ⁇ reaction volume.
• KIE ln(l - yin[(l -j)(R Ro)l
• each peptide was acetylated by reacting the peptidyl-resin with AC 2 O and DIPEA for 0.5-3 hours.
• Amino acids bearing stable isotopes were coupled with 3 equivalents (eq) amino acid, 2.9 eq HCTU, 2.9 eq 6-Cl-HOBt, and 5.8 eq DIPEA.
• Radiolabeled amino acids were coupled with 1.1 eq amino acid (80-125 ⁇ ), 1.1 eq HCTU, 1.1 eq 6-Cl-HOBt, and 2.2 eq DIPEA.
• the 14 C- remotely labeled peptide was acetylated with 1.2 eq [ 14 C]-Ac 2 0 (50-250 ⁇ ) and 2.4 eq DIPEA.
• 3 H-remotely labeled peptides were acetylated with 1.5 eq [ 3 H]-Ac 2 0 (1-5 mCi) and 3 eq DIPEA.
• Peptides not requiring a remote label were acetylated with 10 eq AC 2 O and 20 eq DIPEA.
• Fmoc protection of [ 18 0 2 ]-Phe-OH was carried out by dissolving the amino acid in 1.0 ml H 2 18 0 (97%), followed by addition of NaHC0 3 to bring the reaction pH to 9.0.
• Fmoc-Osu (121 mg, 1.5 eq) was dissolved in 1.5 ml cold acetone and added slowly to the amino acid solution while stirring on ice. Reaction progress was followed by TLC (8:3 :0.4 CH 2 CI3/CH 2 OH/H 2 O) analysis using ninhydrin reactivity for detection, and after two hours no [ 18 0 2 ]-Phe-OH was detected.
• the final yield of [2- 3 H]-Phe-OH was determined to be approximately 80 ⁇ - based on scintillation counting of a small aliquot - corresponding to an estimated chemical yield of 1.0 ⁇ .
• the purified [2- 3 H]-Phe-OH was vacuum dried and dissolved in 1.0 ml water for subsequent Fmoc protection.
• Tables of calculated isotope effects from the fixed parameter methods give examples (from the many exploratory structures calculated) of the redundancies in the predicted IEs for the mechanism of HIV protease.
• the IEs were calculated from theoretical structures using ONIOM (M06-2X/6-31+G**:aml) and (B3LYP/6-31G*:aml) methods in Gaussian 09. The bond making/breaking distance for each step of the reaction was varied and the grid of IEs is shown below.
• IEs with the heading KIE are derived from a structure with one imaginary frequency and those shown under EIE are derived from structures with no negative frequencies.
• KIEs Kinetic Isotope Effects.
• a family of KIEs was measured at atomic positions surrounding the cleavage site of the heptapeptide substrate Acetyl-Ser-Gln-Asn-Phe*Pro-Val- Val-NH2 (*denotes the scissile bond).
• the individual isotopic substitutions and measured KIEs are listed in Table 1 and shown Fig. 2A. Additionally, KIEs were measured for the multi-drug resistant variant (I84V), which is illustrated in Fig. 2B. KIEs were determined by comparing 3 H/ 14 C ratios from isotopic peptides and reference peptides bearing remote radiolabels (Table 1).
• Equation 1 (assuming that any step up to bond cleavage may be isotope-sensitive), Equation 1 can be used to represent the observed KIE (22, 35), where K eq3 represents the equilibrium constant for formation of the 3 and A3 ⁇ 4 is the rate constant for the formation of 7.
• the forward commitment (cf) represents a probability of substrate bound in the 1 being catalytically turned over rather than being released from the Michaelis complex:
• the KIE is measured on V/K and reports on isotope-sensitive steps up to and including the first irreversible step of each catalytic cycle (35, 36).
• a high probability of 1 being turned over to products presents a virtually irreversible step prior to chemistry which can mask expression of the KIE on the chemical step (the intrinsic KIE); the value that reports on the transition state.
• the reportedly high K m for this peptide (37) and the observation that the N KIE value is within the limit of all calculated transition-state models for this reaction suggest that the forward commitment can be considered negligible in the analysis, simplifying the equation of the observed isotope effects to the product of the equilibrium isotope effect (EIE) on formation of 3 and the intrinsic KIE determined by the rate-limiting transition state:
• Theoretical structures used for the final calculation of KIEs include an Ala-Pro dipeptide as the substrate - an alanine residue was used in place of the experimental phenylalanine for simplicity - in addition to several important residues in the active site, including the two catalytic aspartates, the nucleophilic water, as well as the structural water (Fig. 3).
• Theoretical structures were derived from a published crystal structure of HIV-1 protease co-crystallized with a gem-diol intermediate (17). Calculations were performed using both ONIOM (M06-2X/6-31+G**:aml) and (B3LYP/6-31G*:aml) as implemented in Gaussian 09 (38).
• Theoretical structures were located as local minima for the starting peptide and structures 3 and 5.
• IEs were calculated for each of the structures based upon the lowest energy conformation of the starting material peptide and an intermediate or transition structure similar to the conformational geometry found in the published crystal structure.
• all theoretical structures for transition states 2, 4, and 6 were located as first order saddle points on the potential energy surface. By explicitly including a portion of the active site, it is assumed that geometry constraints are not required to mimic the active site functionality and that the saddle points will represent the on-enzyme transition structures.
• Theoretical structures 8, 10, and 12 represent the unconstrained first order saddle points for transition states 2, 4, and 6, respectively (Fig. 3).
• Transition structure 8 corresponds to the first step of the reaction and indicates that attack of the catalytic water is late - (C -o ) is 1.79 A.
• the second step of the reaction involves proton transfer from the aspartyl residue to the nitrogen on proline and occurs at H) of 1.26 A as shown in 10.
• the third chemical step involves the breaking of the scissile C-N bond of protonated amide (11) which occurs relatively late (r ⁇ c-N) is 2.02 A, as shown in transition structure 12).
• a concerted transition structure could not be located for direct formation of 7 from intermediate 3. Therefore, the protonation and cleavage of the amide are considered to be stepwise, though we cannot entirely rule out the possibility of a concerted reaction.
• KIE predictions In accordance with the experimental observation of H2 18 0 exchange (19), IEs for 10, 11, and 12 were calculated as the product of the EIE for formation of 9 and the KIE of the transition structure (Equation 2) (22). Predicted 14 C and 15 N KIEs for the attack of water on the peptide from transition structure 8 are both too large, and are poor matches to the measured values; though it is unlikely that 2 is KIE-determining because of the experimental observation supporting the reversibility of this step (19). Similarly, 14 C and 15 N KIE predictions for the breaking of the C-N bond of the amide peptide in 12 were also too large to match the experimental values.
• the proton transfer transition structure 10 most closely matches the experimentally measured KIEs, though the 14 C and 15 N KIEs are smaller than predicted by approximately 1%. Additionally, the EIEs for the protonated amide intermediate (11) are a relatively close match to the experimentally measured values. This is unsurprising since structures 10 and 11 are nearly identical with the exception of the r ⁇ -H) distance of 1.26 A and 1.12 A, respectively.
• Proton Transfer An important aspect of proton transfer transition structure 13 is that the model contains three protons directly associated with the substrate (Figs. 4 & 5). The most stable conformation of the proline is a five membered boat, placing the lone pair on the nitrogen in position to accept the proton from Asp25. Stretching of the scissile C-N amide bond is associated with the imaginary frequency for the transition structure of proton transfer, suggesting that amide bond cleavage is associated with this step. However, little change occurs between 10, 13, and 11 (in order of decreasing ⁇ ( ⁇ - ⁇ ) ), with i- ( -c-N ) Changing only slightly from 1.51, 1.52, to 1.53 A, respectively. Hydrogen bonding to both oxygens of the gem-diol occurs solely with Asp 125 and is maintained throughout the reaction, consistent with previous dynamic, crystallographic, and neutron diffraction studies (15).
• I84V transition structure is identical to that for the native enzyme indicates that the drug resistant behavior that arises is independent from transition-state interactions.
• a stable mimic of transition structure 13 shown in Fig. 4 should be an effective inhibitor both the native and I84V HIV-1 protease, and likely an effective inhibitor against all biologically relevant HIV-1 protease variants.
• Electrostatic potential maps provide useful blueprints for inhibitor design since they provide shape and charge details of the reactant at the moment of the transition state.
• the map of indinavir reveals a discrepancy common among the transition structure and the existing FDA approved HIV-1 protease inhibitors. All clinically approved inhibitors posses the gem-diol mimic, but lack the charge character that results from the additional proton present in 13 ( Figure 5B and C).
• transition structure 13 has three protons with BO charges of +0.549, +0.564 (diol OH's), and +0.504 (proline N), where the indinavir has one proton within the diol mimic with an NBO charge of +0.514.
• a transition-state analogue scaffold proposed from the electrostatic potential map in Fig. 5B is shown in Figure 5C (right) with a focus on transition structure features. While the catalytic aspartic acids provide a proton to both the transition-state mimic and indinavir upon inhibitor binding, the crucial feature of the proposed transition-state inhibitor scaffold is the increased protonation state of the nitrogen, and should be a focus of future inhibitor design.

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