Péter Boross

Novel bis-Tetrahydrofuranylurethane-Containing Nonpeptidic Protease Inhibitor (PI) UIC-94017 (TMC114) with Potent Activity against Multi-PI-Resistant Human Immunodeficiency Virus In Vitro

ABSTRACT We designed, synthesized, and identified UIC-94017 (TMC114), a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing a 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) and a sulfonamide isostere which is extremely potent against laboratory HIV-1 strains and primary clinical isolates (50% inhibitory concentration [IC50], ∼0.003 μM; IC90, ∼0.009 μM) with minimal cytotoxicity (50% cytotoxic concentration for CD4+ MT-2 cells, 74 μM). UIC-94017 blocked the infectivity and replication of each of HIV-1NL4-3 variants exposed to and selected for resistance to saquinavir, indinavir, nelfinavir, or ritonavir at concentrations up to 5 μM (IC50s, 0.003 to 0.029 μM), although it was less active against HIV-1NL4-3 variants selected for resistance to amprenavir (IC50, 0.22 μM). UIC-94017 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents. Structural analyses revealed that the close contact of UIC-94017 with the main chains of the protease active-site amino acids (Asp-29 and Asp-30) is important for its potency and wide spectrum of activity against multi-PI-resistant HIV-1 variants. Considering the favorable pharmacokinetics of UIC-94017 when administered with ritonavir, the present data warrant that UIC-94017 be further developed as a potential therapeutic agent for the treatment of primary and multi-PI-resistant HIV-1 infections.

Molecular basis for substrate recognition and drug resistance from 1.1 to 1.6 Å resolution crystal structures of HIV-1 protease mutants with substrate analogs

HIV‐1 protease (PR) and two drug‐resistant variants – PR with the V82A mutation (PRV82A) and PR with the I84V mutation (PRI84V) – were studied using reduced peptide analogs of five natural cleavage sites (CA‐p2, p2‐NC, p6pol‐PR, p1‐p6 and NC‐p1) to understand the structural and kinetic changes. The common drug‐resistant mutations V82A and I84V alter residues forming the substrate‐binding site. Eight crystal structures were refined at resolutions of 1.10–1.60 Å. Differences in the PR–analog interactions depended on the peptide sequence and were consistent with the relative inhibition. Analog p6pol‐PR formed more hydrogen bonds of P2 Asn with PR and fewer van der Waals contacts at P1′ Pro compared with those formed by CA‐p2 or p2‐NC in PR complexes. The P3 Gly in p1‐p6 provided fewer van der Waals contacts and hydrogen bonds at P2–P3 and more water‐mediated interactions. PRI84V showed reduced van der Waals interactions with inhibitor compared with PR, which was consistent with kinetic data. The structures suggest that the binding affinity for mutants is modulated by the conformational flexibility of the substrate analogs. The complexes of PRV82A showed smaller shifts of the main chain atoms of Ala82 relative to PR, but more movement of the peptide analog, compared to complexes with clinical inhibitors. PRV82A was able to compensate for the loss of interaction with inhibitor caused by mutation, in agreement with kinetic data, but substrate analogs have more flexibility than the drugs to accommodate the structural changes caused by mutation. Hence, these structures help to explain how HIV can develop drug resistance while retaining the ability of PR to hydrolyze natural substrates.

Atomic resolution crystal structures of HIV-1 protease and mutants V82A and I84V with saquinavir

Saquinavir (SQV), the first antiviral HIV‐1 protease (PR) inhibitor approved for AIDS therapy, has been studied in complexes with PR and the variants PRI84V and PRV82A containing the single mutations I84V and V82A that provide resistance to all the clinical inhibitors. Atomic resolution crystal structures (0.97–1.25 Å) of the SQV complexes were analyzed in comparison to the protease complexes with darunavir, a new drug that targets resistant HIV, in order to understand the molecular basis of drug resistance. PRI84V and PRV82A complexes were obtained in both the space groups P21212 and P212121, which provided experimental limits for the conformational flexibility. The SQV interactions with PR were very similar in the mutant complexes, consistent with the similar inhibition constants. The mutation from bigger to smaller amino acids allows more space to accommodate the large group at P1′ of SQV, unlike the reduced interactions observed in darunavir complexes. The residues 79–82 have adjusted to accommodate the large hydrophobic groups of SQV, suggesting that these residues are intrinsically flexible and their conformation depends more on the nature of the inhibitor than on the mutations in this region. This analysis will assist with development of more effective antiviral inhibitors. Proteins 2007.

A Novel Bis-Tetrahydrofuranylurethane-Containing Nonpeptidic Protease Inhibitor (PI), GRL-98065, Is Potent against Multiple-PI-Resistant Human Immunodeficiency Virus In Vitro

ABSTRACT We designed, synthesized, and identified GRL-98065, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing the structure-based designed privileged cyclic ether-derived nonpeptide P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF), and a sulfonamide isostere, which is highly potent against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0002 to 0.0005 μM) with minimal cytotoxicity (50% cytotoxicity, 35.7 μM in CD4+ MT-2 cells). GRL-98065 blocked the infectivity and replication of each of the HIV-1NL4-3 variants exposed to and selected by up to a 5 μM concentration of saquinavir, indinavir, nelfinavir, or ritonavir and a 1 μM concentration of lopinavir or atazanavir (EC50, 0.0015 to 0.0075 μM), although it was less active against HIV-1NL4-3 selected by amprenavir (EC50, 0.032 μM). GRL-98065 was also potent against multiple-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents, HIV-1 isolates of various subtypes, and HIV-2 isolates examined. Structural analyses revealed that the close contact of GRL-98065 with the main chain of the protease active-site amino acids (Asp29 and Asp30) is important for its potency and wide-spectrum activity against multiple-PI-resistant HIV-1 variants. The present data demonstrate that the privileged nonpeptide P2 ligand, bis-THF, is critical for the binding of GRL-98065 to the HIV protease substrate binding site and that this scaffold can confer highly potent antiviral activity against a wide spectrum of HIV isolates.

IgA EGFR antibodies mediate tumour killing in vivo

Currently all approved anti‐cancer therapeutic monoclonal antibodies (mAbs) are of the IgG isotype, which rely on Fcgamma receptors (FcγRs) to recruit cellular effector functions. In vitro studies showed that targeting of FcαRI (CD89) by bispecific antibodies (bsAbs) or recombinant IgA resulted in more effective elimination of tumour cells by myeloid effector cells than targeting of FcγR. Here we studied the in vivo anti‐tumour activity of IgA EGFR antibodies generated using the variable sequences of the chimeric EGFR antibody cetuximab. Using FcαRI transgenic mice, we demonstrated significant in vivo anti‐tumour activity of IgA2 EGFR against A431 cells in peritoneal and lung xenograft models, as well as against B16F10‐EGFR cells in a lung metastasis model in immunocompetent mice. IgA2 EGFR was more effective than cetuximab in a short‐term syngeneic peritoneal model using EGFR‐transfected Ba/F3 target cells. The in vivo cytotoxic activity of IgA2 EGFR was mediated by macrophages and was significantly decreased in the absence of FcαRI. These results support the potential of targeting FcαRI for effective antibody therapy of cancer.

Quantitative analysis of proteins in the tear fluid of patients with diabetic retinopathy

Diabetic retinopathy is the leading cause of new cases of legal blindness among adults in the developed countries. Approximately 40% of all people with diabetes have diabetic retinopathy and 5% of these have sight-threatening form. As the advanced stage, where there is a high risk for vision loss, can develop without any serious symptoms, sometimes it is hard to detect it. A non invasive method to detect biomarkers characteristic for diabetic retinopathy from the tear fluid was developed. Tear samples from diabetic patients with no retinopathy, non proliferative and proliferative stages of diabetic retinopathy were analyzed and the protein content of each sample was compared to the protein content of tear pool from healthy volunteers. The samples were labeled with iTRAQ fourplex labels and were analyzed with nanoHPLC coupled ESI-MS/MS mass spectrometry. The lipocalin 1, lactotransferrin, lacritin, lysozyme C, lipophilin A and immunoglobulin lambda chain were identified as possible biomarker candidates with significantly higher relative levels in the tear of patients with diabetic retinopathy.

The Therapeutic CD38 Monoclonal Antibody Daratumumab Induces Programmed Cell Death via Fcγ Receptor–Mediated Cross-Linking

Emerging evidence suggests that FcγR-mediated cross-linking of tumor-bound mAbs may induce signaling in tumor cells that contributes to their therapeutic activity. In this study, we show that daratumumab (DARA), a therapeutic human CD38 mAb with a broad-spectrum killing activity, is able to induce programmed cell death (PCD) of CD38+ multiple myeloma tumor cell lines when cross-linked in vitro by secondary Abs or via an FcγR. By comparing DARA efficacy in a syngeneic in vivo tumor model using FcRγ-chain knockout or NOTAM mice carrying a signaling-inactive FcRγ-chain, we found that the inhibitory FcγRIIb as well as activating FcγRs induce DARA cross-linking–mediated PCD. In conclusion, our in vitro and in vivo data show that FcγR-mediated cross-linking of DARA induces PCD of CD38-expressing multiple myeloma tumor cells, which potentially contributes to the depth of response observed in DARA-treated patients and the drug’s multifaceted mechanisms of action.

Combining mutations in HIV-1 protease to understand mechanisms of resistance.

HIV‐1 develops resistance to protease inhibitors predominantly by selecting mutations in the protease gene. Studies of resistant mutants of HIV‐1 protease with single amino acid substitutions have shown a range of independent effects on specificity, inhibition, and stability. Four double mutants, K45I/L90M, K45I/V82S, D30N/V82S, and N88D/L90M were selected for analysis on the basis of observations of increased or decreased stability or enzymatic activity for the respective single mutants. The double mutants were assayed for catalysis, inhibition, and stability. Crystal structures were analyzed for the double mutants at resolutions of 2.2–1.2 Å to determine the associated molecular changes. Sequence‐dependent changes in protease‐inhibitor interactions were observed in the crystal structures. Mutations D30N, K45I, and V82S showed altered interactions with inhibitor residues at P2/P2′, P3/P3′/P4/P4′, and P1/P1′, respectively. One of the conformations of Met90 in K45I/L90M has an unfavorably close contact with the carbonyl oxygen of Asp25, as observed previously in the L90M single mutant. The observed catalytic efficiency and inhibition for the double mutants depended on the specific substrate or inhibitor. In particular, large variation in cleavage of p6pol‐PR substrate was observed, which is likely to result in defects in the maturation of the protease from the Gag‐Pol precursor and hence viral replication. Three of the double mutants showed values for stability that were intermediate between the values observed for the respective single mutants. D30N/V82S mutant showed lower stability than either of the two individual mutations, which is possibly due to concerted changes in the central P2‐P2′ and S2‐S2′ sites. The complex effects of combining mutations are discussed. Proteins 2002;48:107–116.

Amino Acid Preferences for a Critical Substrate Binding Subsite of Retroviral Proteases in Type 1 Cleavage Sites

ABSTRACT The specificities of the proteases of 11 retroviruses representing each of the seven genera of the family Retroviridae were studied using a series of oligopeptides with amino acid substitutions in the P2 position of a naturally occurring type 1 cleavage site (Val-Ser-Gln-Asn-Tyr↓Pro-Ile-Val-Gln; the arrow indicates the site of cleavage) in human immunodeficiency virus type 1 (HIV-1). This position was previously found to be one of the most critical in determining the substrate specificity differences of retroviral proteases. Specificities at this position were compared for HIV-1, HIV-2, equine infectious anemia virus, avian myeloblastosis virus, Mason-Pfizer monkey virus, mouse mammary tumor virus, Moloney murine leukemia virus, human T-cell leukemia virus type 1, bovine leukemia virus, human foamy virus, and walleye dermal sarcoma virus proteases. Three types of P2 preferences were observed: a subgroup of proteases preferred small hydrophobic side chains (Ala and Cys), and another subgroup preferred large hydrophobic residues (Ile and Leu), while the protease of HIV-1 preferred an Asn residue. The specificity distinctions among the proteases correlated well with the phylogenetic tree of retroviruses prepared solely based on the protease sequences. Molecular models for all of the proteases studied were built, and they were used to interpret the results. While size complementarities appear to be the main specificity-determining features of the S2 subsite of retroviral proteases, electrostatic contributions may play a role only in the case of HIV proteases. In most cases the P2 residues of naturally occurring type 1 cleavage site sequences of the studied proteases agreed well with the observed P2 preferences.

Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors.

Clinical inhibitor amprenavir (APV) is less effective on HIV‐2 protease (PR2) than on HIV‐1 protease (PR1). We solved the crystal structure of PR2 with APV at 1.5 Å resolution to identify structural changes associated with the lowered inhibition. Furthermore, we analyzed the PR1 mutant (PR1M) with substitutions V32I, I47V, and V82I that mimic the inhibitor binding site of PR2. PR1M more closely resembled PR2 than PR1 in catalytic efficiency on four substrate peptides and inhibition by APV, whereas few differences were seen for two other substrates and inhibition by saquinavir (SQV) and darunavir (DRV). High resolution crystal structures of PR1M with APV, DRV, and SQV were compared with available PR1 and PR2 complexes. Val/Ile32 and Ile/Val47 showed compensating interactions with SQV in PR1M and PR1, however, Ile82 interacted with a second SQV bound in an extension of the active site cavity of PR1M. Residues 32 and 82 maintained similar interactions with DRV and APV in all the enzymes, whereas Val47 and Ile47 had opposing effects in the two subunits. Significantly diminished interactions were seen for the aniline of APV bound in PR1M and PR2 relative to the strong hydrogen bonds observed in PR1, consistent with 15‐ and 19‐fold weaker inhibition, respectively. Overall, PR1M partially replicates the specificity of PR2 and gives insight into drug resistant mutations at residues 32, 47, and 82. Moreover, this analysis provides a structural explanation for the weaker antiviral effects of APV on HIV‐2.