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Objective: There is considerable uncertainty regarding the optimal use of rifampicin for the treatment of tuberculous (TB) meningitis. A pharmacokinetic modeling and simulation study of rifampicin concentrations in cerebrospinal fluid (CSF) during TB meningitis treatment was performed in this study. Methods: Parameters for rifampicin pharmacokinetics in CSF were estimated using individual-level rifampicin pharmacokinetic data, and the model was externally validated in three separate patient cohorts. Monte Carlo simulations of rifampicin serum and CSF concentrations were performed. The area under the rifampicin CSF concentration-versus-time curve during 24 h (AUC0–24) relative to the minimum inhibitory concentration (MIC) served as the pharmacodynamic target. Results: Across all simulated patients on the first treatment day, 85% attained the target AUC0–24/MIC ratio of 30 under a weight-based dosing scheme approximating 10 mg/kg. At the rifampicin MIC of 0.5 mg/l, the probability of AUC0–24/MIC target attainment was 26%. With an intensified dosing strategy corresponding to 20 mg/kg, target attainment increased to 99%, including 93% with a MIC of 0.5 mg/l. Conclusions: Under standard dosing guidelines, few TB meningitis patients would be expected to attain therapeutic rifampicin exposures in CSF when the MIC is ≥0.5 mg/l. Either downward adjustment of the rifampicin MIC breakpoint in the context of TB meningitis, or intensified rifampicin dosing upwards of 20 mg/kg/day, would reflect the likelihood of pharmacodynamic target attainment in CSF. Keywords: Tuberculosis, Meningitis, Tuberculous meningitis, Pharmacokinetics, Pharmacodynamics

Indian rhesus macaques (Macaca mulatta) are routinely used in preclinical studies to evaluate therapeutic Abs and candidate vaccines. The efficacy of these interventions in many cases is known to rely heavily on the ability of Abs to interact with a set of Ab FcγR expressed on innate immune cells. Yet, despite their presumed functional importance, M. mulatta Ab receptors are largely uncharacterized, posing a fundamental limit to ensuring accurate interpretation and translation of results from studies in this model. In this article, we describe the binding characteristics of the most prevalent allotypic variants of M. mulatta FcγR for binding to both human and M. mulatta IgG of varying subclasses. The resulting determination of the affinity, specificity, and glycan sensitivity of these receptors promises to be useful in designing and evaluating studies of candidate vaccines and therapeutic Abs in this key animal model and exposes significant evolutionary divergence between humans and macaques.

We sought to identify sequences in the monoclonal antibody m18 complementarity determining regions (CDRs) that are responsible for its interaction with HIV-1 gp120 and inhibition of the envelope receptor binding sites. In the accompanying paper (DOI 10.1021/bi101160r), we reported that m18 inhibits CD4 binding through a nonactivating mechanism that, at the same time, induces conformational effects leading to inhibition of the coreceptor site. Here, we sought to define the structural elements in m18 responsible for these actions. Direct binding and competition analyses using surface plasmon resonance showed that YU-2 gp120 binding is stabilized by a broad paratope of residues in the m18 CDRs. Additionally, several m18 residues were identified for which mutants retained high affinity for gp120 but had suppressed CD4 and 17b inhibition activities. A subset of these mutants did, however, neutralize HXBc2 viral infection. The results obtained in this work demonstrate that the combined m18 paratope contains subsets of residues that are differentially important for the binding and inhibition functions of the m18 neutralizing antibody. The data also add to prior observations that high-affinity antibodies that do not inhibit monomeric gp120 receptor site interactions may still exhibit significant antiviral activity.

Structure-activity correlations were investigated for substituted peptide conjugates that function as dual receptor site antagonists of HIV-1 gp120. A series of peptide conjugates were constructed via click reaction of both aryl and alkyl acetylenes with an internally incorporated azidoproline 6 derived from the parent peptide 1 (12p1, RINNIPWSEAMM). Compared to 1, many of these conjugates were found to exhibit several orders of magnitude increase in both affinity for HIV-1 gp120 and inhibition potencies at both the CD4 and coreceptor binding sites of gp120. We sought to determine structural factors in the added triazole grouping responsible for the increased binding affinity and antiviral activity of the dual inhibitor conjugates. We measured peptide conjugate potencies in both kinetic and cell infection assays. High affinity was sterically specific, being exhibited by the cis- but not the trans-triazole. The results demonstrate that aromatic, hydrophobic, and steric features in the residue 6 side-chain are important for increased affinity and inhibition. Optimizing these features provides a basis for developing gp120 dual inhibitors into peptidomimetic and increasingly smaller molecular weight entry antagonist leads.

The development of drug resistance remains a critical problem for current HIV-1 antiviral therapies, creating a need for new inhibitors of HIV-1 replication. We previously reported on a novel anti-HIV-1 compound, N(2)-(phenoxyacetyl)-N-[4-(1-piperidinyl-carbonyl)benzyl]glycinamide (14), that binds to the highly conserved phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2))binding pocket of the HIV-1 matrix (MA) protein. In this study, we re-evaluate the hits from the virtual screen used to identify compound 14 and test them directly in an HIV-1 replication assay using primary human peripheral blood mononuclear cells. This study resulted in the identification of three new compounds with antiviral activity; 2-(4-{[3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl]methyl})-1-piperazinyl)-N-(4-methylphenyl)-acetamide (7), 3-(2-ethoxyphenyl)-5-[[4-(4-nitrophenyl)piperazin-1-yl]methyl]-1,2,4-oxadiazole (17), and N-[4-ethoxy-3-(1-piperidinylsulfonyl)phenyl]-2-(imidazo[2,1-b][1,3]thiazol-6-yl)acetamide (18), with compound 7 being the most potent of these hits. Mechanistic studies on 7 demonstrated that it directly interacts with and functions through HIV-1 MA. In accordance with our drug target, compound 7 competes with PI(4,5)P(2) for MA binding and, as a result, diminishes the production of new virus. Mutation of residues within the PI(4,5)P(2) binding site of MA decreased the antiviral effect of compound 7. Additionally, compound 7 displays a broadly neutralizing anti-HIV activity, with IC(50) values of 7.5–15.6 μm for the group M isolates tested. Taken together, these results point towards a novel chemical probe that can be used to more closely study the biological role of MA and could, through further optimization, lead to a new class of anti-HIV-1 therapeutics.

Due to the emergence of drug-resistant strains and the cumulative toxicities associated with current therapies, demand remains for new inhibitors of HIV-1 replication. The HIV-1 matrix (MA) protein is an essential viral component with established roles in the assembly of the virus. Using virtual and surface plasmon resonance (SPR)-based screening, we describe the identification of the first small molecule to bind to the HIV-1 MA protein and to possess broad range anti-HIV properties.

Many large natural product antibiotics act by specifically binding and sequestering target molecules found on bacterial cells. We have developed a new strategy to expedite the structural analysis of such antibiotic-target complexes, in which we covalently link the target molecules to carrier proteins, and then crystallize the entire carrier/target/antibiotic complex. Using native chemical ligation, we have linked the Lys-d-Ala-d-Ala binding epitope for glycopeptide antibiotics to three different carrier proteins. We show that recognition of this peptide by multiple antibiotics is not compromised by the presence of the carrier protein partner, and use this approach to determine the first-ever crystal structure for the new therapeutic dalbavancin. We also report the first crystal structure of an asymmetric ristocetin antibiotic dimer, as well as the structure of vancomycin bound to a carrier-target fusion. The dalbavancin structure reveals an antibiotic molecule that has closed around its binding partner; it also suggests mechanisms by which the drug can enhance its half-life by binding to serum proteins, and be targeted to bacterial membranes. Notably, the carrier protein approach is not limited to peptide ligands such as Lys-d-Ala-d-Ala, but is applicable to a diverse range of targets. This strategy is likely to yield structural insights that accelerate new therapeutic development.

We investigated the interaction between cross-reactive HIV-1 neutralizing human monoclonal antibody m18 and HIV-1YU-2 gp120 in an effort to understand how this antibody inhibits the entry of virus into cells. m18 binds to gp120 with high affinity (KD≈5 nM) as measured by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR analysis further showed that m18 inhibits interactions of gp120 with both soluble CD4 and CD4-induced antibodies that have epitopes overlapping the coreceptor binding site. This dual receptor site antagonism, which occurs with equal potency for both inhibition effects, argues that m18 is not functioning as a mimic of CD4, in spite of the presence of a putative CD4-like loop formed by HCDR3 in the antibody. Consistent with this view, m18 was found to interact with gp120 in the presence of saturating concentrations of a CD4-mimicking small molecule gp120 inhibitor, suggesting that m18 does not require unoccupied CD4 Phe43 binding cavity residues of gp120. Thermodynamic analysis of the m18-gp120 interaction suggests that m18 stabilizes a conformation of gp120 that is unique from and less structured than the CD4-stabilized conformation. Conformational mutants of gp120 were studied for their impact on m18 interaction. Mutations known to disrupt the coreceptor binding region and to lead to complete suppression of 17b binding had minimal effects on m18 binding. This argues that energetically important epitopes for m18 binding lie outside the disrupted bridging sheet region used for 17b and coreceptor binding. In contrast, mutations in the CD4 region strongly affected m18 binding. Overall, the results obtained in this work argue that m18, rather than mimicking CD4 directly, suppresses both receptor binding site functions of HIV-1 gp120 by stabilizing a nonproductive conformation of the envelope protein. These results can be related to prior findings about the importance of conformational entrapment as a common mode of action for neutralizing CD4bs antibodies, with differences mainly in epitope utilization and the extent of gp120 structuring.

In an effort to identify broadly active inhibitors of HIV-1 entry into host cells, we had previously reported a family of dodecamer triazole-peptide conjugates with nanomolar affinity for viral surface protein gp120. This class of peptides exhibits potent antiviral activity and the capacity to simultaneously inhibit interaction of viral envelope protein with both CD4 and co-receptor. In the current investigation, we used minimization of structural complexity of the lead triazole inhibitor HNG-156 (peptide 1) in order to explore the limits of the pharmacophore that enables dual antagonism and to improve opportunities for peptidomimetic design. Truncations of both carboxyl- and amino-terminal residues of the initial 12 amino acid residues of peptide 1 were found to have minimal effect on both affinity and antiviral activity. In contrast, the central triazole Pro-Trp cluster at residues 6 and 7 with ferrocenyl-triazole-Pro (Ftp) was found to be critical for bioactivity. Amino terminal residues distal to the central triazole Pro-Trp sequence tolerated decreasing degrees of side chain variation upon approaching the central cluster. A peptide fragment containing residues 3-7 (Asn-Asn-Ile-Ftp-Trp) exhibited substantial direct binding affinity, antiviral potency, dual receptor site antagonism and induction of gp120 structuring, all properties defining the functional signature of the parent compound 1. This active core contains a stereochemically specific hydrophobic triazole-Pro-Trp cluster, with a short N-terminal peptide extension providing groups for potential main chain and side chain hydrogen binding. The results of this work argue that the pharmacophore for dual antagonism is structurally limited, enhancing the potential to develop minimized peptidomimetic HIV-1 entry inhibitors that simultaneously suppress binding of envelope protein to both of its host cell receptors. The results also argue that the target epitope on gp120 is relatively small, pointing to a localized allosteric inhibition site in the HIV-1 envelope that could be targeted for small-molecule inhibitor discovery.

In this work, we identified a high affinity and potency metallocene-containing triazole peptide conjugate that suppresses the interactions of HIV-1 envelope gp120 at both its CD4 and co-receptor binding sites. The ferrocene-peptide conjugate, HNG-156, was formed by an on-resin copper-catalysed [2 + 3] cycloaddition reaction. Surface plasmon resonance interaction analysis revealed that, compared to a previously reported phenyl-containing triazole conjugate HNG-105 (105), peptide 156 had a higher direct binding affinity for several subtypes of HIV-1 gp120 due mainly to the decreased dissociation rate of the conjugate-gp120 complex. The ferrocene triazole conjugate bound to gp120 of both clade A (92UG037-08) and clade B (YU-2 and SF162) virus subtypes with nanomolar KD in direct binding and inhibited the binding of gp120 to soluble CD4 and to antibodies that bind to HIV-1YU-2 gp120 at both the CD4 binding site and CD4-induced binding sites. HNG-156 showed a close-to nanomolar IC50 for inhibiting cell infection by HIV-1BaL whole virus. The dual receptor site antagonist activity and potency of HNG-156 make it a promising viral envelope inhibitor lead for developing anti-HIV-1 treatments.