Screening of the NIH Clinical Collection for inhibitors of HIV-1 integrase activity

Drug repurposing offers a validated approach to reduce drug attrition within the drug discovery and development pipeline through the application of known drugs and drug candidates to treat new indications. Full exploitation of this strategy necessitates the screening of a vast number of molecules against an extensive number of diseases of high burden or unmet need and the subsequent dissemination of the findings. In order to contribute to endeavours within this field, we screened the 727 compounds comprising the US National Institutes of Health (NIH) Clinical Collection through an HIV-1 (human immunodeficiency virus type 1) integrase stand transfer inhibition assay on an automated scintillation proximity assay platform. Only two compounds were identified within the initial screen, with cefixime trihydrate and epigallocatechin gallate found to reduce integrase strand transfer activity at IC50 values of 6.03±1.29 ?M and 9.57±1.62 ?M, respectively. However, both cefixime trihydrate and epigallocatechin gallate retained their low micromolar inhibitory activity when tested against a raltegravir-resistant integrase double mutant (FCIC50 values of 0.83 and 0.06, respectively), were ineffective in an orthogonal strand transfer ELISA (<30% inhibition at 100 ?M) and produced negligible selectivity index values (<1) in vitro. While no useful inhibitors of HIV-1 integrase strand transfer activity were found within the NIH Clinical Collection, the identification of two assay-disrupting molecules demonstrates the importance of consideration of non-specific inhibitors in drug repurposing screens. Significance: • This study is the first to screen the US NIH Clinical Collection for potential HIV-1 integrase inhibitors. • The pervasive nature of promiscuous inhibitors is emphasised

Multiple epitope immunogens (MEI) mimic the variability of the V3 loop of HIV-1 subtype C.

M.Sc.Therapeutic and preventative treatment are continually being sought to cease or curtail the worldwide HIV-1 epidemic. At present, therapeutic drug strategies such as highly active anti-retroviral therapy (HAART) have been particularly successful in slowing disease progression and reducing the incidence of AIDS and AIDS related mortality (Detels et al., 1998; Mocroft et al., 1998; Palella et al., 1998). However, the high costs, intricate dosing regimens and limited availability of the HAART drugs (Butera, 2000) has restricted its efficacy in developing and third world countries. As such, available and future drugs will remain inaccessible to the regions that are profoundly affected by the epidemic. An effective vaccine presents a viable solution to the HIV-1 epidemic in these countries. Approximately 70 vaccines are presently in various stages of clinical trials, the majority of which are subtype B specific (Johnston and Flores, 2001). This prevents their use in the predominantly subtype C infected sub-Saharan region of Africa, which accounts for 50% of the global HIV / AIDS population and includes South Africa, statistically the country with the highest number of people living with HIV / AIDS of any country in the world (UNAIDS, 2002). Presently there is no HIV-1 vaccine, regardless of subtype, in clinical use. This owes to several difficulties that hinder the progression of vaccine development, including the lack of predictive animal models, the establishment of viral latency and the difficulty involved in overcoming HIV-1 genetic diversity (Klein, 1999). The expansive HIV-1 genetic variation exhibited by HIV-1 is attributed to a high number of errors made by the reverse transcriptase (RT) enzyme (Coffin, 1992) and the absence of RT proofreading mechanisms during HIV-1 replication (Roberts et al., 1988; Bebenek et al., 1989). The HIV-1 nucleotide sequence drift is most frequently observed in the envelope (env) gene and expressed in env gene products (Shafer et al., 1999). Expression of the variable genome results in the production of progeny strains that are not identical to the parent strain (i.e. HIV-1 exists as a quasispecies within each seropositive individual and between individuals) and contributes to the diverse collection of viral strains in global circulation that vary across and within subtypes. Thus, for an HIV-1 vaccine to be efficient and truly functional it would be required to target this observed hypervariability and be effective against a multitude of currently circulating strains, exhibit cross-clade specificity and remain viable despite the emergence of variant strains. In this study we describe the design, synthesis and immunological ability of a multiple epitope immunogen (MEI) that mimics the hypervariability observed within the third variable (V3) loop of the envelope gp120 region of HIV-1 subtype C. Conjugation to a multiple antigenic peptide (MAP) produces a four -branched (b4) tetrameric peptide construct, designated MEIV3b4. This construct was characterized by theoretical and analytical techniques, tested in a variety of immunological assays and assessed for its potential as a candidate vaccine component. The construct was comparatively analysed through evaluation of three comparison peptides, two of which are hypervariable and based on the V3 region, the other representing a conserved region of HIV-1 envelope. The V3 peptides, named b-MEI-s and poly-L-MEI, differ from the MEIV3b4 construct in that they are less variable and less branched or conjugated to a traditional carrier rather than to a MAP system, respectively. The conserved peptide, designated CCD4 allowed for comparative evaluation between conserved and variable peptides as potential vaccine components.Dr. Debra Meye

HIV-1 subtype C envelope-based peptide constructs as potential vaccine components.

The development of an effective HIV vaccine is hindered by several obstacles. One of the leading challenges is the antigenic variability of HIV-1 that is exhibited throughout all viral gene products but to greatest extent in the viral envelope proteins. This phenomenon is the result of continuous mutations in the HIV genome and is responsible for the immune escape of viral mutants. Many studies have suggested that a multivalent vaccine that elicits broadly cross-reactive antibodies is required to efficiently target antigenic variability. To this end, we have designed and analyzed a synthetic peptide construct that mimicked the major variability exhibited in the V3 loops of HIV-1 subtype C isolates. The peptide construct, described as a multiple epitope immunogen of the V3 loop with 8 branches and termed MEIV3b8, was shown to be non-toxic but highly immunogenic in experimental animals (mice and rabbits) and produced antibodies that were reactive to V3 loop peptides of various subtypes, variant envelope proteins and whole viral isolates [at antibody titers 1000 in enzyme-linked immunosorbent assays (ELISAs)]. Furthermore, functional antibodies were generated in rabbits that mediated neutralization of a neutralization-sensitive HIV-1 isolate and two distinct primary HIV-1 isolates in several different neutralization assays (at antibody titres 1213). Additionally, the MEIV3b8 induced both proliferative and inflammatory immune responses in a murine model.Finally, antibodies in the plasma of individuals (n = 148) infected with HIV-1 subtype C, subtype B and HIV-2 were found to bind to the MEIV3b8 as antigen in ELISAs. Through these findings, this study demonstrated that the variable MEIV3b8 effectively addressed antigenic variability and provided evidence that this peptide construct may hold application in HIV-1 preventative and therapeutic vaccination as well as HIV immunodiagnosis.Dr. D. Meye

Inducing the Degradation of Disease-Related Proteins Using Heterobifunctional Molecules

Current drug development strategies that target either enzymatic or receptor proteins for which specific small molecule ligands can be designed for modulation, result in a large portion of the proteome being overlooked as undruggable. The recruitment of natural degradation cascades for targeted protein removal using heterobifunctional molecules (or degraders) provides a likely avenue to expand the druggable proteome. In this review, we discuss the use of this drug development strategy in relation to degradation cascade-recruiting mechanisms and successfully targeted disease-related proteins. Essential characteristics to be considered in degrader design are deliberated upon and future development challenges mentioned

Modification of HIV-1 reverse transcriptase and integrase activity by gold(III) complexes in direct biochemical assay

Gold(I) and gold(III) complexes have been previously investigated for potential biomedical applications including as anti-HIV agents. The oxidising nature of some gold(III) complexes yields well-documented cellular toxicity in cell-based assays but the effect in direct biochemical assays has not been fully investigated. In this study, gold(III) complexes were evaluated in HIV-1 reverse transcriptase and HIV-1 integrase biochemical assays. The gold(III) tetrachlorides KAuCl4 and HAuCl4 yielded sub-micromolar IC50’s of 0.947 and 0.983 μM in the direct HIV-1 RT assay, respectively, while IC50’s ranging from 0.461 to 8.796 μM were obtained for seven selected gold(III) complexes. The gold(III) tetrachlorides were also effective inhibitors of integrase enzymatic activity with &gt;80% inhibition obtained at a single dose evaluation of 10 μM. RT inhibition was decreased in the presence of a reducing agent (10 mM DTT) and against the M184V HIV-1 RT mutant, while none of the gold(III) complexes were effective inhibitors in cell-based antiviral assays (SI values &lt;5.95). Taken together, the findings of this study demonstrate that gold(III) complexes modify HIV-1 enzyme activity in direct biochemical assays, most likely through protein oxidation

Platinum(II) and gold(I) complexes based on 1,1 '-bis(diphenylphosphino)metallocene derivatives: Synthesis, characterization and biological activity of the gold complexes

The synthesis of series of 1,2,1' substituted bis(diphenylphosphino)- ruthenocenyl (1-4) and ferrocenyl cis-platinum(II) (5-7) and gold(I) (8-12) complexes are described. Crystal structures of 2 and 4, as well as 5, 6 and 10 confirm the molecular geometry of these ligands and their metal complexes. Preliminary investigation of four gold complexes as potential anticancer, antiHIV and antimalaria showed at least one gold compound that has excellent activity towards one of these diseases. The three gold(I) complexes, {1- [1-(dimethylamino)ethyl]-1 ,2-bis(diphenylphosphino)ruthenocene-kappa P-2,P'}bis[chlorogold(I)] (8) (IC50 = 1.40 mu M), {1-[1-(acetoxyethyl)-1',2-bis(diphenylphosphino)ferrocene-kappa P-2,P']bis[chlorogold(1)] (9) (IC50 = 0.51 mu M), {1-[1-(3-carboxypropanamido)ethyl]-1',2-bis(diphenylphosphino)-ruthenocene kappa P-2,P'} bis[chlorogold(I)] (12) (IC50 = 1.784 mu M), have the best activities against cancer, HIV and malaria respectively. (C) 2012 Elsevier B.V. All rights reserved