Michal Maes

Cyclic peptide inhibitors of HIV-1 integrase derived from the LEDGF/p75 protein

Restricting linear peptides to their bioactive conformation is an attractive way of improving their stability and activity. We used a cyclic peptide library with conformational diversity for selecting an active and stable peptide that mimics the structure and activity of the HIV-1 integrase (IN) binding loop from its cellular cofactor LEDGF/p75 (residues 361-370). All peptides in the library had the same primary sequence, and differed only in their conformation. Library screening revealed that the ring size and linker structure had a huge effect on the conformation, binding and activity of the peptides. One of the cyclic peptides, c(MZ 4-1), was a potent and stable inhibitor of IN activity in vitro and in cells even after 8 days. The NMR structure of c(MZ 4-1) showed that it obtains a bioactive conformation that is similar to the parent site in LEDGF/p75.

Peptides derived from HIV-1 Rev inhibit HIV-1 integrase in a shiftide mechanism.

The HIV‐1 Integrase protein (IN) mediates the integration of the viral cDNA into the host genome. IN is an emerging target for anti‐HIV drug design, and the first IN‐inhibitor was recently approved by the FDA. We have developed a new approach for inhibiting IN by “shiftides”: peptides derived from its cellular binding protein LEDGF/p75 that inhibit IN by shifting its oligomerization equilibrium from the active dimer to an inactive tetramer. In addition, we described two peptides derived from the HIV‐1 Rev protein that interact with IN and inhibit its activity in vitro and in cells. In the current study, we show that the Rev‐derived peptides also act as shiftides. Analytical gel filtration and cross‐linking experiments showed that IN was dimeric when bound to the viral DNA, but tetrameric in the presence of the Rev‐derived peptides. Fluorescence anisotropy studies revealed that the Rev‐derived peptides inhibited the DNA binding of IN. The Rev‐derived peptides inhibited IN catalytic activity in vitro in a concentration‐dependent manner. Inhibition was much more significant when the peptides were added to free IN before it bound the viral DNA than when the peptides were added to a preformed IN‐DNA complex. This confirms that the inhibition is due to the ability of the peptides to shift the oligomerization equilibrium of the free IN toward a tetramer that binds much weaker to the viral DNA. We conclude that protein–protein interactions of IN may serve as a general valuable source for shiftide design.

Mechanism of action of the HIV-1 integrase inhibitory peptide LEDGF 361-370

The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361-370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361-370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361-370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361-370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound.

Revealing the Ligand Binding Site of NhaA Na+/H+ Antiporter and its pH Dependence

Background: Cell pH and Na+ homeostasis requires Na+/H+ antiporters such as NhaA. Results: Mutational analysis and ITC measurements revealed the NhaA-Li+ binding site. Conclusion: Binding of Li+ to purified NhaA is enthalpy-driven, highly specific, and pH-dependent and involves a single binding site. Significance: The pH-dependent NhaA-ligand binding is an insight into the mechanism of activity of NhaA and possibly other antiporters. pH and Na+ homeostasis in all cells requires Na+/H+ antiporters. In most cases, their activity is tightly pH-regulated. NhaA, the main antiporter of Escherichia coli, has homologues in all biological kingdoms. The crystal structure of NhaA provided insights into the mechanism of action and pH regulation of an antiporter. However, the active site of NhaA remained elusive because neither Na+ nor Li+, the NhaA ligands, were observed in the structure. Using isothermal titration calorimetry, we show that purified NhaA binds Li+ in detergent micelles. This interaction is driven by an increase in enthalpy (ΔH of −8000 ± 300 cal/mol and ΔS of −15.2 cal/mol/degree at 283 K), involves a single binding site per NhaA molecule, and is highly specific and drastically dependent on pH; Li+ binding was observed only at pH 8.5. Combining mutational analysis with the isothermal titration calorimetry measurements revealed that Asp-163, Asp-164, Thr-132, and Asp-133 form the Li+ binding site, whereas Lys-300 plays an important role in pH regulation of the antiporter.

Peptides that inhibit HIV‐1 integrase by blocking its protein–protein interactions

HIV‐1 integrase (IN) is one of the key enzymes in the viral replication cycle. It mediates the integration of viral cDNA into the host cell genome. IN activity requires interactions with several viral and cellular proteins, as well as IN oligomerization. Inhibition of IN is an important target for the development of anti‐HIV therapies, but there is currently only one anti‐HIV drug used in the clinic that targets IN. Several other small‐molecule anti‐IN drug leads are either undergoing clinical trials or in earlier stages of development. These molecules specifically inhibit one of the IN‐mediated reactions necessary for successful integration. However, small‐molecule inhibitors of protein–protein interactions are difficult to develop. In this review, we focus on peptides that inhibit IN. Peptides have advantages over small‐molecule inhibitors of protein–protein interactions: they can mimic the structures of the binding domains within proteins, and are large enough to competitively inhibit protein–protein interactions. The development of peptides that bind IN and inhibit its protein–protein interactions will increase our understanding of the IN mode of action, and lead to the development of new drug leads, such as small molecules derived from these peptides, for better anti‐HIV therapy.

Peptide inhibitors of HIV-1 integrase: From mechanistic studies to improved lead compounds

The HIV-1 integrase enzyme (IN) catalyzes integration of viral DNA into the host genome. We previously developed peptides that inhibit IN in vitro and HIV-1 replication in cells. Here we present the design, synthesis and evaluation of several derivatives of one of these inhibitory peptides, the 20-mer IN1. The peptide corresponding to the N-terminal half of IN1 (IN1 1-10) was easier to synthesize and much more soluble than the 20-mer IN1. IN1 1-10 bound IN with improved affinity and inhibited IN activity as well as HIV replication and integration in infected cells. While IN1 bound the IN tetramer, its shorter derivatives bound dimeric IN. Mapping the peptide binding sites in IN provided a model that explains this difference. We conclude that IN1 1-10 is an improved lead compound for further development of IN inhibitors.

Cation diffusion facilitators transport initiation and regulation is mediated by cation induced conformational changes of the cytoplasmic domain.

Cation diffusion facilitators (CDF) are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all domains of life. CDFs were shown to be involved in several human diseases, such as Type-II diabetes and neurodegenerative diseases. In this work, we employed a multi-disciplinary approach to study the activation mechanism of the CDF protein family. For this we used MamM, one of the main ion transporters of magnetosomes – bacterial organelles that enable magnetotactic bacteria to orientate along geomagnetic fields. Our results reveal that the cytosolic domain of MamM forms a stable dimer that undergoes distinct conformational changes upon divalent cation binding. MamM conformational change is associated with three metal binding sites that were identified and characterized. Altogether, our results provide a novel auto-regulation mode of action model in which the cytosolic domains conformational changes upon ligand binding allows the priming of the CDF into its transport mode.

The disordered region of Arabidopsis VIP1 binds the Agrobacterium VirE2 protein outside its DNA-binding site

Agrobacterium is a pathogen that genetically transforms plants. The bacterial VirE2 protein envelopes the T-DNA of Agrobacterium and protects it from degradation. Within the transfected cells, VirE2 interacts with the plant VIP1 leading to nuclear transport of the T-DNA complex. Active VirE2 is an oligomer with a tendency to aggregate, hampering its studies at the molecular level. In addition, no structural or quantitative information is available regarding VIP1 or its interactions. The lack of information is mainly because both VIP1 and VirE2 are difficult to express and purify. Here, we present the development of efficient protocols that resulted in pure and stable His-tagged VIP1 and VirE2. Circular dichroism spectroscopy and computational predictions indicated that VIP1 is mostly intrinsically disordered. This may explain the variety of protein-protein interactions it participates in. Size exclusion chromatography revealed that VirE2 exists in a two-state equilibrium between a monomer and an oligomeric form. Using the purified proteins, we performed peptide array screening and revealed the binding sites on both proteins. VirE2 binds the disordered regions of VIP1, while the site in VirE2 that binds VIP1 is different from the VirE2 DNA-binding site. Peptides derived from these sites may be used as lead compounds that block Agrobacterium infection of plants.

Interactions of HIV-1 proteins as targets for developing anti-HIV-1 peptides

Protein-protein interactions (PPI) are essential in every step of the HIV replication cycle. Mapping the interactions between viral and host proteins is a fundamental target for the design and development of new therapeutics. In this review, we focus on rational development of anti-HIV-1 peptides based on mapping viral-host and viral-viral protein interactions all across the HIV-1 replication cycle. We also discuss the mechanism of action, specificity and stability of these peptides, which are designed to inhibit PPI. Some of these peptides are excellent tools to study the mechanisms of PPI in HIV-1 replication cycle and for the development of anti-HIV-1 drug leads that modulate PPI.

A screening methodology for purifying proteins with aggregation problems.

Many proteins are prone to aggregate or insoluble for different reasons. This poses an extraordinary challenge at the expression level, but even more during downstream purification processes. Here we describe a strategy that we developed for purifying prone-to-aggregate proteins. Our methodology can be easily implemented in small laboratories without the need for automated, expensive platforms. This procedure is especially suitable for intrinsically disordered proteins (IDPs) and for proteins with intrinsically disordered regions (IDRs). Such proteins are likely to aggregate due to their lack of tertiary structure and their extended and flexible conformations. Similar methodologies can be applied to other proteins with comparable tendency to aggregate during the expression or purification steps. In this chapter, we will mainly focus on protein solubility and stability issues during purification and storage, on factors that can prevent aggregation or maintain solubility, and on the importance of the early elimination of aggregates during protein purification.