Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist.

The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in cancer, HIV, and COVID-19. Despite its importance as a drug target, the molecular activation mechanism of CCR5, i.e., how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N terminus of agonist chemokines pushes onto specific structural motifs at the bottom of the orthosteric pocket that activate the canonical GPCR microswitch network. This activation mechanism differs substantially from other CC chemokine receptors that bind chemokines with shorter N termini in a shallow binding mode involving unique sequence signatures and a specialized activation mechanism.

Rapid and low-cost multiplex synthesis of chemokine analogs.

Peptides represent a promising source of new medicines, but improved technologies are needed to facilitate discovery and optimization campaigns. In particular, longer peptides with multiple disulfide bridges are challenging to produce, and producing large numbers of structurally related variants is dissuasively costly and time-consuming. The principal cost and time drivers are the multiple column chromatography purification steps that are used during the multistep chemical synthesis procedure, which involves both ligation and oxidative refolding steps. In this study, we developed a method for multiplex parallel synthesis of complex peptide analogs in which the structurally variant region of the molecule is produced as a small peptide on a 384-well synthesizer with subsequent ligation to the longer, structurally invariant region and oxidative refolding carried out in-well without any column purification steps. To test the method, we used a panel of 96 analogs of the chemokine RANTES (regulated on activation normal T cell expressed and secreted)/CCL5 (69 residues, two disulfide bridges), which had been synthesized using standard approaches and characterized pharmacologically in an earlier study. Although, as expected, the multiplex method generated chemokine analogs of lower purity than those produced in the original study, it was nonetheless possible to closely match the pharmacological attributes (anti-HIV potency, capacity to elicit G protein signaling, and capacity to elicit intracellular receptor sequestration) of each chemokine analog to reference data from the earlier study. This rapid, low-cost approach has the potential to support discovery and optimization campaigns based on analogs of other chemokines as well as those of other complex peptide and small protein targets of a similar size.

Stability of 5P12-RANTES, A Candidate Rectal Microbicide, in Human Rectal Lavage.

In the absence of an effective vaccine, strategies to prevent HIV transmission are urgently needed. Condomless receptive anal intercourse represents a major route of transmission, and efforts are being made to develop strategies, in which potent anti-HIV drugs are formulated for topical application to the rectum before sex. 5P12-RANTES is a promising candidate for such a purpose. It is an analog of the human chemokine RANTES/CCL5, which potently blocks CCR5, the principal coreceptor used by HIV to enter and infect target cells. As a protein, 5P12-RANTES is potentially vulnerable to attack by proteases in the rectal environment. In this study, we tested the stability of 5P12-RANTES on exposure to rectal lavage samples obtained from healthy volunteers, using a sensitive HIV entry inhibition assay as an indicator of stability. Varying levels of inactivating activity toward 5P12-RANTES were detected across the different lavage samples. Analysis of even the most aggressive samples indicated that protease activity in the rectal environment is unlikely to impact on the anti-HIV activity of 5P12-RANTES when applied pericoitally at the envisaged clinical dose (1 mM). This study indicates that 5P12-RANTES has adequate stability for further development as an HIV prevention drug for rectal use.

Enhancing Antitumor Immune Responses by Optimized Combinations of Cell-penetrating Peptide-based Vaccines and Adjuvants.

Cell penetrating peptides (CPPs) from the protein ZEBRA are promising candidates to exploit in therapeutic cancer vaccines, since they can transport antigenic cargos into dendritic cells and induce tumor-specific T cells. Employing CPPs for a given cancer indication will require engineering to include relevant tumor-associated epitopes, administration with an appropriate adjuvant, and testing for antitumor immunity. We assessed the importance of structural characteristics, efficiency of in vitro transduction of target cells, and choice of adjuvant in inducing the two key elements in antitumor immunity, CD4 and CD8 T cells, as well as control of tumor growth in vivo. Structural characteristics associated with CPP function varied according to CPP truncations and cargo epitope composition, and correlated with in vitro transduction efficiency. However, subsequent in vivo capacity to induce CD4 and CD8 T cells was not always predicted by in vitro results. We determined that the critical parameter for in vivo efficacy using aggressive mouse tumor models was the choice of adjuvant. Optimal pairing of a particular ZEBRA-CPP sequence and antigenic cargo together with adjuvant induced potent antitumor immunity. Our results highlight the irreplaceable role of in vivo testing of novel vaccine constructs together with adjuvants to select combinations for further development.

Generating Chemokine Analogs with Enhanced Pharmacological Properties Using Phage Display.

Phage display technology, which allows extremely rare ligands to be selected from libraries of variants according to user-defined selection criteria, has made a huge impact on the life sciences. In this chapter, we describe phage display methods for the discovery of chemokine analogs with enhanced pharmacological properties. We discuss strategies for chemokine library design and provide a recommended technique for library construction. We also describe cell-based library selection approaches that we have used to discover chemokine analogs, not only receptor antagonists but also variants with unusual effects on receptor signaling and trafficking. By providing a survey of the different phage chemokine projects that we have undertaken, we comment on the parameters most likely to affect success. Finally, we discuss how phage display-derived chemokine analogs with altered pharmacological activity represent valuable tools to better understand chemokine biology, and why certain among them have the potential to be developed as new medicines.

A scalable low-cost cGMP process for clinical grade production of the HIV inhibitor 5P12-RANTES in Pichia pastoris.

In the continued absence of an effective anti-HIV vaccine, approximately 2 million new HIV infections occur every year, with over 95% of these in developing countries. Calls have been made for the development of anti-HIV drugs that can be formulated for topical use to prevent HIV transmission during sexual intercourse. Because these drugs are principally destined for use in low-resource regions, achieving production costs that are as low as possible is an absolute requirement. 5P12-RANTES, an analog of the human chemokine protein RANTES/CCL5, is a highly potent HIV entry inhibitor which acts by achieving potent blockade of the principal HIV coreceptor, CCR5. Here we describe the development and optimization of a scalable low-cost production process for 5P12-RANTES based on expression in Pichia pastoris. At pilot (150 L) scale, this cGMP compliant process yielded 30 g of clinical grade 5P12-RANTES. As well as providing sufficient material for the first stage of clinical development, this process represents an important step towards achieving production of 5P12-RANTES at a cost and scale appropriate to meet needs for topical HIV prevention worldwide.

The Human Autoantibody Response to Apolipoprotein A-I Is Focused on the C-Terminal Helix: A New Rationale for Diagnosis and Treatment of Cardiovascular Disease?

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death worldwide and new approaches for both diagnosis and treatment are required. Autoantibodies directed against apolipoprotein A-I (ApoA-I) represent promising biomarkers for use in risk stratification of CVD and may also play a direct role in pathogenesis. METHODOLOGY: To characterize the anti-ApoA-I autoantibody response, we measured the immunoreactivity to engineered peptides corresponding to the different alpha-helical regions of ApoA-I, using plasma from acute chest pain cohort patients known to be positive for anti-ApoA-I autoantibodies. PRINCIPAL FINDINGS: Our results indicate that the anti-ApoA-I autoantibody response is strongly biased towards the C-terminal alpha-helix of the protein, with an optimized mimetic peptide corresponding to this part of the protein recapitulating the diagnostic accuracy for an acute ischemic coronary etiology (non-ST segment elevation myocardial infarction and unstable angina) obtainable using intact endogenous ApoA-I in immunoassay. Furthermore, the optimized mimetic peptide strongly inhibits the pathology-associated capacity of anti-ApoA-I antibodies to elicit proinflammatory cytokine release from cultured human macrophages. CONCLUSIONS: In addition to providing a rationale for the development of new approaches for the diagnosis and therapy of CVD, our observations may contribute to the elucidation of how anti-ApoA-I autoantibodies are elicited in individuals without autoimmune disease.

Efficient orthogonal bioconjugation of dendrimers for synthesis of bioactive nanoparticles.

Nanoparticles carrying biologically active functional sets (e.g., targeting moiety, payload, tracer) have potential use in a wide range of clinical applications. Though complex, such constructions should, as far as possible, have a defined molecular architecture and be monodisperse. However, the existing methods to achieve this goal are unsuitable for the incorporation of peptides and proteins, and those that provide for orthogonal introduction of two different types of functional element are incompatible with the use of commercially available materials. In this study, we have developed approaches for the production of nanoparticles based on commercially available polyamidoamine (PAMAM) dendrimers. First, we identified an optimized oxime conjugation strategy under which complex dendrimers can be fully decorated not only with model peptides, but also with recombinant proteins (insulin was taken as an example). Second, we developed a strategy based on a two-chain covalent heterodendrimer (a "diblock") based on cystamine core PAMAM dendrimers and used it to generate heterodendrimers, into which a peptide array and a mannose array were orthogonally introduced. Finally, by incorporating a functionalized linker into the diblock architecture we were able to site-specifically introduce a third functional element into the nanoparticle. We exemplified this approach using fluorescein, a mannose array, and a peptide array as the three functionalities. We showed that incorporation of a mannose array into a nanoparticle strongly and specifically enhances uptake by sentinel cells of the immune system, an important property for vaccine delivery applications. These PAMAM dendrimer-based approaches represent a robust and versatile platform for the development of bioactive nanoparticles.

Chemokine analogues show suitable stability for development as microbicides.

New prevention strategies are urgently needed to slow the spread of the HIV/AIDS pandemic, and in the absence of an effective vaccine, there is hope that "microbicides"-HIV inhibitors applied to mucosal surfaces before sexual intercourse-may be able to make an impact. Because developing countries are at the center of the epidemic, affordability and stability during storage are key criteria for candidate microbicides. Furthermore, because formulation strategies that provide long-duration protection after a single dose may enhance acceptability and compliance, stability in the vaginal environment and in the presence of semen should also be considered. PSC-RANTES, a human chemokine analog, has shown promise as a candidate microbicide, but because it contains nonnatural structures that necessitate chemical synthesis steps, it is not suitable for production at a feasible cost and scale for general distribution in developing countries. We have recently developed 2 new fully recombinant chemokine analogs, 5P12-RANTES and 6P4-RANTES, which show equivalent anti-HIV activity to PSC-RANTES. In this study, we tested the stability of these molecules under conditions related to use as microbicides. Our results suggest that stability issues will not present a major obstacle to the further development of these promising molecules as microbicides.

Highly potent, fully recombinant anti-HIV chemokines: reengineering a low-cost microbicide.

New prevention strategies for use in developing countries are urgently needed to curb the worldwide HIV/AIDS epidemic. The N-terminally modified chemokine PSC-RANTES is a highly potent entry inhibitor against R5-tropic HIV-1 strains, with an inhibitory mechanism involving long-term intracellular sequestration of the HIV coreceptor, CCR5. PSC-RANTES is fully protective when applied topically in a macaque model of vaginal HIV transmission, but it has 2 potential disadvantages related to further development: the requirement for chemical synthesis adds to production costs, and its strong CCR5 agonist activity might induce local inflammation. It would thus be preferable to find a recombinant analogue that retained the high potency of PSC-RANTES but lacked its agonist activity. Using a strategy based on phage display, we set out to discover PSC-RANTES analogs that contain only natural amino acids. We sought molecules that retain the potency and inhibitory mechanism of PSC-RANTES, while trying to reduce CCR5 signaling to as low a level as possible. We identified 3 analogues, all of which exhibit in vitro potency against HIV-1 comparable to that of PSC-RANTES. The first, 6P4-RANTES, resembles PSC-RANTES in that it is a strong agonist that induces prolonged intracellular sequestration of CCR5. The second, 5P12-RANTES, has no detectable G protein-linked signaling activity and does not bring about receptor sequestration. The third, 5P14-RANTES, induces significant levels of CCR5 internalization without detectable G protein-linked signaling activity. These 3 molecules represent promising candidates for further development as topical HIV prevention strategies.

Highly potent HIV inhibition: engineering a key anti-HIV structure from PSC-RANTES into MIP-1 beta/CCL4.

The HIV coreceptor CCR5 is a validated target for both the prevention and therapy of HIV infection. PSC-RANTES, an N-terminally modified analogue of one of the natural chemokine ligands of CCR5 (RANTES/CCL5), is a potent inhibitor of HIV entry into target cells. Here, we set out to engineer the anti-HIV activity of PSC-RANTES into another natural CCR5 ligand (MIP-1beta/CCL4), by grafting into it the key N-terminal pharmacophore region from PSC-RANTES. We were able to identify MIP-1beta/CCL4 analogues that retain the receptor binding profile of MIP-1beta/CCL4, but acquire the very high anti-HIV potency and characteristic inhibitory mechanism of PSC-RANTES. Unexpectedly, we discovered that in addition to N-terminal structures from PSC-RANTES, the side chain of Lys33 is also necessary for full anti-HIV potency.

Medicinal chemistry applied to a synthetic protein: development of highly potent HIV entry inhibitors.

We have used total chemical synthesis to perform high-resolution dissection of the pharmacophore of a potent anti-HIV protein, the aminooxypentane oxime of [glyoxylyl1]RANTES(2-68), known as AOP-RANTES, of which we designed and made 37 analogs. All involved incorporation of one or more rationally chosen nonnatural noncoded structures, for which we found a clear comparative advantage over coded ones. We investigated structure-activity relationships in the pharmacophore by screening the analogs for their ability to block the HIV entry process and produced a derivative, PSC-RANTES [N-nonanoyl, des-Ser1[L-thioproline2, L-cyclohexylglycine3]-RANTES(2-68)], which is 50 times more potent than AOP-RANTES. This promising group of compounds might be optimized yet further as potential prophylactic and therapeutic anti-HIV agents. The remarkable potency of our RANTES analogs probably involves the unusual mechanism of intracellular sequestration of CC-chemokine receptor 5 (CCR5), and it has been suggested that this arises from enhanced affinity for the receptor. We found that inhibitory potency and capacity to induce CCR5 down-modulation do appear to be correlated, but that unexpectedly, inhibitory potency and affinity for CCR5 do not. We believe this study represents the proof of principle for the use of a medicinal chemistry approach, above all one showing the advantage of noncoded structures, to the optimization of the pharmacological properties of a protein. Medicinal chemistry of small molecules is the foundation of modern pharmaceutical practice, and we believe we have shown that techniques have now reached the point at which the approach could also be applied to the many macromolecular drugs now in common use.

Human immunodeficiency virus type 1 entry inhibitors selected on living cells from a library of phage chemokines.

The chemokine receptors CCR5 and CXCR4 are promising non-virus-encoded targets for human immunodeficiency virus (HIV) therapy. We describe a selection procedure to isolate mutant forms of RANTES (CCL5) with antiviral activity considerably in excess of that of the native chemokine. The phage-displayed library of randomly mutated and N-terminally extended variants was screened by using live CCR5-expressing cells, and two of the selected mutants, P1 and P2, were further characterized. Both were significantly more potent HIV inhibitors than RANTES, with P2 being the most active (50% inhibitory concentration of 600 pM in a viral coat-mediated cell fusion assay, complete protection of target cells against primary HIV type 1 strains at a concentration of 10 nM). P2 resembles AOP-RANTES in that it is a superagonist of CCR5 and potently induces receptor sequestration. P1, while less potent than P2, has the advantage of significantly reduced signaling activity via CCR5 (30% of that of RANTES). Additionally, both P1 and P2 exhibit not only significantly increased affinity for CCR5 but also enhanced receptor selectivity, retaining only trace levels of signaling activity via CCR1 and CCR3. The phage chemokine approach that was successfully applied here could be adapted to other chemokine-chemokine receptor systems and used to further improve the first-generation mutants reported in this paper.