A highly potent and safe pyrrolopyridine-based allosteric HIV-1 integrase inhibitor targeting host LEDGF/p75-integrase interaction site

Abstract

Allosteric integrase inhibitors (ALLINIs) are a class of experimental anti-HIV agents that target the noncatalytic sites of the viral integrase (IN) and interfere with the IN-viral RNA interaction during viral maturation. Here, we report a highly potent and safe pyrrolopyridine-based ALLINI, STP0404, displaying picomolar IC50 in human PBMCs with a >24,000 therapeutic index against HIV-1. X-ray structural and biochemical analyses revealed that STP0404 binds to the host LEDGF/p75 protein binding pocket of the IN dimer, which induces aberrant IN oligomerization and blocks the IN-RNA interaction. Consequently, STP0404 inhibits proper localization of HIV-1 RNA genomes in viral particles during viral maturation. Y99H and A128T mutations at the LEDGF/p75 binding pocket render resistance to STP0404. Extensive in vivo pharmacological and toxicity investigations demonstrate that STP0404 harbors outstanding therapeutic and safety properties. Overall, STP0404 is a potent and first-in-class ALLINI that targets LEDGF/p75 binding site and has advanced to a human trial.

Fig 1. ALLINI structures and STP0404 activities.

A. Quinoline-based BI224436, B. Benzothiazole-based GS-9822, C. Pyridine-based KF116, D. Pyrrolopyridine-based STP0404. The conserved chemical moiety containing tert-butoxy and carboxylic acid groups are marked by “]”. E. In vitro anti-HIV-1 efficacy of STP0404 against HIV-1_NL4-3 and 89.6 strains in indicated cell lines. TI: Minimal therapeutic index values were calculated with 50% in vitro inhibitory concentration values (IC₅₀) and 10 μM where no sign of cytotoxicity was observed during tissue culture cytotoxicity measurements (TC₅₀) of all tested compounds in both MT4 and CEMx174 cell lines.

Fig 2. Selection of STP0404-resistant HIV-1 variants.

Two independent in vitro serial passages (grey and blue lines) of HIV-1_89.6 in CEMx174 cells were initiated at STP0404 IC₉₀ concentration (black dotted line). Viral production at each passage was monitored by p24 ELISA of collected media, and STP0404 concentration (red line) was elevated when p24 level spikes were observed. 20 cloned IN sequences were determined for three viral populations (passages, 2, 10, and 14) in both viral selections, with percentages of resistance Y99H and A128T mutation populations noted. Fold increases in IC₅₀ values of the viral populations collected at passage 4 against STP0404 and passage 14 against STP0404 and BI224436 are indicated.

Fig 3. Characterization of STP0404 resistant viral replication kinetics, efficacy against common polymorphisms, antiviral efficacy of enantiomers, and X-ray structure of IN CCD–STP0404 complex.

A. Replication kinetics of STP0404 resistant HIV-1 variants in CEMx174 cells. p24 levels of wild type (WT) HIV-1_89.6 or indicated IN variant in the collected culture media are noted. Fold differences of the variants in p24 levels, compared to wild type virus culture at day 4 were calculated. B. IC₅₀ values of STP0404 against wild type HIV-1_89.6 or indicated IN variants in CEMx174 cells. C. IC₅₀ values of (R) and (S) enantiomers of STP0404 against wild type HIV-1_89.6 in CEMx174 cells were determined, and their fold differences were calculated. Data in panels a-c are presented as means of triplicates and error bars indicate the standard deviations from the means. D. X-ray structure of HIV-1 IN CCD dimer bound to STP0404 (yellow/mesh). Two interacting CCDs are colored green (subunit 1) and cyan (subunit 2). Fo-Fc omit map calculated while omitting STP0404 is contoured at 3σ at resolution of 2.2 Å. A close up of STP0404 (mesh) binding to the LEDGF/p75 binding pocket was shown in S2 Fig. E. Superposition of our STP0404 structure (yellow, PDB: 7KE0) with quinoline based BI-D (pink, PDB: 4ID1) and pyridine based KF116 (gray, PDB: 4O55). F. Principal STP0404 interactions with the CCD-CCD dimer. W132, E170, H171 and T174 side chains are shown as sticks. Dashed lines indicate hydrogen bonding contacts. G. The side chains of A128 and Y99 are shown in the context of bound STP0404. Data collection and refinement statistics are described in S1 Table.

Fig 4. Effects of STP0404 on IN-RNA binding, HIV-1 particle morphology, IN multimerization, LEDGF/p75 binding, and early versus late stages of HIV-1 replication.

A. Inhibition of IN-RNA binding. B. TEM images of HIV-1 particles. C. Effect of STP0404 on IN multimerization. D. Effect of IN binding to LEDGF/p75. Data in panels A, C and D are means of triplicates with error bars indicating standard deviations from the means. E. STP0404 effect on early versus late stages of HIV-1 replication. Jurkat cells were transduced with HIV-1 GFP vector produced from 239T cells treated with and without STP0404 (0 and 60 nM), indicated as “producing cells”. Jurkat cells pre-treated with STP0404 (0 and 60 nM) were transduced with HIV-1 GFP vector produced from untreated 293T cells, indicated as “infecting cells”. The transduction efficiency was determined by FACS for GFP expression. F. LEDGF/p75 effect on STP0404 efficacy. Two independent LEDGF/p75 knockout Jurkat cell lines (1-F10 and 2-C10; cite PMID 32994325) were pretreated with STP0404 (0 and 60 nM) and transduced with HIV-GFP. Transduction efficiency was determined by FACS. Data in panels e and f are presented as means of triplicates and error bars indicate the standard deviations from the means. P-value < 0.05 is represented as *; p-value < 0.001 is represented as ***; ns indicates not significant.

Fig 5. In vivo pharmacokinetics and histology of STP0404 treated subjects.

A. SD rats were administrated with STP0404 by IV (1 and 5 mg/kg) and PO (2 and 10 mg/kg). B. Beagle dogs were administrated IV and PO (2 mg/kg). Plasma concentrations were determined by LC-MS for 24 h post administration (see Methods). For A and B, data are presented as means of three independent experiments and error bars indicate the standard deviations from the means. C. Half-lives (T_1/2), area under the curve (AUC), maximum concentration (C_max), and bioavailability (F_t) from the administrated animals (A and B) were calculated. D. Histology of beagle dogs orally administrated with STP0404 (90 mg/kg) or vehicle for bladder, ureter, and kidney in 4-week GLP toxicology study. The entire GLP toxicology study’s results were summarized in S3 Table.