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• The human immunodeficiency virus (HIV) is the etiological agent responsible for acquired immune deficiency syndrome (AIDS), posing a significant global public health challenge. Oral antiretroviral (ARV) drugs have served as a fundamental component of HIV treatment for over three decades, playing a pivotal role in reversing the initially devastating trajectory of the HIV epidemic. The current oral medications are characterized by their ease of administration, high efficacy, and safety for both preventing and treating HIV-1 infection [1]. However, ensuring lifelong adherence to once-daily oral medications remains a significant challenge for many individuals living with HIV (persons with HIV (PWH)) and those who could benefit from pre-exposure prophylaxis. In such cases, the availability of effective longer-acting treatment and prevention options administered less frequently would offer alternatives that are advantageous compared to existing oral ARV agents. These advantages encompass an enhanced quality of life, improved adherence rates, and overall success rates, including prevention options that may yield life-saving reductions in individual and population-level HIV transmission rates while significantly contributing towards ending the global HIV epidemic.

  Recognizing the multifaceted roles of the capsid (CA) protein in the HIV-1 lifecycle, it has become an appealing molecular target for antiretroviral drug development. Recent discoveries have underscored the N-terminal domain and C-terminal domain (NTD-CTD) interprotomer pocket as a binding site for small molecule modulators [2, 3]. On December 22, 2022, Gilead's long-acting HIV drug lenacapavir (LEN, Sunlenca), an HIV-1 capsid inhibitor, received the Food and Drug Administration approval for the treatment of AIDS. Noteworthy aspects of this drug include: (a) its long-acting nature with only two doses required per year by subcutaneous injection (s.c.); (b) its pioneering role as an HIV-1 capsid inhibitor; (c) a molecular weight of 968, surpassing the rule of five proposed by Lipinski; (d) containing 10 fluorine atoms, breaking Merck's Aprepitant (Emend)'s record of seven fluorine atoms. Recently, Gilead Sciences announced that in the third phase of clinical trials for HIV pre-exposure prophylaxis in women, the drug achieved a remarkable effectiveness rate of 100%, making it the world's first HIV prevention drug to achieve such high efficacy [4].

  Looking back at the discovery process of LEN, its origins can be traced back to 2010 when Pfizer unveiled an HIV-1 capsid inhibitor PF-74 (Fig. 1) during an academic conference [5-7]. This compound was identified through phenotypic screening conducted by Agouron, a subsidiary of Pfizer. PF-74 disrupts capsid assembly in vitro, induces premature uncoating, and impairs nuclear entry of pre-integration complexes by blocking the binding of cellular cofactors in HIV-1 infected cells. Despite exhibiting some anti-retroviral activity, Pfizer opted not to pursue further development of this peptidoid drug due to the challenge associated with enhancing its oral bioavailability. The compound, however, garnered significant attention from Gilead. Utilizing combinatorial chemical parallel synthesis, Gilead has synthesized numerous dipeptides, among which dipeptide 2 emerges as exhibiting a potency that is 25 times greater than the prototype PF-74 (median effective concentration (EC₅₀) = 52 nmol/L). Nevertheless, due to its peptide-like nature, dipeptide 2 is susceptible to hydrolysis. Upon cyclization of the amide into pyridine 3, only one amide bond remains intact resulting in a substantial enhancement in activity (EC₅₀ = 0.65 µmol/L). Subsequent parallel synthesis yields an even more potent amide 4 (EC₅₀ = 0.15 µmol/L). However, amide 4 presents its own set of challenges as it is susceptible to metabolism by the hepatic enzyme CYP3A. Specifically, it may undergo oxidation by CYP3A leading to the formation of alcohol 4a which is subsequently eliminated giving rise to enamide 4b. Therefore, they subsequently synthesized various replacements for hydroxyl indole groups, including cyclohexylpyrazole 5, pyrazole 6, tetrafluorocyclohexylpyrazole 7, and cyclopropylpyrazole 8. After persistent efforts, cyclopropylpyrazole 8 was ultimately identified as the optimal choice due to its significantly reduced clearance rate (EC₅₀ = 167 nmol/L, plasma clearance (CL) < 0.17 L h^-1 kg^-1). Subsequently, the presence of amides on the right side emerged as a significant constraint on bioavailability. Consequently, Gilead replaced numerous bioisomers, among which aminoindazole 9 exhibited favorable potency and low clearance. Further optimization efforts led to the identification of sulfanilamide 10 as a solution to this issue. To enhance compound potency, Gilead discovered that norethisterone 11 provided additional interaction with the capsid binding site. The final optimized sulfone 12 has reached the range of 200 picomoles. Subsequent structural modifications eventually led Gilead to discover LEN (half-maximal effective concentration (EC₅₀) = 105 pmol/L, half-maximal cytotoxic concentration (CC₅₀) > 50 µmol/L). By targeting the HIV capsid, LEN disrupts various processes throughout the viral life cycle, including capsid uncoating, nuclear transport, viral assembly and release, and capsid formation. LEN primarily inhibited HIV-1 replication in the early stage by stabilizing capsid and preventing its breakdown in infected T-cells. Additionally, it inhibits the binding of cellular cofactors to the HIV-1 capsid, such as NUP153 and CPSF6. In the late stage of the HIV life cycle, LEN disrupts the capsid lattice, thereby inhibiting the maturation of the HIV virus. Due to its mechanism of action targeting the HIV capsid, LEN has exhibited remarkable potency against a range of HIV-1 subtypes (including A, A1, AE, AG, B, BF, C, D, E, F, G and H), HIV-2 in vitro and in patients with multidrug-resistant HIV-1 infection in clinical trials, highlighting LEN's considerable therapeutic value for patients with clinical drug resistance. Although capsid mutantions were observed in approximately 6% patents (including M66I, M66I+N74D, Q67H/K/N, K70H/N/R/S, N74D/H, Q67H+K70R, A105T/S, and T107A/C/N/S) in patients, most of these mutants incur significant fitness costs. The presence of hindrance isomerism results in LEN being capable of existing as either a weak acid or a weak base in solution, with a composition ratio of two hindrance isomers ranging from 1:5 to 1:8 depending on temperature and pH (WO2019035904).

  Figure 1

  

  Figure 1.  The discovery process of LEN. The figure was created using ChemDraw.

  DownLoad: Full-Size Img PowerPoint

  The pharmacokinetic (PK) studies in Table 1 suggest that LEN has limited water solubility and low systemic clearance in both non-clinical species (rat, dog) and humans [8]. Whether administered as an aqueous suspension (2% Poloxam 188 in normal saline) or a formulation containing polyethylene glycol (PEG) in an aqueous solution (68.2% PEG 300 + 31.8% water), LEN maintains sustained plasma exposure levels after s.c. administration without unexpected rapid release of the drug. Additionally, it exhibits two-phase absorption kinetics with an initial phase of rapid-release absorption followed by a subsequent phase of slow-release absorption. Clinical studies have shown that LEN provides slow and sustained drug release after a single subcutaneous administration in healthy volunteers, with estimated half-lives ranging from 32 days to 45 days. Due to its high potency, exceptional stability, and optimal release rates from injection warehouses, LEN is an ideal choice for long-acting injectable dosage forms administered every 6 months in humans for HIV-1 prevention and treatment. Furthermore, long-acting LEN offers a significant therapeutic alternative for individuals living with HIV who want to alleviate the burden of daily oral medications or circumvent potential stigma associated with daily oral regimens. Due to its prolonged duration in the body and biannual administration requirement, it will significantly improve patient compliance while also serving as a long-acting preparation for pre-exposure prophylaxis (PrEP). Undoubtedly, this contributes significantly towards achieving World Health Organization's goal of ending the AIDS pandemic by 2030.

  Table 1

  

  Table 1.  PK model-informed release rates and total fraction dose release of LEN.

  DownLoad: CSV

  Formulation	Species	Dose	Kindirect (h−1)	Tlag/MTT (day)	T1/2 (day)	Fracindirect	F (%)
  Aqueous suspension (100 mg/mL)	Rat	10 mg/kg	0.00309	6.5	9.3	0.91	136
  	Dog	6 mg/kg	0.00326	0.6	8.8	0.87	92
  	Human	30–450 mg	0.000623	0.3	46.3	0.86	112
  PEG/water solution (309 mg/mL)	Rat	50 mg/kg	0.000818	15	35.3	0.98	75
  	Dog	12 mg/kg	0.0015	7.3	19.2	0.94	80

  As an innovative first-in-class modulator of HIV-1 CA, LEN demonstrates potent activity in the picomolar range and exhibits low toxicity as well as prolonged metabolic clearance, thereby enabling sustained therapeutic blood concentrations for up to six months following subcutaneous administration. The comprehensive synthesis of information on LEN provides valuable insights into the future direction of anti-HIV-1 agents, encompassing its discovery, mechanism of action, and clinical trials. This compilation offers critical data for guiding future drug design endeavors. The observed nonclinical pharmacokinetic properties strongly support the selection of LEN as a candidate for long-acting administration. The combination of high potency, exceptional stability, and optimal release rate from the injection depot renders LEN highly suitable for a parenteral long-acting formulation.

  Although LEN has benefited from established modification strategies that enhance its potency and pharmacokinetics, there is potential for innovative approaches. For instance, the incorporation of reversible or irreversible covalent modifiers could further refine drug exposure and action duration, a concept also suitable for exploration in HIV-1 CA modulators. Moreover, targeted protein degradation (TPD), exemplified by proteolysis-targeting chimeras (PROTACs), presents a novel pharmacological strategy. Importantly, it offers several advantages that promote its utilization, including low dosage requirements, a wide range of target options, and minimal catalyst usage. This principle has been successfully applied in the fields of influenza and hepatitis C virus to cleave and eliminate specific proteins by exploiting potent cellular degradation systems. Applying TPD principles to LEN-like scaffolds may establish a new class of small molecules designed to completely eradicate HIV-1 CA functions. Such inhibitors can significantly contribute to our understanding of targeted-based drug design and facilitate the development of novel therapeutic strategies. Lastly, while the NTD-CTD interprotomer pocket is an attractive target in CA, it is not the sole viable option. As our comprehension of CA biology deepens, new therapeutic targets emerge. Recent insights into the nuclear uncoating of CA cores suggest novel intervention points associated with viral integration processes. Uncovering new host dependency factors that govern uncoating and integration will expand our fundamental knowledge about CA mechanisms and provide fresh avenues for therapeutic innovation.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Kai Tang: Writing – original draft. Guochao Wei: Writing – review & editing. Peng Zhan: Writing – review & editing.

  Acknowledgments

  This work was supported by National Key R & D Program of China (No. 2023YFC2306800), Natural Science Foundation of Shandong Province (No. ZR2024QH201) the Postdoctoral Fellowship Program of CPSF (No. GZC20231489) and China Postdoctoral Science Foundation (No. 2023M742101), the Key Research and Development Program, Ministry of Science and Technology of the People's Republic of China (No. 2023YFC2606500), the Shandong Laboratory Program (No. SYS202205), and Sanming Project of Medicine in Shenzhen (No. SZSM202311032).

  
  
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  Figure 1  The discovery process of LEN. The figure was created using ChemDraw.

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  Table 1.  PK model-informed release rates and total fraction dose release of LEN.

  Formulation	Species	Dose	Kindirect (h−1)	Tlag/MTT (day)	T1/2 (day)	Fracindirect	F (%)
  Aqueous suspension (100 mg/mL)	Rat	10 mg/kg	0.00309	6.5	9.3	0.91	136
  	Dog	6 mg/kg	0.00326	0.6	8.8	0.87	92
  	Human	30–450 mg	0.000623	0.3	46.3	0.86	112
  PEG/water solution (309 mg/mL)	Rat	50 mg/kg	0.000818	15	35.3	0.98	75
  	Dog	12 mg/kg	0.0015	7.3	19.2	0.94	80

  下载: 导出CSV
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