Introduction

The human immunodeficiency virus type‐1 (HIV‐1) is a retrovirus that progressively attacks and weakens the immune system, specifically targeting CD4+T cells. If left untreated, HIV infection leads to acquired immunodeficiency syndrome (AIDS), the clinical stage defined by profound immunosuppression and susceptibility to opportunistic infections [1]. Since the start of the epidemic, an estimated 91.4 million people have acquired HIV, with approximately 40.8 million people living with the virus globally by the end of 2024 [2]. Despite these, significant advances in the antiretroviral therapy (ART) have transitioned AIDS from a fatal diagnosis to a manageable chronic condition, significantly extending the lifespan and improving the quality of life of affected patients [3].

In the search for effective antiretroviral agents, targeting the viral enzyme integrase (IN) has emerged as a cornerstone of modern ART, offering a vital alternative for patients who have developed resistance to traditional ART regimens. The discovery of drugs targeting a specific step of the integration process, referred to as IN strand transfer inhibitors (INSTIs), has marked a key breakthrough in this area. Subsequent optimization of first‐generation inhibitors led to the development of second‐generation INSTIs, which offer superior genetic barriers to resistance and enhanced pharmacokinetic (PK) profiles, establishing them as frontline components of modern ART [4, 5, 6]. However, despite these great achievements, HIV treatment remains a substantial burden. The virus's exceptionally high mutation rate frequently renders established therapeutic regimens ineffective over time. This concern is further underscored by recent findings that second‐generation INSTIs can select for resistant mutants [7, 8, 9, 10]. Some of these variants exhibit cross‐resistance to other antiretrovirals, including INSTIs, highlighting the impelling need to explore alternative mechanisms to inhibit HIV‐1 effectively.

Over the last two decades, targeting IN beyond its active site through noncatalytic, allosteric IN inhibitors (ALLINIs) has emerged as a promising, complementary strategy to enhance antiviral potency and limit the selection of mutant strains. In this scenario, disrupting the interaction between IN and the host cofactor lens epithelium–derived growth factor (LEDGF/p75) led to the development of early protein–protein interaction ALLINIs with significant antiviral effects and notable genetic barriers to resistance. Notably, they showed no cross‐resistance with existing antiretrovirals, making them ideal candidates for supplementing current ART regimens. In addition to elucidating the underlying basis for ALLINI activity, insightful studies have aided in unveiling previously unrecognized aspects of HIV‐1 biology, opening new avenues for therapeutic intervention and highlighting new opportunities to target IN outside its catalytic site [11, 12, 13]. Continuous research has since identified several promising ALLINIs with both in vitro and in vivo efficacy, some of which have advanced in clinical trials as next‐generation therapeutic options.

This review provides an extensive overview of ALLINIs. Following a stepwise historical perspective, we discuss the discovery and development of this class of inhibitors, highlighting both the key insights clarifying their unique mechanism of action, and the medicinal chemistry efforts which have ultimately led to some of the most promising and advanced anti‐HIV‐1 candidates. Furthermore, we highlight a set of recent inhibitors which, despite sharing similarities with ALLINIs, demonstrate a distinct dual‐mode of inhibition by targeting both the catalytic activity of IN and IN–RNA binding.

HIV‐1 Life Cycle

HIV‐1 belongs to the Retroviridae family and is an enveloped virus characterized by an icosahedral capsid enclosing the viral genome. As with all retroviruses, the genome consists of two copies of a positive sense single‐stranded RNA (ssRNA) [14]. HIV‐1 selectively packages the viral genome and introduces specific modifications to the +ssRNA [15, 16]. The viral genome encodes three structural polyproteins (Gag, Pol, and Env) as well as six accessory proteins (Tat, Rev, Nef, Vpr, Vif, and Vpu), all of which play essential roles in viral replication and pathogenesis. HIV‐1 entry into host cells is mediated by the viral envelope glycoproteins gp120 and gp41, which recognize the CD4 receptor in combination with the CCR5 or CXCR4 coreceptors, expressed on target cells such as T helper lymphocytes and microglial cells [15, 16, 17]. Following receptor engagement and membrane fusion, the viral core is released into the cytoplasm, where the viral replication cycle begins.

A hallmark of HIV‐1 replication is the reverse transcription of the ssRNA genome into double‐stranded DNA (dsDNA). Reverse transcription is carried out by the virally encoded reverse transcriptase (RT), a multifunctional enzyme that exhibits both RNA‐dependent DNA polymerase activity, responsible for the synthesis of the RNA:DNA intermediate, and ribonuclease H (RNase H) activity, which selectively degrades the RNA strand of the RNA:DNA hybrid [18, 19]. Following reverse transcription, the newly synthesized viral dsDNA is transported to the nucleus, where another viral enzyme, IN, catalyzes its insertion into the host genome through a tightly regulated integration process. Finally, as observed for other retroviruses, viral maturation requires the activity of the HIV‐1 protease (PR), which cleaves the Gag and Gag‐Pol polyproteins into functional viral components, yielding infectious virions.

HIV‐1 IN

A defining hallmark of retroviral replication is the obligatory integration of the viral genome into the host DNA, a feature that clearly distinguishes retroviruses from all other viral families. This critical step is mediated by the viral enzyme IN, which, together with RT and PR, constitutes the canonical enzymatic triad encoded by replication‐competent retroviruses [11, 12, 20].

HIV‐1 IN is a member of the polynucleotidyl transferase superfamily and is a 32 kDa protein composed of 288 amino acids, functioning as a higher‐order multimer and organized into three distinct structural domains [12]. The N‐terminal domain (NTD) contains a conserved zinc‐binding motif that coordinates a Zn2+ ion, ensuring proper protein folding and multimerization. The catalytic core domain (CCD) harbors the conserved DDE catalytic triad (D64, D116, and E152), which coordinates two Mg2+ ions essential for IN catalytic activity. Finally, the C‐terminal domain (CTD) is involved in specific interactions with both viral and host DNA.

HIV‐1 IN functions as a multimer, and all three domains contribute to the formation and stabilization of its functional oligomeric state (Figure 1) [12, 18, 21, 22].

FIGURE 1: Structure of tetrameric HIV‐1 strand transfer complex intasome. Cartoon representation of the HIV‐1 stable synaptic complex after strand transfer reaction, as determined by cryoEM (PDB: 5U1C). The viral DNA is presented as ladders in red, whereas the target DNA is colored in orange. The IN protomers bound to DNA (inner INs) are shown as ribbons in light blue and pink, the other IN protomers (outer INs) are rendered as ribbons in light green and yellow. Magnesium ions are shown as gray spheres.

To gain detailed insights into the mechanism of HIV‐1 DNA integration, prominent efforts have been devoted to obtaining high‐resolution structures of key integration intermediate nucleoprotein complexes. However, structural studies of HIV‐1 intasomes have proven challenging, largely due to the propensity of HIV‐1 IN and the assembled complexes to undergo aggregation [21, 22, 23].

While early studies on intasomes relied on related retroviruses (such as prototype foamy virus, PFV) [24, 25, 26, 27] and homology modeling for HIV‐1 [28, 29], advances in cryo‐EM have ultimately enabled the determination of high‐resolution structures [22]. These revealed a dynamic organization containing a minimum of four IN subunits and as many as 16 subunits, all arranged around the central viral DNA ends. Notably, this has marked a key milestone in the field, highlighting unprecedented features of the unique architecture of the HIV‐1 intasome (Figure 1) [11, 12, 22, 30].

In addition, this achievement has catalyzed the resolution of related structures encompassing bound INSTIs, yielding key insights into their underlying mechanism of action [31]. Notably, these structures revealed key differences in drug binding that were previously unappreciated in studies using PFV intasome surrogates. By revealing in‐depth details at near‐atomic resolution, these findings have helped to conclusively rationalize how such inhibitors bind to their native target, providing key insights for rationally designing next‐generation inhibitors while explaining the molecular basis for drug‐resistant HIV‐1 phenotypes [4, 11, 12, 31, 32, 33].

HIV‐1 IN mediates the insertion of the retrotranscribed viral dsDNA into the host cell genome through two sequential and spatially distinct catalytic steps. In its multimeric form, the enzyme assembles a stable synaptic complex (SSC) with viral DNA [12, 22]. The first reaction occurs in the cytoplasm within the preintegration complex (PIC), a nucleoprotein assembly that includes viral enzymes and host factors to shield the viral genome from degradation. This step, termed 3′‐processing (3′‐P), involves the removal of a GT dinucleotide from each 3′‐end of the viral long terminal repeats (LTRs). The second reaction, known as strand transfer (ST), takes place in the nucleus and results in the covalent integration of the processed viral DNA into host chromosomal DNA, followed by gap repair mediated by the host DNA repair machinery (Figure 2) [11, 12, 20, 30, 34].

FIGURE 2: The linear viral reverse transcript (orange lines; plus‐strands shaded more darkly than the yellow minus‐strands throughout the diagram) contains a copy of the LTR at each end. These LTRs are composed of blue U3, yellow R repeat, and light‐orange U5 sequences. The upstream LTR is bordered by the primer‐binding site (PBS), while the downstream element is adjacent to the polypurine tract (PPT). During the 3′‐P, IN hydrolyzes the DNA strands immediately next to the invariant CA dinucleotides. In the case of HIV‐1, this reaction liberates the pGT‐OH dinucleotide from the ends of the viral DNA. Following nuclear translocation, the intasome engages with the host target DNA (represented by red lines) to mediate DNA strand transfer. The remaining DNA gaps are subsequently repaired by the host cell machinery, yielding a target site duplication (thin green lines) that flanks the integrated provirus.

Among the cellular cofactors involved in facilitating the integration process, LEDGF/p75 [35, 36] plays a key role by tethering IN to chromatin through specific interactions between its IN binding domain (IBD) and the V‐shaped pocket at the CCD dimer interface [37, 38]. LEDGF/p75 is a nuclear protein belonging to the hepatoma‐derived growth factor (HDGF)‐related protein (HRP) family [39]. A defining feature of this family is the presence of a highly conserved N‐terminal PWWP domain, which is involved in chromatin binding and protein–protein interactions. In the LEDGF/HRP family, this specifically appears as a modified proline–histidine–tryptophan–proline (PHWP) motif. Its key functional role, however, resides in the C‐terminal region, which contains the IBD. This domain enables direct interaction with HIV‐1 IN, acting as a molecular tether that anchors the PIC to host chromatin and facilitates its targeting to transcriptionally active regions, thereby directing viral DNA integration across active gene bodies (Figure 3). The interaction is mediated by approximately 80 residues within the IBD, which primarily engage the CCD of IN, with additional contributions from the NTD in enhancing binding affinity. Furthermore, LEDGF/p75 stabilizes IN by shielding it from proteasomal degradation, thereby promoting efficient viral integration and productive replication [36, 40, 41, 42, 43].

FIGURE 3: LEDGF/p75 recruits the HIV PIC and promotes HIV integration at a nearby site. The PIC, depicted in purple, is composed of IN and additional viral/host proteins associated with the viral DNA. Viral IN interacts with LEDGF/p75 (shown in light blue), forming a stable complex.

HIV‐1 INSTIs

Owing to its essential function in the viral replication cycle and its well‐established druggability, HIV‐1 IN has become a major target for antiretroviral drug discovery. Since the emergence of HIV infection, extensive efforts have led to the development of numerous IN inhibitors, several of which are now routinely employed in therapy. All IN inhibitors currently approved for clinical use belong to the class of INSTIs, which selectively block the ST reaction by chelating the two Mg2+ ions within the CCD, thereby preventing the stable integration of the viral dsDNA into the host genome [4, 5, 6, 12, 32, 44, 45]. The discovery of diketoacid (DKA) derivatives as selective INSTIs represented an important milestone in the field, providing early evidence for validating IN as a viable pharmacological target. These compounds feature a DKA pharmacophore consisting of a coplanar, metal‐coordinating oxygen triad linked to a heteroaromatic core and a halobenzyl moiety. The latter occupies a narrow pocket (normally reserved for the 3′‐terminal base of the viral DNA) created by the ejection of the invariant GT dinucleotide after the 3′‐P. Here, the halobenzyl moiety interacts with the penultimate cytidine at the 3′ end of the DNA reactive strand, displacing the terminal 3′‐adenosine through an induced‐fit mechanism [5, 24, 28, 45, 46, 47, 48, 49, 50, 51].

Optimization efforts focused on the modification of the DKA scaffold to lock the pharmacophore into a coplanar conformation in DKA bioisosteric analogs. These structural refinements led to the development of potent in vitro and in vivo inhibitors, eventually resulting in the first approved INSTIs raltegravir (RAL, 1, Figure 4) and elvitegravir (EVG, 2, Figure 4) [4, 5, 45, 48, 52, 53, 54, 55].

FIGURE 4: Chemical structures of INSTIs. First‐generation INSTIs: RAL (1) and EVG (2) and second‐generation INSTIs: DTG (3), BIC (4) and CAB (5).

Although the discovery of RAL and EVG marked a major milestone in ART, their clinical utility was frequently hampered by suboptimal PK profiles and a low genetic barrier to resistance. Specifically, the rapid development of single or combined IN mutations often led to relevant cross‐resistance, compromising the extensive use of these first‐generation INSTIs [4, 5, 30, 33, 45, 56, 57, 58].

To address these limitations, prominent medicinal chemistry efforts were undertaken to identify effective INSTIs with improved antiviral efficacy, a higher genetic barrier to resistance, and favorable PK properties. These investigations focused on the identification of inhibitors bearing a more conformationally constrained metal‐binding pharmacophore, exemplified by the tricyclic carbamoyl‐pyridone scaffold of the second‐generation INSTI dolutegravir (DTG, 3, Figure 4). Of note, this scaffold retains the essential triad of heteroatoms required for Mg2+ chelation while improving geometric preorganization and reducing entropic penalties upon binding. Subsequent optimizations focused on fine‐tuning interactions within the IN–viral DNA complex through the introduction of optimized hydrophobic substituents capable of occupying adjacent pockets and reinforcing π–π stacking with viral DNA bases. These efforts led to the development of structurally related analogus such as bictegravir (BIC, 4, Figure 4) and cabotegravir (CAB, 5, Figure 4), which share the same metal‐chelating scaffold as DTG but incorporate additional structural refinements, including expanded bicyclic systems and tailored side chains [4, 5, 12, 30, 33, 44, 57, 58, 59, 60, 61, 62, 63, 64].

Such modifications have significantly broadened the efficacy of INSTIs against challenging viral strains resistant to first‐generation INSTIs, while also providing slower dissociation kinetics from the intasome [5, 30, 64, 65, 66]. Importantly, the improved PK characteristics of second‐generation INSTIs have translated into significant clinical advantages, including once‐daily unboosted regimens and, in the case of CAB, long‐acting injectable formulations administered monthly or at even longer intervals. This reduced dosing frequency markedly improves treatment adherence and represents a major advancement not only for therapeutic regimens but also for preventive strategies. Notably, long‐acting CAB has been approved for pre‐exposure prophylaxis (PrEP), underscoring the transformative impact of second‐generation INSTIs beyond treatment and into HIV prevention [4, 5, 6, 63, 64, 65, 66, 67, 68, 69, 70].

Noteworthy, the successful optimization of these second‐generation agents was crucially informed by the extensive structure–activity relationship (SAR) and resistance data gathered from first‐generation INSTIs, alongside advancements in the structural and functional understanding of IN [4, 5, 11, 12, 24, 28, 30, 31, 32, 33, 44, 60, 61, 62, 64].

Despite these, the emergence of viral strains with reduced susceptibility to INSTIs remains a critical challenge [7, 8, 9, 10, 71, 72, 73]. Beyond the primary mutations identified under selective pressure from second‐generation INSTIs [5, 12, 71, 72, 73, 74, 75], significant concern has shifted toward distal substitutions located away from the active site. Structural studies have indicated that these residues are not directly involved in INSTI binding; instead, they contribute to the secondary coordination sphere of the catalytic Mg2+. Mutations at these sites can perturb the precise coordination geometry of the metal ions, thereby indirectly modifying the electronic and spatial environment of the catalytic core and resulting in impaired inhibitor affinity [4, 12, 32, 33].