Summary and Outlook

As a hallmark of HIV‐1 replication, efforts to discover new anti‐HIV agents have focused extensively on the search for therapeutic agents targeting IN. These efforts have culminated in the development of first‐ and second‐generation INSTIs, which, owing to their remarkable antiviral efficacy and favorable PK profiles, have dramatically transformed ART regimens to become a cornerstone. However, the continuous selection of resistant variants remains a significant challenge, hindering the broad therapeutic utility of even these highly effective drugs. While resistance to second‐generation INSTIs has emerged clinically only recently, the decline in efficacy of first‐generation INSTIs occurred soon after their approval, limiting their long‐term use. These findings have prompted the exploration of alternative strategies to target IN through mechanisms of inhibition different from those of INSTIs.

Given the complex interplay between IN and human cofactors during integration, research has focused on targeting IN noncatalytically through the development of allosteric inhibitors capable of circumventing cross resistance issues and escape mutations within the catalytic site. In this context, ALLINIs have provided fundamental insights in the field, proving that targeting IN beyond its active site is a viable antiviral strategy. Indeed, ALLINIs have demonstrated efficacy against a broad range of viral clades, including those resistant to approved INSTIs. Furthermore, the fact that ALLINIs are not antagonistic to INSTIs, but rather demonstrate a likely synergistic or additive effect, suggests the great promise for their use in combination regimens. Targeting both the catalytic and allosteric functions of IN could likely delay the onset of resistance, indicating that INSTI‐ALLINI combinations could provide great therapeutic utility in the near future.

To establish ALLINIs as viable therapeutic options, extensive studies were conducted to optimize their drug‐like properties. This encompassed investigating not only their biochemical properties, but also their antiviral efficacy, genetic barrier to resistance, PKs, and toxicological properties. In this context, SAR studies, supported by biochemical and structural investigations, have been central to elucidating the key structural features impacting ALLINI activity. Exploration of chemical modifications has proven fundamental to understanding how specific characteristics impact the multimodal mode of action of these inhibitors.

While the 2‐(tert‐butoxy)‐2‐(4‐substituted aryl‐3‐yl)acetic acid moiety has been established as key pharmacophore of ALLINIs, fine‐tuning of structural features significantly impacts biological activity. This is specifically highlighted by compounds 27 and 29 (Figure 17), which, despite featuring alternative central scaffolds relative to the previously described ALLINIs, have shown prominent properties in both in vitro and in vivo models. Importantly, given their exceptional profiles, these ALLINIs represent a breakthrough in the field and are currently under investigations in clinical trials, further corroborating the great potential of ALLINIs as next‐generation therapeutics.

Research on ALLINIs has highlighted unexpected features in their mode of action. Although initial studies were primarily focused on developing protein–protein interaction inhibitors to disrupt the IN–LEDGF/p75 binding, the mechanism of action of ALLINIs has proven more complex. While disrupting protein–protein interaction could contribute to their efficacy, studies have found a major impact on IN multimerization. Notably, these investigations have been supported by recent advances in biophysical and structural biology, which have facilitated in‐depth investigations of the molecular basis of ALLINI function at near‐atomic detail.

Given their role in stabilizing CCD–CTD interactions to ultimately yield nonfunctional IN, ALLINIs have been recently categorized as molecular glues. However, differently from molecular glue degraders, which promote target degradation by recruiting E3 ligases, ALLINIs are more accurately described as nondegrading molecular glues [146]. Indeed, instead of exploiting the ubiquitin–proteasome system to eliminate disease‐associated proteins, ALLINIs work by stabilizing protein interfaces. This disrupts the structural and functional features of IN, inducing rapid, higher‐order multimerization and hyper‐aggregation of IN that ultimately impairs virion morphogenesis. Exploiting such mechanism is of great interest in modern drug discovery, with the development of nondegrading molecular glues becoming a major focus in the field. These findings, combined with the unprecedented structural insights from recent studies and extensive SAR data from past years, hold great promise for the rational design and optimization of next‐generation ALLINIs in the near future.

Furthermore, given their multimodal nature, ALLINIs have served as relevant investigational tools to probe the inherently complex structural and functional features of IN. A paradigmatic example of this is found in structural studies that characterized the enzyme's complex multimerization states [134]. By using these inhibitors to stabilize IN complexes, it was revealed that the enzyme possesses an intrinsic propensity to form not only linear aggregates but also complex branched polymers with fractal‐like properties. These studies demonstrated that homomeric interactions between the CTD act as critical branching points that transform the enzyme into an inactive, gel‐like state. Without the stabilizing effect of ALLINIs as research tools, the full extent of this structural plasticity and the CTD's role in driving higher‐order multimerization might have remained hidden.

This deep understanding of the viral structural network highlights potential avenues for future drug design targeting specific atomic vulnerabilities. A particularly strategic prospect lies in the identification of a novel binding pocket at the CCD–CTD interface, created by a significant conformational shift of W131 [116]. This pocket, found to be occupied by an ethylene glycol molecule in crystal structures, offers a strategic opportunity to modify traditional scaffolds. Such refinements could increase binding affinity and raise the genetic barrier against common resistance mutations like T125 and A128, which disrupt the complex interplay between IN, the capsid, and the inhibitor.

In addition to their therapeutic potential, ALLINIs have served as fundamental tools for unveiling previously unrecognized aspects of HIV‐1 biology. The discovery that ALLINIs primarily affect the late stages of the HIV‐1 life cycle has aided in unraveling their major effect on maturation of viral particles through the inhibition of IN–RNA interaction. This feature, which was previously unknown in the field, has allowed researchers to gain additional insights into mechanisms underlying ALLINI function, revealing that the binding of IN to RNA is fundamental for virion infectivity. These structural insights provided the conceptual foundation for the latest breakthroughs in high‐resolution cryo‐EM [153]. Consequently, it is now clear that the ability of IN to multimerize is not merely a drug‐induced artifact but a critical native function. In mature virions, IN forms ordered filaments that serve as a structural scaffold to organize and anchor the viral RNA genome. The fundamental insights provided by ALLINI‐based studies allowed researchers to visualize IN's polymeric potential, enabling cryo‐EM to eventually confirm that this architecture is fundamental for proper virion morphogenesis and genome packaging. Overall, these impactful studies have provided key benchmarks for innovative, structure‐based drug discovery campaigns focused on unprecedented mechanisms.

Looking forward, the search for innovative mechanisms has led to the development of dual‐action inhibitors [148] capable of targeting both the IN catalytic site and the IN–RNA interface, offering a bifunctional model that could drastically limit viral escape routes.

The future of HIV‐1 therapy lies in this continued integration of medicinal chemistry and structural biology, turning every new insight into IN multimerization and IN–RNA binding interaction into a more effective therapeutic weapon. Ultimately, the true legacy of ALLINIs lies in their ability to bridge the gap between molecular inhibition and structural orchestration, turning the virus's own complexity into its ultimate vulnerability and providing the blueprint for a future where HIV‐1 is systematically outsmarted.